CN110122482B - Preparation process of plant immune protein soluble powder of alternaria tenuissima PeaT1 - Google Patents
Preparation process of plant immune protein soluble powder of alternaria tenuissima PeaT1 Download PDFInfo
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Abstract
The invention discloses a preparation process of a plant immune protein soluble powder of Alternaria tenuissima PeaT1, belonging to the field of powder production, and comprising the following steps of A, inoculating hypha to a culture medium flat plate and putting the culture medium flat plate into a constant-temperature incubator for culturing for at least 72 hours; b, selecting a small amount of thalli from the culture medium flat plate, inoculating the selected thalli into a shake flask liquid culture medium, and performing seed fermentation and shaking culture; c, inoculating the seed shake flask into a fermentation tank for at least 2-stage fermentation; d, adding 0.5M Tris HCl buffer solution to protect protein after fermentation is completed, and then breaking the wall; and E, filtering the crude protein solution obtained by breaking the walls of the mycelium cells by using a 200-mesh screen, removing residues and the tissue of the mycelium cells, retaining the crude protein solution, adding a carrier filler aid, and drying to obtain soluble powder. The invention improves the formula of the culture medium by optimizing the fermentation process and parameters, reduces the cost of fermentation raw materials, shortens the fermentation time, reduces the fermentation pollution probability and reduces the fermentation energy consumption cost.
Description
Technical Field
The invention belongs to the field of powder production, and particularly relates to a preparation process of a plant immune protein soluble powder of alternaria tenuissima PeaT 1.
Background
PeaT1 is an acidic protein with thermal stability separated and purified from Alternaria tenuisma (Alternaria Tenuissma), no relevant report exists at home and abroad, PeaT1 is an originally created new functional microbial protein in China, and a coding gene of the protein is cloned (GenBank accession number: CH 445335) (Longsonghua, Zhang, Qidwin, and the like. PeaT1 can stimulate the tobacco production system to acquire resistance (Mao J, Liu Q, Yang X, et al. Purification and expression of a protein enzyme from Alternaria tenuissima and an enzyme-mediated destination stresses in tobacaco [ J ]. Annals of Applied Biology, 2010, 156(3):411 < - > 420.), improve the activity of protective enzymes such as POD, SOD and the like in crops, promote the growth of crops, and improve the yield and quality (Zhanggangqi, Qiuden, Qiuchun, and the like. Alternaria alternata protein elicitor researches on the main properties and related enzyme changes of cotton [ J ]. Cotton bulletin, 2008, 20(3):186 < - > 191). Besides tobacco, the protein elicitor PeaT1 can also improve the yield of crops such as tomato, rice, cotton and the like, and promote the growth of Arabidopsis (Li, Qiu De Wen, Liu Zheng, etc.. plant activator protein has an induction effect on the disease resistance of tomato [ J ]. Chinese biological prevention and control academy 2005, 21(4): 265) 268.; left bin, Qiu De Wen, Luo Wida. plant activator protein has an effect on the growth of rice seedlings and related enzyme activities [ J ]. science and technology and engineering 2005, 5(17 1260): 1262.; Zhang Jun, Meizheng Ding, Yang Dun, etc.. the fungal activator protein has an effect on the main yield traits and fiber quality traits of cotton plants [ C ], 2006). The PeaT1 has the condition for development as a biopesticide, but no research on the large-scale production thereof has been carried out. Pilot plant of pesticide in corridor of plant protection department of Chinese academy of agricultural sciences firstly registers pesticide, and prepares 6% oligosaccharide-catenin wettable powder (trade name: Altailing) by matching with amino-oligosaccharin, and obtains formal registration certificate of pesticide (registration certificate number: PD 20171725) in 2017 and 8 months.
The wettable powder is a dosage form which is prepared by fully mixing and crushing original pesticide, inert filler and a certain amount of auxiliary agent according to a proportion to achieve a certain powder fineness. The product has no difference from powder in shape, but can be wetted and dispersed by water to form suspension after being added into water due to the addition of auxiliary agents such as wetting agent and dispersing agent, and can be sprayed for application.
The soluble powder refers to powder and pesticide which are completely soluble in water. Is a processing agent of pesticide. The pesticide is prepared by mixing and grinding a pesticide raw material with high water solubility or a pesticide raw material with low water solubility with hydrophilic groups, water-soluble inorganic salt, an adsorbent and the like. The fineness of the powder particles is required to be 98 percent and pass through a 80-mesh sieve. The preparation can be diluted with water, quickly dissolved in water and dispersed and suspended in water, and does not block a spray head during spraying. The dosage form has higher control effect than wettable powder, is convenient to use and is convenient to package and transport.
The soluble powder can form true solution in water without precipitation or long-term standing without precipitation. The spraying device is suitable for spraying high-concentration liquid and is used for preventing and treating airplane spraying and drip irrigation without blocking a spray head. The wettable powder has poor suspension performance, more sediment is generated during use, dust pollution is caused, and for manufacturers with the general production technology which is not too hard, the wettable powder has long wetting time, poor suspension rate and generation of sediment after long-term storage, is used for airplane spraying and drip irrigation, is easy to block a spray head, causes phytotoxicity or has poor effect, and is a dosage form to be eliminated.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation process of the plant immunoprotein soluble powder of the Alternaria tenuissima PeaT1, which is characterized in that the large-scale production cost is obviously reduced and the product yield is obviously improved by optimizing the fermentation process, improving the culture medium formula and improving the extraction process, and the added filler components are changed to prepare a new soluble powder formulation by a new process of high-pressure homogenizing and crushing and filtering residues with a 200-mesh sieve, so that the product solubility, the suspension rate and the wettability are obviously improved, no precipitate is generated, and the preparation process can be widely applied to modern agricultural use scenes such as airplane spraying, drip irrigation and the like, thereby the market of the product is wider.
In order to solve the technical problems, the invention adopts the technical scheme that: a preparation process of a plant immune protein soluble powder of Alternaria tenuissima PeaT1 is characterized by comprising the following steps,
step A, inoculating hyphae to a culture medium flat plate and putting the culture medium flat plate into a constant-temperature incubator for culturing for at least 72 hours;
b, selecting a small amount of thalli from the culture medium flat plate, inoculating the selected thalli into a shake flask liquid culture medium, and performing seed fermentation and shaking culture;
c, inoculating the seed shake flask into a fermentation tank for at least 2-stage fermentation;
d, adding 0.5M Tris HCl buffer solution to protect protein after fermentation is completed, and then breaking the wall;
and E, filtering the crude protein liquid obtained by breaking the cell walls of the mycelia through a 200-mesh screen, removing residues of a fermentation culture medium and cell wall tissues of the mycelia, retaining the crude protein liquid, adding a carrier filler aid, and drying to obtain soluble powder.
The beneficial technical effects of the invention are as follows:
1. by optimizing the fermentation process and parameters and improving the formula of the culture medium, the cost of fermentation raw materials is reduced; the fermentation time is shortened to 74h, the fermentation pollution probability is reduced, the fermentation energy consumption cost is reduced, and the mycelium yield is increased to 54 g/L;
2. the extraction process is improved, so that the product yield is obviously improved: the cell wall breaking rate reaches more than 95 percent after high-pressure homogenization and crushing, so that the yield of the protein reaches 3.3g/L, and the cost for producing the protein product with the same concentration in the future is indirectly greatly reduced;
3. high-pressure homogenizing and cell breaking and 200-mesh screen filtering process are added to remove impurities. The physical properties of the product are improved, and the water-insoluble substances are greatly reduced; the fermentation liquor is reserved, and the reserved fermentation clear liquid contains nutrients such as soluble polysaccharide which is not utilized in fermentation and metabolites obtained by fermentation of bacteria, so that the product effect is improved to a certain extent;
4. a detection method for measuring the plant immune protein PeaT1 by a spectrophotometer is established;
5. the formula of the carrier filler assistant is researched, the plant immune protein PeaT1 soluble powder is prepared, the product character of the plant immune protein PeaT1 soluble powder is obviously improved in the indexes of solubility, suspension rate, wettability and water-insoluble substances compared with the character of wettable powder, the plant immune protein PeaT1 soluble powder is a brand new formulation, and favorable conditions are created for popularizing modern agricultural markets (flying prevention and drip irrigation) in the future.
The present invention will be described in detail with reference to the accompanying drawings.
Drawings
FIG. 1 is a flow chart of the process for preparing the soluble powder of Alternaria tenuissima PeaT1 plant immune protein of the present invention;
FIG. 2 is a PeaT1 standard curve;
FIG. 3 is a wheat root vigor standard curve;
FIG. 4 is a graph showing the root activity.
Detailed Description
Referring to the attached figure 1, the invention provides a preparation process of a plant immune protein soluble powder of Alternaria tenuissima PeaT1, which comprises the following steps.
And step A, inoculating the hyphae to a culture medium plate and putting the culture medium plate into a constant-temperature incubator for culturing for at least 72 hours.
Specifically, the plate of the medium inoculated with the mycelia was cultured in a constant temperature incubator at 28-32 ℃ for at least 72 hours. The culture medium plate is a potato agar culture medium plate, and the formula of the culture medium comprises 200g of potatoes, 20g of sugar, 1L of water and 18g of agar powder.
And step B, selecting a small amount of thalli from the culture medium flat plate, inoculating the thalli into a shake flask liquid culture medium, and performing seed fermentation and shaking culture.
Specifically, the formula of the shake flask liquid culture medium comprises, by mass, 3.0-5.5% of corn starch, 1.0-4.5% of soybean meal, 0.1-1.0% of sugar, 0.01-1.00% of ammonium sulfate, 0.01-1.00% of a protein production inducer, 0.01-0.50% of a protein activator I, 0.01-0.50% of a protein activator II and the balance of water. The shaking culture condition is that the shaking culture is carried out for at least 40h under the conditions of 180 rpm-200 rpm and 28-32 ℃.
And step C, inoculating the shake flask of the seeds into a fermentation tank for at least 2-stage fermentation.
In particular, a stage 2 or 3 fermentation may be performed in this step.
The first embodiment is as follows:
when performing a level 2 fermentation, a level 1 fermentation comprises the following steps: inoculating the seed into a 500L fermentation tank for fermentation, wherein the liquid loading of the culture medium is 65%, the sterilization temperature is 121 ℃, the sterilization time is 30min, the fermentation culture temperature is 28-32 ℃, the pH value is 6.0-7.2, and the fermentation time is 36 h; the 2-stage fermentation comprises the following steps: fermenting the strain in 500L fermentation tank, inoculating into 5000L fermentation tank, and fermenting for 20 hr, wherein the liquid loading of culture medium is 70%, the sterilization temperature is 121 deg.C, the sterilization time is 30min, the fermentation culture temperature is 28-32 deg.C, the pH is 6.0-7.2.
In detail, the formula of the culture medium during the 1-level fermentation comprises, by mass, 3.0-5.5% of corn starch, 1.0-4.5% of soybean meal, 0.1-1.0% of sugar, 0.01-1.00% of ammonium sulfate, 0.01-1.00% of a protein production inducer, 0.01-0.50% of a protein activator I, 0.01-0.50% of a protein activator II and the balance of water; the formula of the culture medium for 2-level fermentation comprises, by mass, 3.0-5.5% of corn starch, 1.0-4.5% of soybean meal, 0.1-1.0% of sugar, 0.01-1.00% of ammonium sulfate, 0.01-1.00% of a protein production inducer, 0.01-0.50% of a protein activator I, 0.01-0.50% of a protein activator II and the balance of water.
Example two:
when performing a stage 3 fermentation, a stage 1 fermentation comprises the following steps: inoculating the seed into a 500L fermentation tank for fermentation, wherein the liquid loading of the culture medium is 65%, the sterilization temperature is 121 ℃, the sterilization time is 30min, the fermentation culture temperature is 28-32 ℃, the pH is 6.0-7.2, and the fermentation time is 36 h; the 2-stage fermentation comprises the following steps: fermenting the strain in 500L fermentation tank, inoculating into 5000L fermentation tank, and fermenting for 20 hr, wherein the liquid loading of culture medium is 70%, the sterilization temperature is 121 deg.C, the sterilization time is 30min, the fermentation culture temperature is 28-32 deg.C, the pH is 6.0-7.2; the 3-stage fermentation comprises the following steps: the strain is inoculated into a 40000L fermentation tank for 3-level fermentation after being fermented in a 5000L fermentation tank, wherein the liquid loading of a culture medium is 75%, the sterilization temperature is 121 ℃, the sterilization time is 30min, the fermentation culture temperature is 28-32 ℃, the pH value is 6.0-7.2, and the fermentation time is 18 h.
In detail, during the level 1 fermentation, the formula of the culture medium comprises, by mass, 3.0-5.5% of corn starch, 1.0-4.5% of soybean meal, 0.1-1.0% of sugar, 0.01-1.00% of ammonium sulfate, 0.01-1.00% of a protein production inducer, 0.01-0.50% of a protein activator I, 0.01-0.50% of a protein activator II and the balance of water; during 2-level fermentation, the formula of the culture medium comprises, by mass, 3.0-5.5% of corn starch, 1.0-4.5% of soybean meal, 0.1-1.0% of sugar, 0.01-1.00% of ammonium sulfate, 0.01-1.00% of a protein production inducer, 0.01-0.50% of a protein activator I, 0.01-0.50% of a protein activator II and the balance of water; during 3-level fermentation, the formula of the culture medium comprises, by mass, 3.0-5.5% of corn starch, 1.0-4.5% of soybean meal, 0.1-1.0% of sugar, 0.01-1.00% of ammonium sulfate, 0.01-1.00% of a protein production inducer, 0.01-0.50% of a protein activator I, 0.01-0.50% of a protein activator II and the balance of water.
Because the fermentation period existing in the fungus fermentation process is long, the fermentation process is easy to pollute and other problems, a large number of fermentation small and medium tests are carried out on Alterneria tenuissima in factories and laboratories, and the optimized fermentation medium formula removes expensive peptone, potato starch and soybean meal; the use amount of several trace elements in the culture medium is increased, the corn starch and the soybean meal powder are selected and used as agricultural and sideline products, the soybean meal is the remainder of the soybean after oil extraction, the price is low, large-scale and nearby purchase is convenient, and the raw material cost and purchase cost of large-scale production are greatly saved; the dry weight of the mycelium obtained after fermentation is obviously improved by optimizing the formula of the culture medium, and reaches 54 g/L. Through adjusting tests on the fermentation temperature, pH, ventilation capacity and liquid loading capacity of the culture medium of the fermentation tank, optimal fermentation production parameters and optimal fermentation production process are obtained. Greatly shortens the fermentation time, shortens the fermentation time of the strain in the tank from 86 hours to 74 hours, and greatly reduces the fermentation cost and the contamination risk of the factory scale production.
And D, adding 0.5M Tris HCl buffer solution to protect protein after fermentation is completed, and then breaking the wall.
Adding Tris HCl buffer solution into the bacterial liquid, introducing into a high-pressure homogenizer to break the cell wall of the mycelium, and crushing for 1min at the crushing pressure of 80 MPa. The high-pressure homogenizer utilizes materials to enter a valve group with adjustable pressure under the action of a special plunger, after passing through a flow-limiting gap with a specific width, the materials with instantaneous pressure loss are sprayed out at a very high flow rate (1000-1500 m/s) and collide on an impact ring of one of collision valve components, so as to generate three effects: cavitation effects, impact effects, shear effects. After the three effects are carried out, the particle size of the material can be uniformly thinned to be below 100nm, and the cell wall breaking rate is more than 95%. The PeaT1 is an intracellular protein expressed by Alternaria Tenuissima, but the characteristics of the intracellular protein have considerable requirements on extraction and preparation processes, and compared with the cell wall breaking rate of mycelium grinding by using a colloid mill, the cell wall breaking rate is improved from about 50% to 95% at present by using a high-pressure homogenizer instead of the conventional process of using colloid mill to break cells, the protein yield is obviously improved after the cell wall breaking rate is improved, and the protein yield reaches 3.3 g/L. After homogenization, the fineness of the product is finer and more uniform, and the physical properties of the product are improved. The step greatly reduces the production cost and obviously improves the quality and the yield of the product.
And E, filtering the crude protein liquid obtained by breaking the cell walls of the mycelia through a 200-mesh screen, removing residues of a fermentation culture medium and cell wall tissues of the mycelia, retaining the crude protein liquid, adding a carrier filler aid, and drying to obtain soluble powder.
In the traditional process, plate-frame filtration is carried out after fermentation is finished, supernatant is removed, and mycelia are reserved for cell disruption; the improved process comprises the following steps: filtering with 200 mesh sieve after cell wall breaking to remove mycelium cell wall and fermentation medium residue, and retaining fermentation supernatant; the removal of the residues can obviously improve the quality and physical indexes of the products in the later period: the water insoluble substance of the product is greatly reduced, the solubility is better, the impurities are less, the suspension property and the wettability of the preparation are obviously improved, the product can be used for spraying by a superfine atomizing nozzle used for spraying by an airplane, the blockage of the nozzle is avoided, and the market application range is wider; the reserved fermentation clear liquid contains nutrient substances such as soluble polysaccharide which are not utilized in fermentation and metabolites fermented by bacteria, and the product effect is improved to a certain extent.
The carrier filler additive comprises, by mass, 30-40% of biochemical fulvic acid, 10-15% of white carbon black, 25-40% of humic acid, 3-9% of organic silicon, 4-9% of protein adsorbent, 3-6% of protein protective agent and 2-7% of protein stabilizing agent.
In the above steps, the protein production inducer may be IPTG, lactose, pyruvic acid, etc., or a combination of one or more of them.
The protein activator I and the protein activator II can be metal ions such as potassium ions, magnesium ions, calcium ions and the like; and organic compounds such as bromide ions, anions such as hydrogen ions and the like, glutathione, ethylene diamine tetraacetic acid and the like, and also can be one or a combination of more of the organic compounds.
The protein adsorbent can be activated carbon, silica gel, ion exchange resin, etc., and can also be one or a combination of several of the activated carbon, silica gel, ion exchange resin, etc.
The protein protective agent can be glycine-hydrochloric acid buffer solution, phthalic acid-hydrochloric acid buffer solution, disodium hydrogen phosphate-citric acid buffer solution and the like, and can also be one or a combination of several of the buffer solutions.
The protein stabilizer can be hyaluronic acid-trehalose, sucrose-glycerol-sodium chloride, gelatin-glycerol-sodium chloride, EDTA-glycerol-gelatin-sodium chloride, etc., and can also be one or more of the combination.
The present invention will be described in detail below with reference to the example of the stage 3 fermentation in step C.
A, selecting a small amount of hyphae from a seed tube of Alterneria tenuissima (rotten Chinese cabbage diseased leaves are screened, separated and purified), inoculating the hyphae to a solid potato agar culture medium plate (a culture medium formula is that 200g of potatoes, 20g of sugar, 1L of water and 18g of agar powder), and culturing for at least 72 hours in a constant-temperature incubator at 28 ℃.
And step B, selecting a small amount of thalli from the solid culture medium, inoculating the selected thalli into a shake flask liquid culture medium (the formula of the culture medium comprises, by mass, 3.0% of corn starch, 2% of soybean meal, 0.5% of sugar, 0.3% of ammonium sulfate, 0.5% of a protein production inducer (lactose is adopted in the embodiment), 0.04% of a protein activator I (ethylene diamine tetraacetic acid is adopted in the embodiment), 0.04% of a protein activator II (glutathione is adopted in the embodiment) and the balance of water) which is sterilized at 121 ℃ for 30min, performing seed fermentation, and performing shaking culture for at least 40h under the conditions of 180-200 rpm and 30 ℃.
And step C, inoculating the shake flask of the seeds into a fermentation tank for 3-level fermentation.
1, fermentation at the level of 1: after 40h of culture, the seed shake flask is inoculated into a 500L fermentation tank for 1-stage fermentation, wherein the formula of a culture medium comprises, by mass, 3.0% of corn starch, 2% of soybean meal, 0.5% of sugar, 0.3% of ammonium sulfate, 0.5% of a protein production inducer (lactose is adopted in the embodiment), 0.04% of a protein activator I (ethylene diamine tetraacetic acid is adopted in the embodiment), 0.04% of a protein activator II (glutathione is adopted in the embodiment), and the balance of water, the liquid loading amount of the culture medium is 65%, the sterilization temperature is 121 ℃, the sterilization temperature is 30min, the fermentation culture temperature is 30 +/-1 ℃, the pH is 6.8, and the fermentation time is 36 h.
2, fermenting: the strain is fermented for 36 hours in a 500L fermentation tank and then inoculated into a 5000L fermentation tank for 2-stage fermentation, wherein the formula of a culture medium comprises, by mass, 3% of corn starch, 2% of soybean meal, 0.5% of sugar, 0.3% of ammonium sulfate, 0.5% of a protein production inducer (lactose is adopted in the embodiment), 0.04% of a protein activator I (ethylene diamine tetraacetic acid is adopted in the embodiment), 0.04% of a protein activator II (glutathione is adopted in the embodiment), and the balance of water, the liquid loading amount of the culture medium is 70%, the sterilization temperature is 121 ℃, the fermentation culture temperature is 30 ℃ +/-1 ℃, the pH is 6.8, and the fermentation time is 20 hours.
And (3) fermenting: the strain is fermented for 20 hours in a 5000L fermentation tank and then inoculated into a 40000L fermentation tank for 3-grade fermentation, wherein the formula of a culture medium comprises, by mass, 3% of corn starch, 2% of soybean meal, 0.5% of sugar, 0.3% of ammonium sulfate, 0.5% of a protein production inducer (lactose is adopted in the embodiment), 0.04% of a protein activator I (ethylene diamine tetraacetic acid is adopted in the embodiment), 0.04% of a protein activator II (glutathione is adopted in the embodiment), and the balance of water, the liquid loading amount of the culture medium is 75%, the sterilization temperature is 121 ℃, the sterilization temperature is 30 ℃ plus or minus 1 ℃, the pH is 6.8, and the fermentation time is 18 hours.
And D, adding 0.5M Tris HCl buffer solution to protect protein after fermentation is completed, and then breaking the wall.
And E, filtering the crude protein liquid obtained by breaking the cell wall of the mycelium through a 200-mesh screen. Removing residues of a fermentation culture medium and mycelium cell wall tissues, reserving a crude protein solution, adding a carrier filler auxiliary agent, and drying to obtain soluble powder.
And (3) measuring the content of the crude protein solution: the Coomassie brilliant blue dyeing method is used for detecting the content of crude protein liquid by utilizing the characteristics that Coomassie brilliant blue G-250 dye is combined with basic amino acid and aromatic amino acid residues of protein in an acid solution to cause that the maximum absorption peak of free dye is changed from 465nm to 595nm, and the light absorption value of the protein-dye compound under 595nm is in direct proportion to the protein concentration.
Taking the test tube with the plug, pressing the sample adding table to add samples, reacting for 2min, performing colorimetric determination on a spectrophotometer, recording the optical density value at 595nm, and drawing a standard curve; the concentration of the crude protein liquid is determined by calculating the optical density value of the crude protein liquid on a standard curve, and then the amount of the added carrier and the filler is calculated.
And (3) drawing a sample adding table by using a standard curve:
and (3) batch detection results:
measuring the average value of the absorbance values of the sample at the wavelength of 595nm to be 0.143, and calculating by using a PeaT1 standard curve (see figure 2) to obtain the protein content of 5.1614 mug/mL in the reaction solution; because the dilution multiple is 6 after the dyeing solution is added, the total volume of the solution to be measured is 100ml, the sample volume of the sample to be measured is 500mg, and the protein content in the crude protein solution can be calculated to be 0.62%, the total mass of the carrier and the filler can be about 5T for 30T fermentation liquor, and the quality of the final product can be ensured to be qualified.
Adding carrier filler auxiliary agent, wherein the carrier filler auxiliary agent comprises, by mass, 40% of biochemical fulvic acid, 10% of white carbon black, 30% of humic acid, 5% of organic silicon, 6% of protein adsorbent (specifically adopting activated carbon), 6% of protein protective agent (specifically adopting phthalic acid-hydrochloric acid buffer solution) and 3% of protein stabilizing agent (specifically adopting hyaluronic acid-trehalose).
And (3) drying: adding the protein liquid into a carrier and a filler, and then drying in a centrifugal spray dryer, wherein the material inlet temperature is as follows: 180 ℃ and 200 ℃, outlet temperature: 70-80 ℃. The material is dispersed into mist by a high-speed centrifugal atomizer and is fully contacted with hot air, so that the moisture of the material is instantly evaporated to finish drying, and a powdery finished product with the fineness of 100-200 meshes is formed.
Indoor biological activity assay of the product: soaking seeds of wheat and corn with the product and clear water, comparing the influence of the product and the clear water on the seed germination rate and the root activity, and measuring the seed germination rate by using a counting method; by utilizing the characteristic that 2, 3, 5-triphenyltetrazolium chloride (TTC) can be reduced into insoluble red triphenylmethyle (TTF) by hydrogen, if the root system has vitality, dehydrogenase in the method can use the TTC as a hydrogen acceptor to enable the TTC to become TTF and become red, and the reduction amount is in direct proportion to the vitality of the root system, and the vitality of the root system is measured by using a TTC method.
0.2ml of 0.4% TTC solution was added to a beaker, and a little Na was added2S2O4The red TTF was produced immediately after shaking the powder. Then ethyl acetate is added to the solution to reach the constant volume of 10mL, and the solution is shaken up. Then, 0.25ml, 0.50ml, 1.00ml, 1.50ml and 2.00ml of the solution are respectively taken and placed in a 10ml volumetric flask, ethyl acetate is used for carrying out constant volume to a scale, and then standard colorimetric solution containing 25 microgram, 50 microgram, 100 microgram, 150 microgram and 200 microgram of TTF is obtained, blank is used as a reference, absorbance is measured at the wavelength of 485nm, and a wheat root activity standard curve is drawn (see attached figure 3).
And (3) batch detection results:
measuring the average of the light absorption values of the control group sample under the wavelength of 485nm to be 0.331, and calculating the root activity of the control group sample to be 0.213mg/g/h through a standard curve; the average of the light absorption values of the treatment group samples under the wavelength of 485nm is 0.523, and the root activity of the treatment group samples is calculated to be 0.319mg/g/h through a standard curve, so that the root activity of the treatment group is improved by 50% compared with that of a control group.
Referring to figure 4, 180 wheat seeds were obtained by counting, the germination number of the control group was 156, and the germination rate was 86.7%; the germination number of the treated group is 175, and the germination rate is 97.2%; the germination growth rate of the treated group compared to the control group was 12.2%.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will understand that: modifications to the specific embodiments of the invention or equivalent substitutions for parts of the technical features may be made; without departing from the spirit of the present invention, it is intended to cover all aspects of the invention as defined by the appended claims.
Claims (6)
1. A preparation process of a plant immune protein soluble powder of Alternaria tenuissima PeaT1 is characterized by comprising the following steps,
step A, inoculating hyphae to a culture medium flat plate and putting the culture medium flat plate into a constant-temperature incubator for culturing for at least 72 hours;
b, selecting a small amount of thalli from the culture medium flat plate, inoculating the selected thalli into a shake flask liquid culture medium, and performing seed fermentation and shaking culture;
c, inoculating the seed shake flask into a fermentation tank for at least 2-stage fermentation;
d, adding 0.5M Tris HCl buffer solution to protect protein after fermentation is completed, and then breaking the wall;
e, filtering the crude protein liquid obtained by breaking the cell walls of the mycelia through a 200-mesh screen, removing residues of a fermentation culture medium and cell wall tissues of the mycelia, retaining the crude protein liquid, adding a carrier filler aid, and drying to obtain soluble powder;
and C, performing 2-stage fermentation: the grade 1 fermentation comprises the following steps: inoculating a seed shake flask into a 500L fermentation tank for fermentation, wherein the liquid loading of a culture medium is 65%, the sterilization temperature is 121 ℃, the sterilization time is 30min, the fermentation culture temperature is 28-32 ℃, the pH is 6.0-7.2, the fermentation time is 36h, and the formula of the culture medium comprises, by mass, 3.0-5.5% of corn starch, 1.0-4.5% of soybean meal, 0.1-1.0% of sugar, 0.01-1.00% of ammonium sulfate, 0.01-1.00% of a protein production inducer, 0.01-0.50% of a protein activator I, 0.01-0.50% of a protein activator II and the balance of water; the 2-stage fermentation comprises the following steps: fermenting the strain in a 500L fermentation tank, and then inoculating the strain into a 5000L fermentation tank for fermentation, wherein the liquid loading of a culture medium is 70%, the sterilization temperature is 121 ℃, the sterilization time is 30min, the fermentation culture temperature is 28-32 ℃, the pH is 6.0-7.2, the fermentation time is 20h, and the formula of the culture medium comprises, by mass, 3.0-5.5% of corn starch, 1.0-4.5% of soybean meal, 0.1-1.0% of sugar, 0.01-1.00% of ammonium sulfate, 0.01-1.00% of a protein production inducer, 0.01-0.50% of a protein activator I, 0.01-0.50% of a protein activator II and the balance of water;
or performing 3-stage fermentation in the step C: the grade 1 fermentation comprises the following steps: inoculating a seed shake flask into a 500L fermentation tank for fermentation, wherein the liquid loading of a culture medium is 65%, the sterilization temperature is 121 ℃, 30min, the fermentation culture temperature is 28-32 ℃, the pH is 6.0-7.2, the fermentation time is 36h, and the formula of the culture medium comprises, by mass, 3.0-5.5% of corn starch, 1.0-4.5% of soybean meal, 0.1-1.0% of sugar, 0.01-1.00% of ammonium sulfate, 0.01-1.00% of a protein production inducer, 0.01-0.50% of a protein activator I, 0.01-0.50% of a protein activator II and the balance of water;
the 2-stage fermentation comprises the following steps: fermenting strains in a 500L fermentation tank, and then inoculating the strains in a 5000L fermentation tank for fermentation, wherein the liquid loading of a culture medium is 70%, the sterilization temperature is 121 ℃, the sterilization time is 30min, the fermentation culture temperature is 28-32 ℃, the pH is 6.0-7.2, the fermentation time is 20h, and the formula of the culture medium comprises, by mass, 3.0-5.5% of corn starch, 1.0-4.5% of soybean meal, 0.1-1.0% of sugar, 0.01-1.00% of ammonium sulfate, 0.01-1.00% of a protein production inducer, 0.01-0.50% of a protein activator I, 0.01-0.50% of a protein activator II and the balance of water;
the 3-stage fermentation comprises the following steps: fermenting the strain in a 5000L fermentation tank, and then inoculating the strain into a 40000L fermentation tank for 3-level fermentation, wherein the liquid loading of a culture medium is 75%, the sterilization temperature is 121 ℃, the sterilization temperature is 30min, the fermentation culture temperature is 28-32 ℃, the pH is 6.0-7.2, the fermentation time is 18h, and the formula of the culture medium comprises, by mass, 3.0-5.5% of corn starch, 1.0-4.5% of soybean meal, 0.1-1.0% of sugar, 0.01-1.00% of ammonium sulfate, 0.01-1.00% of a protein production inducer, 0.01-0.50% of a protein activator I, 0.01-0.50% of a protein activator II and the balance of water;
the protein production inducer is one or a combination of IPTG, lactose and pyruvic acid;
the protein activator I and the protein activator II are one or a combination of more of potassium ions, magnesium ions, calcium ions, bromide ions, hydrogen ions, glutathione and ethylene diamine tetraacetic acid.
2. The process for preparing a soluble powder of a plant immunoprotein of PeaT1, according to claim 1, wherein the plate of the medium inoculated with mycelia in step A is cultured in a thermostatic incubator at 28 ℃ for at least 72 h.
3. The process for preparing the soluble powder of alternaria tenuissima peaT1 plant immunoprotein as claimed in claim 1, wherein the culture medium plate in step A is a potato agar culture medium plate, and the culture medium comprises 200g of potato, 20g of sugar, 1L of water and 18g of agar powder.
4. The process for preparing the soluble powder of Alternaria tenuissima PeaT1 plant immune protein according to claim 1, wherein the formulation of the shake flask liquid culture medium in step B comprises 3.0-5.5% by mass of corn starch, 1.0-4.5% by mass of soybean meal, 0.1-1.0% by mass of sugar, 0.01-1.00% by mass of ammonium sulfate, 0.01-1.00% by mass of protein production inducer, 0.01-0.50% by mass of protein activator I, 0.01-0.50% by mass of protein activator II, and the balance of water.
5. The process for preparing the soluble powder of alternaria tenuissima peeT 1 plant immune protein according to claim 1, wherein the shaking culture conditions in step B are that the shaking culture is carried out at 180-200 rpm and 28-32 ℃ for at least 40 h.
6. The process for preparing the plant immunity protein soluble powder of Alternaria tenuissima PeaT1 as claimed in claim 1, wherein in step E, the carrier filler auxiliary agent comprises 30-40% by mass of fulvic acid, 10-15% by mass of white carbon black, 25-40% by mass of humic acid, 3-9% by mass of organosilicon, 4-9% by mass of protein adsorbent, 3-6% by mass of protein protective agent and 2-7% by mass of protein stabilizer.
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| CN113439745A (en) * | 2021-05-28 | 2021-09-28 | 苏州农业职业技术学院 | Novel plant immune protein and production process thereof |
| CN113647516A (en) * | 2021-07-26 | 2021-11-16 | 中农科生物工程技术(苏州)有限公司 | Preparation method and application of streptozochytrium induced resistance protein LiiP1 |
| CN113603535B (en) * | 2021-08-19 | 2022-09-13 | 中农科生物工程技术(苏州)有限公司 | Combined water-soluble fertilizer and preparation method thereof |
| CN115340425A (en) * | 2022-08-17 | 2022-11-15 | 河南柏裕植物免疫科技有限公司 | Immune protein for activating plant secondary metabolism |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103918716A (en) * | 2013-01-10 | 2014-07-16 | 陕西汤普森生物科技有限公司 | Brevibacillus laterosporus-containing efficient pesticide composition |
| CN105638744A (en) * | 2015-12-19 | 2016-06-08 | 佛山市艳晖生物科技有限公司 | Preparation method of Brevibacillus brevis wettable powder |
| CN105875651A (en) * | 2014-10-17 | 2016-08-24 | 中国农科院植保所廊坊农药中试厂 | Application of activator protein prepared from Alternaria tenuissima by microbial fermentation to pesticide preparation |
| CN106085869A (en) * | 2016-06-20 | 2016-11-09 | 浙江龙游东方阿纳萨克作物科技有限公司 | A kind of method for increasing of Alternaria tenuissima activator protein |
| CN106106528A (en) * | 2016-06-30 | 2016-11-16 | 京博农化科技股份有限公司 | A kind of bactericidal composition containing toVerticilliumdahliaActivitie Activitie S of Relative activator protein |
| CN107624771A (en) * | 2017-09-30 | 2018-01-26 | 京博农化科技股份有限公司 | A kind of bactericidal composition of the activator protein containing toVerticilliumdahliaActivitie Activitie S of Relative |
| CN107969423A (en) * | 2017-11-22 | 2018-05-01 | 青海省农林科学院 | A kind of toVerticilliumdahliaActivitie Activitie S of Relative bacterial strain wettable powder and preparation method thereof |
| CN107988084A (en) * | 2017-11-22 | 2018-05-04 | 青海省农林科学院 | A kind of fermentation process of Alternaria tenuissima |
| CN109601537A (en) * | 2018-04-01 | 2019-04-12 | 河北中保绿农作物科技有限公司 | One plant growth regulators and its method of administration |
-
2019
- 2019-05-08 CN CN201910380645.5A patent/CN110122482B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103918716A (en) * | 2013-01-10 | 2014-07-16 | 陕西汤普森生物科技有限公司 | Brevibacillus laterosporus-containing efficient pesticide composition |
| CN105875651A (en) * | 2014-10-17 | 2016-08-24 | 中国农科院植保所廊坊农药中试厂 | Application of activator protein prepared from Alternaria tenuissima by microbial fermentation to pesticide preparation |
| CN105638744A (en) * | 2015-12-19 | 2016-06-08 | 佛山市艳晖生物科技有限公司 | Preparation method of Brevibacillus brevis wettable powder |
| CN106085869A (en) * | 2016-06-20 | 2016-11-09 | 浙江龙游东方阿纳萨克作物科技有限公司 | A kind of method for increasing of Alternaria tenuissima activator protein |
| CN106106528A (en) * | 2016-06-30 | 2016-11-16 | 京博农化科技股份有限公司 | A kind of bactericidal composition containing toVerticilliumdahliaActivitie Activitie S of Relative activator protein |
| CN107624771A (en) * | 2017-09-30 | 2018-01-26 | 京博农化科技股份有限公司 | A kind of bactericidal composition of the activator protein containing toVerticilliumdahliaActivitie Activitie S of Relative |
| CN107969423A (en) * | 2017-11-22 | 2018-05-01 | 青海省农林科学院 | A kind of toVerticilliumdahliaActivitie Activitie S of Relative bacterial strain wettable powder and preparation method thereof |
| CN107988084A (en) * | 2017-11-22 | 2018-05-04 | 青海省农林科学院 | A kind of fermentation process of Alternaria tenuissima |
| CN109601537A (en) * | 2018-04-01 | 2019-04-12 | 河北中保绿农作物科技有限公司 | One plant growth regulators and its method of administration |
Non-Patent Citations (6)
| Title |
|---|
| 7%井冈霉素·极细链格孢激活蛋白可湿性粉剂对烟草病毒病的防效研究;李宏光 等;《现代农业科技》;20141231(第24期);第126、131页 * |
| 产激活蛋白极细链格孢菌发酵工艺优化和中试生产技术;金鑫;《中国农业科学院硕士学位论文》;20090430;第7-34页 * |
| 提高蛋白激发子产量的培养基及发酵条件的优化;金鑫 等;《微生物学杂志》;20090331;第29卷(第2期);第6-11页 * |
| 极细链格孢菌peaT1 基因在毕赤酵母中的表达与功能分析;刘延锋 等;《生物工程学报》;20090331;第25卷(第3期);第413-417页 * |
| 真菌蛋白激发子PeaTl规模化发酵工艺及产品质量检测技术研究;彭智超;《中国农业科学院硕士学位论文》;20190115;第6-17页 * |
| 蛋白激发子PeaT1的高密度发酵及生理功能检测;刘权 等;《生物技术通报》;20090831(第8期);第162-165页 * |
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