CN112889842B - Wettable bacterium powder and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of microbial agents, and particularly relates to wettable bacteria powder and a preparation method and application thereof. The wettable bacteria powder comprises the following raw materials in percentage by mass: 75-90% of microbial inoculum mother powder, 5-10% of dispersing agent, 5-10% of wetting agent, 1-4% of stabilizing agent and 0.1-1.0% of protective agent; the microbial inoculum mother powder is obtained by uniformly mixing and drying concentrated bacterial liquid and a carrier. Through the screening of the carrier, the dispersing agent, the wetting agent, the stabilizing agent and the protective agent and the setting of the mixture ratio of the materials, the wettable bacteria powder with good wetting effect, high suspension rate and high viable count is obtained, and the wettable bacteria powder can be widely used for pesticide wettable bacteria powder.

Description

A kind of wettable bacteria powder and its preparation method and application

technical field

The invention relates to the technical field of microbial inoculants, in particular to a wettable inoculum powder and a preparation method and application thereof.

Background technique

Microbial inoculants are beneficial microorganisms isolated from nature. After screening and identification, they are processed into viable bacterial preparations by fermentation, mixing, drying and other processes, and are widely used in the prior art.

Tobacco scab and wildfire are the main diseases that occur when tobacco leaves mature, and the two causes are caused by different microorganisms, and the control agents are quite different. For wildfire disease, agricultural streptomycin is mainly used, and antibiotics are mainly used for control, so as to enhance drug resistance; as microbial inoculants are widely used in pesticides, microbial inoculants can be used to control tobacco leaves. For the treatment of tobacco scab and wild fire, in the traditional technology, the powder is the main method, and the powder spraying method is used for application, but the efficacy of the control target is poor, the dosage of the main agent per unit area is large, the recovery rate is low, and the drift pollution In recent years, the research on wettable powder has become a new trend in the development of biological inoculum processing technology. It does not require mailing of solvents during processing, and is resistant to rain and wind, and is convenient for packaging, storage and transportation. The effect is better than that of powder, and the residual effect is long.

However, in the prior art, wettable bacteria powder mainly realizes effects such as wetting through the addition of auxiliary agents, but its wetting effect is poor, and the number of viable bacteria in the bacteria powder is greatly reduced, so that the control effect is greatly reduced.

SUMMARY OF THE INVENTION

In view of the above problems existing in the prior art, the present invention adopts a bacterial screening method to obtain a compound high-efficiency bacterial group. After the bacterial group is amplified, cultured and fermented, a concentrated bacterial liquid is obtained by centrifugal pressure filtration, and then a specific carrier and a dispersant are added. , wetting agent, stabilizer and protective agent, and obtain wettable powder after drying.

In order to achieve the above-mentioned purpose of the invention, the present invention provides a wettable bacteria powder, and the mass percentage of each raw material of the wettable bacteria powder is:

75%-90% of bacterial agent mother powder, 5-10% of dispersant, 5-10% of wetting agent, 1-4% of stabilizer, 0.1-1.0% of protective agent;

Described bacterial agent mother powder is obtained by mixing and drying concentrated bacterial liquid and carrier;

The dispersing agent is one or more of sodium tripolyphosphate, PEG-8000, and sodium lignosulfonate;

Described wetting agent is one or both in sodium lauryl sulfonate, stearate;

Described stabilizer is dipotassium hydrogen phosphate or potassium dihydrogen phosphate;

The protective agent is one or more of carboxymethyl cellulose, vitamin C, and sodium lauryl sulfate.

Further, the number of viable bacteria in the wettable powder is 3-5*10 ¹⁰ cfu/ml.

Further, the mass ratio of the concentrated bacterial liquid and the carrier is 1:8-9.5.

Further, the carrier is one or both of white carbon black and diatomaceous earth.

Based on the same inventive concept, the present invention also provides a preparation method of wettable bacterial powder, which specifically includes:

S1: the bacterial classification is inoculated into the fermentation liquid after the slant cultivation, and the supernatant is removed by centrifugation after the fermentation, and the concentrated bacterial liquid is obtained by pressure filtration;

S2: the concentrated bacterial liquid and the carrier are stirred, and spray-drying is carried out to obtain the inoculum mother powder;

S3: stirring the inoculum mother powder with dispersant, wetting agent, stabilizer and protective agent and drying to obtain wettable inoculum powder.

Further, the process parameters of the pressure filtration are: the filtration pressure is 0.1-0.5MPa, the filtration time is 12-35min, and the top water content of the filter cake after filtration is 10-20% of the total volume of the concentrated bacterial solution.

Further, the temperature of the drying process in the step S2 and the step S3 is 55-65°C.

Based on the same inventive concept, the above wettable fungus powder or the application of the wettable fungus powder prepared by the above method in pesticides.

Further, the bacterial group in the concentrated bacterial liquid is a compound bacterial agent, which specifically includes: Bacillus, Lactobacillus, and Leukella, which are used to combat tobacco scab and wildfire.

Beneficial effects:

(1) In the present invention, after the bacterial agent is amplified, cultured and fermented, and the bacterial liquid is concentrated by the method of centrifugation and pressure filtration, the bacterial liquid of high viable bacterial content is obtained, and the bacterial liquid is obtained by means of a carrier, a dispersant, a wetting agent and a wetting agent. , the screening of stabilizers and protective agents and the setting of the ratio of each material, the wettable bacterial powder with good wetting effect, high suspension rate and high viable count was obtained, which can be widely used in pesticide wettable bacterial powder.

(2) the present invention is compounded by adopting compound bacterial agents, such as Bacillus, Lactobacillus, Leukella, and adopts the preparation method of the wettable powder of the present invention to prepare and obtain a method for preventing and treating tobacco scab. And the wettable fungus powder of wild fire disease, which has a good inhibitory effect on the target bacteria of tobacco scab and wild fire disease.

Description of drawings

Fig. 1 is the microscopic examination diagram of the inoculum mother powder of different carriers provided in the embodiment of the present invention;

Fig. 2 is the actual figure of the antibacterial circle of inoculum mother powder of different carriers provided by the embodiment of the present invention;

Fig. 3 is the microscopic examination diagram of the inoculum mother powder of adding different dispersants provided by the embodiment of the present invention;

Fig. 4 is the physical map of the inhibition zone of the bacterial agent mother powder adding different dispersants provided by the embodiment of the present invention;

Fig. 5 is the microscopic examination diagram of the inoculum mother powder of adding different wetting agents provided by the embodiment of the present invention;

Fig. 6 is the physical map of the inhibition zone of the bacterial agent mother powder adding different wetting agents provided by the embodiment of the present invention;

Fig. 7 is the microscopic examination diagram of the inoculum mother powder adding different stabilizers provided by the embodiment of the present invention;

Fig. 8 is the physical map of the inhibition zone of the bacterial agent mother powder adding different stabilizers provided by the embodiment of the present invention;

Fig. 9 is the microscopic examination diagram of the inoculum mother powder of adding different protective agents provided by the embodiment of the present invention;

Fig. 10 is the physical map of the inhibition zone of the bacterial agent mother powder adding different protective agents provided in the embodiment of the present invention;

11 is a microscopic view of the wettable bacteria powder provided in the embodiment of the present invention;

Fig. 12 is the microscopic examination diagram of the wettable bacteria powder diluted with different multiples provided in the embodiment of the present invention;

Figure 13 is a physical diagram of the inhibition zone of the wettable bacteria powder provided in the embodiment of the present invention.

Detailed ways

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to specific embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

Unless otherwise defined, all technical terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the protection scope of the present invention.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or can be prepared by existing methods. In the present invention, the microbial flora is screened from the microbial flora that antagonizes grass scab and wild fire to obtain strains with obvious antagonism, and the compound flora includes: Bacillus 69.7%, Lactobacillus 23.9%, Luc spp. 6.45%.

Example 1

Preparation of concentrated fermentation broth:

The fermented seeds are obtained by cultivating the bacterial strain of the compound bacteria for many times, and inoculated into the fermenter to obtain a fermented bacterial liquid, wherein the culture temperature is 37 ° C, and the slant medium and the fermentation liquid are conventional bacterial culture components. The fermented bacteria liquid is first centrifuged, the supernatant is removed, and the deposited mixture is subjected to pressure filtration, and the plate and frame pressure filtration is used for filtration, and the pressures are 0.1MPa, 0.2MPa, 0.3MPa, 0.4MPa respectively. , 0.5MPa, 0.6MPa, record the time required for complete filtration of each group. In order to separate the bacteria remaining in the filter cake, after the filtration is completed, add water to the plate frame, which is called the amount of top water, where the amount of top water is The percentage content of the total volume of the concentrated bacterial solution is 0%, 5%, 10%, 15%, and 20%, and the content of bacteria in the filtrate is detected to obtain the yield. The results are shown in Table 1 and Table 2. 1 is the comparison of the filtration time under different pressure conditions, and Table 2 shows the content and yield of the bacterial flora when the filtration pressure is 0.4MPa and the amount of top water is different.

Table 1 Influence of filtration pressure on filtration speed

Table 2 Influence of filter cake top water on yield

It can be seen from Table 1 that the greater the filtering pressure during the filtering process, the less filtering time is required, that is, the faster the filtering speed is. During production, the maximum filtration pressure of the diaphragm plate and frame filter is 0.6MPa. The pressure is too high, the production process is dangerous, the energy consumption is large, and the equipment loss is large. Considering the production cost and safety, the filtration pressure is selected to be 0.1-0.5MPa, and the filter-pressing time is 12-35min. As can be seen from Figure 2, the larger the amount of top water, the higher the yield after filtration, but the larger the volume of the filtrate, the greater the workload of the subsequent drying process, so the amount of top water can be selected to be 10-20%, and the yield is 96%. around, and the subsequent processing process is relatively simple.

Example 2

Preparation of inoculum master powder:

The concentrated bacterial liquid obtained in Example 1 and the carrier material were mixed according to a mass ratio of 1:9, and spray-dried at 60°C to obtain a bacterial agent mother powder, wherein the carrier material was selected as white carbon black (500 mesh) , talcum powder (800 mesh), kaolin (1250 mesh) and diatomite (800 mesh), determine the wettability characteristics of the inoculum master powder obtained by different carriers and the inhibitory effect on the target bacteria of tobacco scab and wildfire disease: the The target bacteria suspension is added to the molten medium to make a mixed bacteria plate. After coagulation, 0.2 ml of antagonistic bacterial fermentation filtrate was added, and the diameter of the inhibition zone was measured by the cross method after culturing at 30°C for 72 hours. Table 3 shows the wettability characteristics of the inoculum powder, and Figures 1 and 2 are the microscopic examination results and the inhibition zone of the inoculum mother powder with different carriers.

Table 3 Wetability results of different carrier inoculum powders

It can be seen from Table 3 that the wetting time of silica and diatomite is short, the suspension rate is high, and the number of viable bacteria is high, which can be used as the carrier of wettable bacteria powder. The inoculant mother powder with soil as the carrier has a high bacterial density and a larger diameter of the bacteriostatic circle, both of which have higher bacteriostatic effect.

Example 3

Dispersant selection:

The inoculum mother powder obtained in Example 2 with silica as a carrier is mixed and dried with a dispersant, wherein the addition of the dispersant is 10%, and the dispersant is sodium tripolyphosphate, sodium lignosulfonate, carboxymethyl Cellulose sodium, PEG800, measure the wettability characteristics of the inoculum mother powder after adding the dispersant and the inhibitory effect (concrete method is identical with embodiment 2) to tobacco scab and wildfire target bacteria, table 4 is the wettable bacterial powder Screening of dispersants, Figures 3 and 4 are the microscopic examination results and the inhibition zone of adding different dispersants, respectively.

Table 4 Screening of wettable powder dispersants

As can be seen from Table 4, the wetting time of sodium tripolyphosphate and PEG-800 is short, the suspension rate of sodium lignin sulfamate and sodium tripolyphosphate is high, the number of viable bacteria of sodium lignosulfonate and sodium tripolyphosphate is high, The carrier that can be used for wettable bacteria powder, according to Fig. 3, it can be seen from Figure 3 that the content of the number of viable bacteria diluted 10 times of the mixed preparation is determined by a hemocytometer, and the fermentation broth bacteria concentration in sodium lignosulfonate and sodium tripolyphosphate is the highest. , followed by sodium carboxymethyl cellulose and sodium tripolyphosphate. According to Figure 4, the diameter of the inhibition zone of sodium tripolyphosphate and sodium lignosulfonate is large and has a high antibacterial effect. Therefore, tripolyphosphate Sodium, PEG-8000 and sodium lignosulfonate can be used as dispersants for wettable powder.

Example 4

Wetting agent selection:

The inoculum mother powder and wetting agent obtained in Example 2 are mixed and dried with white carbon black as carrier, wherein the wetting agent addition is 10%, and the wetting agent is specifically stearate, dodecylbenzenesulfonic acid Sodium, Tween 80, measure the wettability characteristics of the microbial inoculum mother powder after adding the wetting agent and the inhibitory effect (concrete method is identical with embodiment 2) to tobacco scab and wildfire target bacteria, and table 5 is the wettable bacteria Screening of powder wetting agents, Figures 5 and 6 are the microscopic examination results and the inhibition zone of adding different wetting agents, respectively.

Table 5 Screening of wettable bacteria powder wetting agent

As can be known from Table 5 and Fig. 5, the wetting time of sodium dodecyl benzene sulfonate and stearate is short, the suspension rate of Tween-80 is higher, and the viable count of stearic acid is high, as can be seen from Fig. 6, The diameter of the inhibition zone of the three wetting agents is relatively close. Based on the wettability results of the above wetting agents and the diameter of the inhibition zone, sodium dodecyl benzene sulfonate, stearic acid and Tween-80 can be used as Wetting agent for wettable bacteria powder, preferably Tween-80.

Example 5

Stabilizer selection

The stabilizer was added to the concentrated bacterial solution obtained in Example 1, and the mixture was uniformly diluted and coated on the NA plate. One part was placed under a 254nm UV lamp at 40cm for 1 hour, and the other part was directly cultivated at 30°C. The control did not add protective agent. , wherein the amount of stabilizer added is 2%, and the stabilizer is specifically CaCO ₃ , K ₂ HPO ₄ , KH ₂ PO ₄ , and the number of viable bacteria after culture and the inhibitory effect on the target bacteria of tobacco scab and wildfire disease are determined (the specific method is related to the Example 2 is the same), Table 6 is the screening of wettable bacterial powder stabilizer, and Figures 7 and 8 are the microscopic examination results and the antibacterial zone of adding different stabilizers, respectively.

Table 6 Screening of Wettable Stabilizers

It can be seen from Table 7 and Figure 8 that the number of viable bacteria is high when the stabilizers K ₂ HPO ₄ and KH ₂ PO ₄ are used. According to Figure 8, the diameter of the inhibition zone of K ₂ HPO ₄ and KH ₂ PO ₄ is larger than that of CaCO ₃ . Based on the above-mentioned results of the number of viable bacteria of the stabilizer and the diameter of the inhibition zone, the stabilizers of the wettable bacterial powder are preferably K ₂ HPO ₄ , KH ₂ PO ₄ .

Example 6

Choice of protective agent

The protective agent was added to the concentrated bacterial solution obtained in Example 1, mixed uniformly, and then applied by gradient dilution on the NA plate. One part was placed under a 254 nm UV lamp at 40 cm for 1 hour, and the other part was directly cultivated at 30 °C. The control did not add protective agent. , wherein the protective agent add-on is 2%, and the protective agent is specifically vitamin C, carboxymethyl cellulose, sodium lauryl sulfate, the number of viable bacteria after the measurement is cultivated and the inhibitory effect ( The specific method is the same as in Example 2), Table 7 is the screening of the wettable bacterial powder stabilizer, and Figures 9 and 10 are the microscopic examination results and the bacteriostatic zone of adding different protective agents, respectively.

Table 7 Screening of wettable powder protective agents

It can be seen from Table 7, Figures 9 and 10 that the number of viable bacteria is high when the protective agent vitamin C and carboxymethyl cellulose are used, and the diameter of the inhibition zone of vitamin C and carboxymethyl cellulose is larger than that of sodium lauryl sulfate. The results of the viable count of the above protective agent and the diameter of the inhibition zone, the protective agents of the wettable bacteria powder are preferably vitamin C and carboxymethyl cellulose.

Example 7

After the concentrated bacterial liquid obtained in Example 1 and the carrier white carbon black were mixed and stirred at a ratio of 1:9 by mass ratio, and spray-dried at 60 ° C to obtain a bacterial agent mother powder; choose dispersant sodium tripolyphosphate ( 10%), wetting agent Tween-80 (10%), stabilizer K ₂ HPO ₄ (2%), protective agent sodium carboxymethyl cellulose (0.1%) and the bacterial agent mother powder (77.9%) Stir and mix evenly and dry at 60°C to obtain wettable bacterial powder.

Microscopic examination of the obtained wettable bacteria powder, and 10 times, 100 times and 10000 times of dilution after compliance with the microscopic examination to observe the concentration of bacteria, see Figures 11 and 12 for details, and the plate confrontation method was used to determine the wettable bacteria. Antagonistic effect of powder on pathogens: carried out indoors, for wildfire and scab disease, the pure cultured pathogenic bacteria were placed in LB and PDA liquid medium, shaken at 28°C for 12 hours, and prepared into a bacterial suspension diluted 5000 times For use; put the isolated antagonistic microbial flora to be tested in 1/2 LB liquid medium, and culture with shaking at 28° C. for 5 d. Mix the diluted pathogenic bacteria suspension with 1/2 solid medium at a ratio of 3:2000 and pour it into the plate. Then use a sterile hole punch with a radius of 5mm to punch holes in a cross shape, add 100 μl of the bacterial solution to be tested into the holes, and mark the number of the tested bacteria group and the inoculation time correspondingly on the bottom of the petri dish. At the same time, a plate was taken, and only tobacco pathogens were added as a control. Incubate at a constant temperature of 28°C. When the control group covers the entire petri dish, observe whether the inhibition zone is formed in the experimental group and measure the size of the inhibition zone; the results are shown in Figure 13.

According to Figures 11 and 12, (a), (b) and (c) in Figure 12 are the microscopic examination results of the 10-fold, 100-fold, and 10,000-fold dilution of the fermented stock solution after the resurrection of the WP, respectively, it can be seen that the embodiment 7. The wettable powder obtained has a higher concentration of viable bacteria. According to Figure 13, 1-4 are parallel 4 samples. It can be seen that the wettable powder samples grow better, and the whole plate is in a transparent state, indicating that the antibacterial effect is good and can be As a wettable fungus powder pesticide for the control of tobacco scab and wildfire.

The above-mentioned embodiments are only preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. The equivalent replacement or change of the solution and its concept shall be included within the protection scope of the present invention.

Claims (4)

1. The wettable bacteria powder is characterized by comprising the following raw materials in percentage by mass:

75-90% of microbial inoculum mother powder, 5-10% of dispersing agent, 5-10% of wetting agent, 1-4% of stabilizing agent and 0.1-1.0% of protective agent, wherein the sum of the mass percentages of the raw materials is 100%;

the microbial inoculum mother powder is obtained by uniformly mixing and drying concentrated bacterial liquid and a carrier, the mass ratio of the concentrated bacterial liquid to the carrier is 1:8-9.5, and the microbial community in the concentrated bacterial liquid is a compound microbial inoculum and specifically comprises the following components: the carrier is one or two of white carbon black and diatomite;

the dispersing agent is one or more of sodium tripolyphosphate, PEG-8000 and sodium lignosulfonate;

the wetting agent is one or two of sodium dodecyl sulfate and stearate;

the stabilizer is dipotassium hydrogen phosphate or potassium dihydrogen phosphate;

the protective agent is one or two of carboxymethyl cellulose and vitamin C;

the viable count of the wettable powder is 3-5 x 10¹⁰cfu/ml。

2. A method for preparing wettable powder of claim 1, which comprises:

s1, inoculating the strain into the fermentation liquor after slant culture, centrifuging after fermentation to remove the supernatant, and obtaining concentrated bacterial liquid by pressure filtration;

s2, uniformly stirring the concentrated bacterial liquid and the carrier, and performing spray drying to obtain bacterial agent mother powder;

s3, uniformly stirring the microbial inoculum mother powder, a dispersing agent, a wetting agent, a stabilizing agent and a protective agent, and drying to obtain wettable bacterial powder.

3. The method for preparing wettable powder of claim 2, wherein the temperature of the drying process in steps S2 and S3 is 55-65 ℃.

4. Use of the wettable powder of claim 1 or the wettable powder obtained by the preparation method of any of claims 2 to 3 in pesticides.

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