CN111925971B - Environment-responsive bacillus emulsion and preparation method thereof - Google Patents

Abstract

The invention relates to an environment-responsive bacillus emulsion, and belongs to the technical field of agricultural microbial agent development. The emulsion comprises an inner oil phase and an outer water phase, wherein bacterial powder, grease and a surfactant are added into the oil phase to form an emulsifiable type inner oil phase, a polysaccharide compound formed by porous starch and chitosan is added into the outer water phase to form an electrostatic response type emulsifier, and the oil phase and the water phase are mixed to form a gel type emulsion. In the land with higher salinization degree, the polysaccharide complex in the emulsion is rapidly disintegrated due to the too high pH value or the electrostatic shielding effect of salt ions, and the micropore structure of the porous starch can increase the contact between the external environment and the oil phase and improve the sensitivity of the emulsion. The dispersed oil drops are in a small droplet state due to the contained oil phase emulsifier, so that the release of the internally embedded strains is further promoted, and the soil environment is improved in a short time. In meta-acid or neutral soil, the emulsion can effectively slow down the release speed of strains, prolong the action duration of the strains and improve the utilization efficiency and time of agricultural microorganisms.

Description

Environment-responsive bacillus emulsion and preparation method thereof

Technical field:

The invention relates to an environment-responsive bacillus emulsion and a preparation method thereof, belonging to the technical field of agricultural microbial agent development.

The background technology is as follows:

Bacillus subtilis is an aerobic gram-positive bacterium, has stronger stress resistance compared with common strains, can resist heat and drought, and is also proved by related documents to be capable of effectively preventing and treating diseases caused by plant fungi when being applied to fields. Bacillus subtilis is widely distributed in various environments of nature, is distributed in soil, on the surfaces of crops and on the surfaces of animals, exists even in part of plant bodies, and is a typical widely accepted bacterium which can be used for promoting and maintaining normal growth of plant bodies.

In recent years, due to abuse of artificial synthetic fertilizers and pesticides, partial agricultural lands in China have the problems of too high content of soil meta-acid, meta-alkali or salt ions and the like. And the physicochemical properties of the soil have great influence on the growth of crops and the fertilizer efficiency of the soil. For example, salinized farmlands are unsuitable for normal growth of crops, while meta-acid soil contains a large amount of free hydrogen ions, which can lead to loss of activity of fertilizers and bacterial species, greatly affecting the utilization rate of the agricultural microbial agents used.

In the existing bacterial fertilizer technology, the treatment of bacillus subtilis is mainly concentrated on spray drying, fluidized bed granulation and the like, and even the form of directly diluting bacterial stock solution and then applying the bacterial stock solution to farmlands is adopted. The agricultural microbial agents produced by the processes can only play a short-time role in farmlands, because most strains are inactivated or lost under severe conditions, the action time is relatively short, the growth conditions of crops can not be effectively improved, antagonism with pathogenic bacteria is generated, and the amplification and the propagation of the crops are inhibited.

The invention comprises the following steps:

The invention aims to overcome the defects of the prior art and provide an environment-responsive bacillus emulsion, and the emulsion stabilized by a porous starch/chitosan electrostatic complex provided by the invention can regulate the number of living bacteria released by the emulsion according to the acid-base value of the soil environment. In agricultural lands with higher salinization degree, the polysaccharide complex in the emulsion is rapidly disintegrated due to the too high pH value or the electrostatic shielding effect of salt ions, so that the emulsion is rapidly demulsified and the oil phase is released. The porous structure of the porous starch can further accelerate the contact between the external environment and the oil phase and increase the sensitivity of the emulsion to the external environment, so that the bacteria are released in a short time to improve the soil environment. Meanwhile, oil drops in the soil can concentrate on the plant roots along with the absorption of the water by the plant roots and release thalli in a concentrated manner, so that the loss of strains is reduced, and the drug effect is enhanced. In the meta-acid or neutral soil, the emulsion can effectively slow down the release speed of the strain, prolong the action duration of the strain and improve the utilization efficiency and the action time of the agricultural microbial agent.

In order to achieve the above purpose, the invention provides an environment-responsive bacillus emulsion, which comprises an inner oil phase and an outer water phase, wherein bacterial powder, grease and a surfactant are added into the oil phase to form an emulsifiable concentrate type inner oil phase, a polysaccharide compound formed by porous starch and chitosan is added into the outer water phase and is dissolved in polyethylene glycol aqueous solution to form an electrostatic-responsive emulsifier, the oil phase and the water phase are dispersed or homogeneously mixed to form a gel type emulsion, and porous starch absorbed by an emulsion interface can realize the emulsifiable concentrate type oil phase to be in responsive contact with the environment outside saline-alkali soil;

Further, the bacterial powder can be at least one of bacillus subtilis bacterial powder, bacillus licheniformis bacterial powder, bacillus thuringiensis bacterial powder or bacillus amyloliquefaciens bacterial powder;

further, the addition amount of the bacterial powder accounts for 20-30% of the mass of the oil phase;

Further, the addition amount of the surfactant accounts for 1% -2% of the oil phase;

further, the addition amount of the porous starch is 2% -7% of the mass of the external water phase;

The pore diameter of the porous starch is 0.5-1.5 mu m, preferably 1 mu m;

the porous starch can be rice porous starch, corn porous starch, tapioca porous starch, potato porous starch or wheat porous starch, etc.;

preferably, the porous starch is corn porous starch;

Further, the addition amount of the chitosan is 1% -2% of the mass of the external water phase;

Further, the solution in the water phase is 40-60% (m: m) polyethylene glycol water solution, and the electrostatic response type external water phase is obtained after the polysaccharide compound formed by the porous starch and the chitosan is dissolved;

Preferably, the solution in the aqueous phase is a 50% aqueous polyethylene glycol solution.

The preparation method of the environment-responsive bacillus emulsion comprises the following steps:

(1) Preparation of an oil phase: adding the fungus powder and the grease in proportion, stirring for 25-30min at room temperature, and then adding the surfactant in proportion to obtain an oil phase;

further, the grease is corn oil, rapeseed oil, palm oil or soybean oil and the like;

further, the surfactant is an anionic emulsifier or a nonionic emulsifier;

Preferably, the surfactant is a mixture of alkylphenol ethoxylates, fatty alcohol ethoxylates and sodium dodecyl sulfate, and the mass ratio is 2:2:1;

(2) Preparation of an aqueous phase: mixing the materials according to the mass ratio of 2-7:1-2 adding porous starch and chitosan, dissolving in 40-60% polyethylene glycol solution according to the ratio of 6-9% (m: v), adding acetic acid accounting for 1-2% (v: v) of water phase, stirring for 20-30min, and then regulating pH to 6.5-6.0 with alkali liquor;

Preferably, the alkali liquor is 0.5mol/L sodium hydroxide aqueous solution;

(3) Preparation of emulsion: mixing the oil phase and the water phase, wherein the inner oil phase accounts for 75% -90% (m: m) of the emulsion, and packaging after emulsification to obtain a product;

preferably, the internal oil phase is 90%.

The invention also provides application of the bacillus emulsion in the field of agricultural microbial agents.

The beneficial effects are that:

1. The invention adopts pH and salt ion response type polysaccharide electrostatic complex formed by electrostatic complexation of porous starch/chitosan as an emulsifier for the first time, the complex can be rapidly adsorbed at the interface of oil drops in the process of shearing the oil drops, and can be simultaneously adsorbed at the interface of two or more oil drops to form a stable emulsion oil drop network, so that the emulsion is converted into gel form from fluid form (shown in figure 1). The contact between the strain and oxygen is reduced, the premature resuscitation of the strain is avoided, the physiological activity of the strain is protected to the greatest extent in the storage and transportation processes, and the bacterial activity of the strain is ensured;

2. The bacillus agent with acid-base salt sensitivity is obtained through the difference of charges of the emulsifier in different acid-base environments, and the release rate of the strain can be adjusted according to the acid-base degree and the salt ion content of the soil environment: in an acidic or neutral environment, the emulsion exists in the form of emulsion gel with anti-dilution capability, and the strain release rate is reduced; in an alkaline environment, the chitosan has extremely low charge quantity, and reduces the electrostatic action with porous starch, so that the embedded strain is released rapidly; in an environment with higher salt ion concentration, the salt ion shields the charge of the emulsifier, reduces the electrostatic effect and increases the release of strains;

3. In the releasing process of the emulsion, the micropore structure of the porous starch can increase the contact between the external environment and the oil phase, and improve the environmental sensitivity of the emulsion. Meanwhile, the oil phase is used and the stabilizing process and components of oil drops are optimized, under the premise of reducing environmental pollution, the oil drops can be quickly changed into nano-sized small oil drops from micro-sized large oil drops to nano-sized small oil drops after being released from an emulsion system, when the particle size of the oil drops is smaller than the size of a strain, the strain can be quickly leaked from the oil drops, and the technology and the mechanism can accelerate the release of internal strains;

4. The external water phase adopts 40-60% polyethylene glycol aqueous solution as solution, can construct micron-sized emulsion with the emulsifiable type internal phase, successfully constructs the internal environment of the oil phase, loads strains and improves the stress resistance and storage stability of the strains;

5. The water content of the emulsion is relatively low, the emulsion is pasty, compared with a common powdery product, the damage of dust flying to a user is reduced, and meanwhile, the emulsion is easier to store and transport than a liquid product;

6. The bacillus emulsion provided by the invention has the advantages of simple preparation process and environmental protection, and is suitable for industrial mass production and application.

Description of the drawings:

FIG. 1 is a schematic diagram of the structure of the product;

FIG. 2 is a field test physical diagram;

FIG. 3 shows the microstructure under a cryo-scanning electron microscope;

FIG. 4 storage modulus and loss modulus plot;

FIG. 5 product release schematic;

figure 6 product instability factor.

Specific embodiments:

in order to make the objects, technical solutions and advantages of the present patent more apparent, the present patent will be described in further detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the present invention.

The structure of the environmental response type microbial inoculum provided by the invention is shown in fig. 1, and the invention is further explained below by combining with a specific embodiment.

Example 1: environment-responsive bacillus subtilis emulsion

(1) Preparation of an oil phase: stirring 150g of bacillus subtilis powder and 450g of rapeseed oil at room temperature for 30min, and then adding 10g (mass ratio 2:2:1) of a mixture of surfactant alkylphenol ethoxylates, fatty alcohol ethoxylates and sodium dodecyl sulfate to obtain an oil phase;

(2) Preparation of an aqueous phase: dissolving 2g of corn porous starch (porous starch pore diameter 0.5-1.5 μm) and 1g of chitosan in 50ml of 50% polyethylene glycol solution (polyethylene glycol 400 is selected as polyethylene glycol in the embodiment of the invention), adding 1ml of acetic acid, stirring for 30min, and regulating pH to 6.5 with 0.5mol/L sodium hydroxide;

(3) Preparation of emulsion: mixing the oil phase and the water phase, adding the inner oil phase with a proportion of 90% into a dispersing machine, dispersing for 3-5 minutes for emulsification, and packaging after emulsification to obtain the product.

Example 2: environment-responsive bacillus subtilis emulsion

(1) Preparation of an oil phase: stirring 150g of bacillus subtilis powder and 450g of rapeseed oil at room temperature for 30min, and then adding 10g (mass ratio 2:2:1) of a mixture of surfactant alkylphenol ethoxylates, fatty alcohol ethoxylates and sodium dodecyl sulfate to obtain an oil phase;

(2) Preparation of an aqueous phase: 2g of corn porous starch (porous starch pore diameter 0.5-1.5 μm) was dissolved in 50ml of 50% polyethylene glycol solution, 1ml of acetic acid was added, and pH was adjusted to 6.5 with 0.5mol/l sodium hydroxide;

(3) Preparation of emulsion: mixing the oil phase and the water phase, adding the inner oil phase with a proportion of 90% into a dispersing machine, dispersing for 3-5 minutes for emulsification, and packaging after emulsification to obtain the product.

Example 3: environment-responsive bacillus subtilis emulsion

(1) Preparation of an oil phase: 150g of bacillus subtilis powder and 450g of rapeseed oil are stirred for 30min at room temperature to obtain an oil phase; (2) preparation of aqueous phase: 2g of corn porous starch (porous starch pore diameter 0.5-1.5 μm) and 1g of chitosan are dissolved in 50ml of 50% polyethylene glycol solution, 1ml of acetic acid is added, and after stirring for 30min, pH is adjusted to 6.5 by 0.5mol/l of sodium hydroxide;

(3) Preparation of emulsion: mixing the oil phase and the water phase, adding the inner oil phase with a proportion of 90% into a dispersing machine, dispersing for 3-5 minutes for emulsification, and packaging after emulsification to obtain the product.

And (3) case effect detection:

(1) Detecting the release amount of living bacteria under different pH environments:

Taking 15g of each sample prepared by the three schemes of examples 1-3, dividing the sample into three parts, respectively placing the three parts in 100ml of sterile water with pH values of 5, 7 and 9 for 1 day, and then detecting the number of strains in the sterile water under different pH environments.

The detection results are shown in table 1, the environment-responsive bacillus subtilis emulsion prepared according to the method of embodiment 1 can adjust the release rate of the wrapped living bacteria according to different pH values, when the pH is alkaline, the release amount of the living bacteria is obviously higher than that of other environments, and in an acidic environment, the release of strains is relatively minimum. The result shows that the environment-responsive bacillus subtilis emulsion provided by the invention can adjust the release amount of the living bacteria wrapped by the bacillus subtilis emulsion according to the pH value of the environment, so that the fertilizer efficiency is prolonged and the environment of the saline-alkali soil is improved.

(2) Detecting the release amount of viable bacteria in a salt ion environment:

Taking 15g of each sample prepared by the three schemes of examples 1-3, dividing the sample into three parts, respectively placing the three parts in 100ml of sterile water with the salt ion concentration of 0-120mM for 1 day, and then detecting the number of strains in the sterile water under the environments with different salt ion concentrations.

The detection results are shown in table 2, the environment-responsive bacillus subtilis emulsion prepared by the method according to the embodiment 1 can adjust the release rate of the wrapped living bacteria according to different salt ion concentrations, when the salt ion concentration is low, the release amount of the living bacteria is obviously lower than that of other embodiment cases, and under the environment with high salt ion concentration, the strain release is obviously increased. The result shows that the environment-responsive bacillus subtilis emulsion provided by the invention can regulate the release amount of living bacteria wrapped by the bacillus subtilis emulsion according to the salt ion concentration of the environment, so that the fertilizer efficiency is prolonged and the environment of saline-alkali soil is improved.

(3) Detecting the influence of the storage time on the activity of the strain:

Samples prepared by the three schemes of examples 1-3 were placed in a sealed storage at room temperature for 0-180 days, and after storage 15g of the samples were diluted in 100g of physiological saline, 0.5wt% tween 80 was added, and placed in a conical flask and shaken with glass beads (flat bottom) in a shaker (200 rpm) for 30min-1h. The strain was then diluted with physiological saline according to the activity and number of strains, the diluted solution was spread on LB medium, and cultured in an incubator at 37℃for 12-16 hours, and counted. The results are shown in Table 3, and show that the strain inactivation of the product prepared in example 1 is significantly lower than that of other samples during storage, thus indicating that the bacterial powder prepared by the method can better protect bacterial cells during storage.

(4) Field test

Fertilizer efficiency experiments are carried out on celery seedlings (plant height is 15 cm), and data acquisition and analysis are carried out on experimental samples. Experiments were carried out in a sunlight greenhouse, sowing was carried out on day 7 and day 15 of 2019, transplanting was carried out on day 9 and day 8, and prepared samples were added to each soil pit before transplanting, with about 5g of each plant. And then conventional crop watering and fertilizing culture is carried out, and experimental results are collected and processed. The results are shown in Table 4, it is obvious that example 1 can promote the growth of celery, the yield of celery is relatively increased, the plant height is obviously higher than that of other experimental groups, the root system is more developed, and the product release principle is shown in FIG. 5.

TABLE 1

TABLE 2

TABLE 3 Table 3

TABLE 4 Table 4

Example 4 physical Properties

(1) Micro and macro structure

Samples prepared according to the three schemes of examples 1-3 were taken at 2g each, and the samples were placed on a plate and covered with a film. The images were then observed and recorded under a scanning electron microscope after rapid freezing with liquid nitrogen.

The detection results are shown in fig. 3, and the samples prepared according to the methods of embodiments 1 and 3 have relatively uniform oil drop sizes, no obvious aggregation phenomenon of oil drops, and form a polysaccharide network structure in the water phase, and the oil drops are embedded into the polysaccharide network structure to form a gel structure. In comparison with the sample prepared in example 2, the oil drops are not uniform in size, obvious large oil drops appear, and no obvious network structure exists, so that the emulsion structure is unstable.

(2) Rheological Properties

5G of each sample prepared by the three schemes of examples 1-3 is placed on a rheometer for frequency scanning to obtain the storage modulus and loss modulus of the sample, the frequency range is 0.1-100rad/s, and the testing temperature is constant at 25 ℃. The test results are shown in FIG. 4, and the samples obtained by the methods described in examples 1 and 3 have a significantly higher storage modulus (G ') and loss modulus (G') than the other samples, because of the stronger gel characteristics imparted by the gel network formed therein.

Example 5 determination of the instability index of emulsion

The emulsion was prepared by the same method as in example 1, except that the aqueous phase solution used was replaced, and the following was adopted:

group A: 30% aqueous polyethylene glycol; group B: 50% aqueous polyethylene glycol; group C: 70% aqueous polyethylene glycol solution; group D: glycerol; group E: propylene glycol;

The prepared 5 groups of emulsion samples were subjected to stability determination by LUMiSizer. After 0.4 g of the sample was added to the sample cell, centrifugation was performed at 3000 rpm for 30 minutes, and during this time, light was irradiated with near infrared light and the intensity of light was evaluated to calculate the instability index of the emulsion, and the obtained instability index was analyzed and compared.

As a result, as shown in fig. 6, the instability index of the emulsion using 50% aqueous polyethylene glycol as the external aqueous phase solution was significantly lower than those of other proportions of polyethylene glycol solution, and glycerin and propylene glycol, indicating that 50% polyethylene glycol was preferable as the external aqueous phase when the emulsion used the emulsion oil phase as the internal phase during production and preparation. The results are also responsive to the stability of the emulsion during storage and transport.

Example 6: environment-responsive bacillus emulsion

(1) Preparation of an oil phase: 150g of bacillus licheniformis bacteria powder and 340g of rapeseed oil are stirred at room temperature for 25min, and then 10g of surfactant calcium dodecyl sulfate is added to obtain an oil phase;

(2) Preparation of an aqueous phase: dissolving 3g of rice porous starch (porous starch pore diameter 0.5-1.5 μm) and 1g of chitosan in 50ml of 40% polyethylene glycol solution, adding 1ml of acetic acid, stirring for 20min, and adjusting pH to 6.5 with 0.5mol/L sodium hydroxide;

(3) Preparation of emulsion: mixing the oil phase and the water phase, adding the oil phase with the ratio of 88 percent into a dispersing machine, dispersing for 3-5 minutes for emulsification, and packaging after emulsification to obtain the product.

Example 7: environment-responsive bacillus emulsion

(1) Preparation of an oil phase: stirring 122.5g of bacillus thuringiensis powder and 350g of soybean oil at room temperature for 25min, and then adding 7.5g (mass ratio of 2:2:1) of a mixture of surfactant alkylphenol ethoxylates, fatty alcohol ethoxylates and sodium dodecyl sulfate to obtain an oil phase;

(2) Preparation of an aqueous phase: 3g of potato porous starch (porous starch pore diameter 0.5-1.5 μm) and 1g of chitosan are dissolved in 50ml of 60% polyethylene glycol solution, 1ml of acetic acid is added, stirring is carried out for 20min, and then the pH is regulated to 6.5 by 0.5mol/L sodium hydroxide;

(3) Preparation of emulsion: mixing the oil phase and the water phase, adding the oil phase with the ratio of 87.5% into a dispersing machine, dispersing for 3-5 minutes for emulsification, and packaging after emulsification to obtain the product.

The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that, for a person skilled in the art, the above embodiments may also make several variations, combinations and improvements, without departing from the scope of the present patent. Therefore, the protection scope of the patent is subject to the claims.

Claims (4)

1. An environment-responsive bacillus emulsion is characterized by comprising an inner oil phase and an outer water phase, wherein bacterial powder, grease and a surfactant are added into the oil phase to form an emulsifiable concentrate type inner oil phase, a polysaccharide compound formed by porous starch and chitosan is added into the outer water phase, and the polysaccharide compound is dissolved in a polyethylene glycol aqueous solution to form an electrostatic-responsive emulsifier, and the oil phase and the water phase are dispersed or mixed homogenously to form a gel type emulsion;

in the oil phase, the addition amount of the bacterial powder accounts for 20-30% of the mass of the oil phase, and the addition amount of the surfactant accounts for 1-2% of the mass of the oil phase;

In the water phase, the addition amount of the porous starch is 2% -7% of the mass of the external water phase, and the addition amount of the chitosan is 1% -2% of the mass of the external water phase;

The grease is corn oil, rapeseed oil, palm oil or soybean oil; the surfactant is an anionic emulsifier or a nonionic emulsifier;

the polyethylene glycol aqueous solution is 40-60% by mass;

The porous starch is rice porous starch, corn porous starch, tapioca porous starch, potato porous starch or wheat porous starch; the diameter of the small holes of the porous starch is 0.5-1.5 mu m;

The bacterial powder is at least one of bacillus subtilis bacterial powder, bacillus licheniformis bacterial powder, bacillus thuringiensis bacterial powder or bacillus amyloliquefaciens bacterial powder;

the preparation method of the environment-responsive bacillus emulsion comprises the following steps:

(1) Preparation of an oil phase: adding the fungus powder and the grease in proportion, stirring for 25-30min at room temperature, and then adding the surfactant in proportion to obtain an oil phase;

(2) Preparation of an aqueous phase: according to the mass ratio of 2-7:1-2 adding porous starch and chitosan, dissolving in 40-60% polyethylene glycol solution according to the ratio of 6-9%, adding acetic acid accounting for 1-2% of the volume of the water phase, stirring for 20-30min, and then regulating the pH to 6.5-6.0 with alkali liquor;

(3) Preparation of emulsion: mixing the oil phase and the water phase, wherein the oil phase accounts for 75-90% of the emulsion, emulsifying and packaging to obtain the product.

2. The environmentally responsive bacillus emulsion of claim 1, prepared by the method of:

(1) Preparation of an oil phase: adding the fungus powder and the grease in proportion, stirring for 25-30min at room temperature, and then adding the surfactant in proportion to obtain an oil phase;

The surfactant is a mixture of alkylphenol ethoxylates, fatty alcohol ethoxylates and sodium dodecyl sulfate, and the mass ratio is 2:2:1;

(2) Preparation of an aqueous phase: according to the mass ratio of 2-7:1-2 adding corn porous starch and chitosan, dissolving in 50% polyethylene glycol solution according to the feed liquid ratio of 6-9%, adding acetic acid accounting for 1-2% of the water phase volume ratio, stirring for 20-30min, and then adjusting the pH to 6.5-6.0 by using 0.5mol/L sodium hydroxide solution;

(3) Preparation of emulsion: mixing the oil phase and the water phase, wherein the internal oil phase accounts for 90 percent, emulsifying and packaging to obtain the product.

3. The environmentally responsive bacillus emulsion of claim 1, prepared by the method of:

(1) Preparation of an oil phase: 150g of bacillus subtilis powder and 450g of rapeseed oil are stirred at room temperature for 30min, and then added with a mass ratio of 2:2:1, 10g of a mixture of alkylphenol ethoxylates, fatty alcohol ethoxylates and sodium dodecyl sulfate to obtain an oil phase;

(2) Preparation of an aqueous phase: 2g of corn porous starch and 1g of chitosan are dissolved in 50ml of 50% polyethylene glycol solution, 1ml of acetic acid is added, and after stirring is carried out for 30min, the pH is regulated to 6.5 by 0.5mol/L sodium hydroxide;

(3) Preparation of emulsion: mixing the oil phase and the water phase, adding the inner oil phase with a proportion of 90% into a dispersing machine, dispersing for 3-5 minutes for emulsification, and packaging after emulsification to obtain the product.

4. Use of the environmentally-responsive bacillus emulsion of any one of claims 1-3 in the field of agricultural microbial agents.