Oil suspending agent for improving survival rate of bacillus and preparation method of oil suspending agent

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an oil suspending agent for improving the survival rate of bacillus and a preparation method of the oil suspending agent. The oil suspending agent consists of an oil phase and a water phase, wherein the oil phase consists of the following raw materials in parts by weight: 35-45 parts of coconut oil, 20-25 parts of white oil, 10-15 parts of modified isopropyl stearate, 2-3 parts of Tween-80 and 0.2-0.3 part of an ultraviolet absorbent; the water phase is prepared from the following raw materials in parts by weight: 14 to 18 parts of bacillus, 0.8 to 1.2 parts of graft modified hydroxypropyl methyl cellulose, 0.04 to 0.06 part of citric acid, 0.05 to 0.06 part of polydimethylsiloxane, 0.1 to 0.2 part of leucine, 0.5 to 0.8 part of glucose and 0.5 to 0.8 part of trehalose. The emulsion stability is enhanced through modified isopropyl stearate, the hydroxypropyl methyl cellulose grafted with carboxymethyl is thickened, a charge repulsion layer is formed, and the survival rate of bacillus is remarkably increased.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to an oil suspension for improving the survival rate of bacillus and a preparation method thereof. Background Art

[0002] Bacillus spp. has attracted people's attention because of its widespread presence in soil, water, animals and plants, and its ability to form highly tolerant spores. These spores can survive for many years in extreme environments until they encounter suitable conditions and germinate into viable bacteria again. Based on this characteristic, Bacillus is widely used in the agricultural field, for example, as a biological control agent to combat plant pathogens, or as a biological fertilizer to promote plant growth. Although Bacillus has a strong survival ability, it still faces the problem of decreased survival rate during actual application. Especially when directly sprayed in the field, exposed to strong sunlight, high temperature, drought and the influence of chemical pesticides, the survival rate of Bacillus will be significantly reduced, thus affecting its efficacy. Therefore, how to improve the survival rate of Bacillus under adverse environmental conditions has become a technical problem that needs to be solved urgently. In recent years, researchers have been committed to developing new Bacillus carrier materials in order to enhance their stress resistance by improving their physical and chemical properties.

[0003] As a new type of dosage form, oil suspension has aroused people's interest with its good adhesion and protective effect. This dosage form can not only make Bacillus uniformly distributed on the treated surface, but also isolate the influence of external harsh environmental factors to a certain extent, thereby improving the survival rate of Bacillus. Oil suspension is usually composed of active ingredients (i.e. Bacillus), carrier oil, dispersant, stabilizer and other additives. The selection of carrier oil needs to take into account its compatibility with Bacillus and its stability under different environmental conditions; dispersants and stabilizers help to ensure the uniform distribution of Bacillus in the oil phase and prevent sedimentation. Therefore, it is extremely necessary to develop an oil suspension that can effectively improve the survival rate of Bacillus. Summary of the invention

[0004] In view of the defects of the prior art, the object of the present invention is to provide an oil suspension concentrate for improving the survival rate of Bacillus and a preparation method thereof.

[0005] The technical effect of the present invention is achieved by the following technical scheme: an oil suspension for improving the survival rate of bacillus, which consists of an oil phase and an aqueous phase, wherein the oil phase comprises the following raw materials in parts by weight: 35-45 parts of coconut oil, 20-25 parts of white oil, 10-15 parts of modified isopropyl stearate, 2-3 parts of Tween-80 and 0.2-0.3 parts of ultraviolet absorber; and the aqueous phase comprises the following raw materials in parts by weight: 14-18 parts of bacillus, 0.8-1.2 parts of grafted modified hydroxypropyl methylcellulose, 0.04-0.06 parts of citric acid, 0.05-0.06 parts of polydimethylsiloxane, 0.1-0.2 parts of leucine, 0.5-0.8 parts of glucose and 0.5-0.8 parts of trehalose.

[0006] Preferably, the specific preparation steps of the modified isopropyl stearate are as follows:

[0007] A1: add isopropyl stearate to a 50wt% ethanol solution, heat in a water bath to control the temperature at 50°C, stir and dissolve evenly, slowly add concentrated sulfuric acid solution, stir and mix evenly, and slowly add propylene oxide dropwise during the reaction, heat in a water bath to control the temperature at 50-70°C, and continue the reaction for 2-6h;

[0008] A2: After the reaction in step A1 is completed, the reaction solution is cooled to room temperature, 1M NaOH solution is added to adjust the pH of the solution to neutral, and the solution is allowed to stand for 10 to 30 minutes. The organic layer is separated by centrifugation at 6000 rpm for 10 minutes, washed three times with distilled water, concentrated to 1 / 4 to 1 / 5 of the original volume by a rotary evaporator, and vacuum dried at 50°C for 12 to 16 hours to obtain modified isopropyl stearate.

[0009] Preferably, the ratio of the amount of isopropyl stearate, ethanol solution, concentrated sulfuric acid solution and propylene oxide used in step A1 is 1 g: 8-10 mL: 0.03-0.05 mL: 0.2-0.3 mL.

[0010] Preferably, the specific preparation steps of the grafted modified hydroxypropyl methylcellulose are as follows:

[0011] B1: Add hydroxypropyl methylcellulose to 50wt% ethanol solution, stir and dissolve evenly at room temperature, slowly add NaOH, continue stirring for 30min, then slowly drop 10wt% sodium chloroacetate solution at a rate of 2-5mL / min, heat in a water bath to control the temperature at 50-70℃, and stir to react for 3-6h;

[0012] B2: After the reaction in step B1 is completed, cool to room temperature, adjust the pH to neutral with 1M HCl solution, add excess ethanol three times the volume of the original solution, centrifuge at 5000 rpm for 10 min to separate the precipitate, wash the precipitate three times with distilled water, and vacuum dry at 50-60°C for 12 h to obtain grafted modified hydroxypropyl methylcellulose.

[0013] Preferably, the ratio of the amount of hydroxypropyl methylcellulose, ethanol solution, NaOH and sodium chloroacetate solution used in step B1 is 1 g: 10-15 mL: 0.2-0.25 g: 8-10 mL.

[0014] Preferably, the ultraviolet absorber is any one of phenylbenzimidazole sulfonic acid and hydroxyphenylbenzotriazole.

[0015] Preferably, the Bacillus is Bacillus thuringiensis.

[0016] Preferably, another aspect of the present invention is to provide a method for preparing an oil suspension concentrate for improving the survival rate of Bacillus, and the specific preparation steps are as follows:

[0017] S1: Add modified isopropyl stearate to a reactor, then add coconut oil and white oil, heat and control the temperature to 50-60°C, stir and mix evenly, slowly add Tween-80, stir and disperse evenly, add ultraviolet absorber, stir and mix evenly at 200 rpm to obtain oil phase, and maintain the temperature at 50-60°C for standby use;

[0018] S2: suspend the Bacillus in sterile water, maintain a low temperature of 4-10°C, add grafted modified hydroxypropyl methylcellulose, stir at 300-500 rpm for 20-30 min, add citric acid, continue to stir and disperse evenly, then add polydimethylsiloxane and leucine, stir and disperse evenly at 300 rpm, add glucose and trehalose, stir and disperse evenly at 500 rpm to obtain an aqueous phase;

[0019] S3: lowering the temperature of the oil phase prepared in step S1 to 35-40°C, raising the temperature of the water phase to 20-25°C, and slowly dropping 20% ​​of the water phase into the oil phase, stirring at 300 rpm for 15-20 minutes, controlling the temperature at 35°C during stirring, and obtaining a pretreated emulsion after the stirring is completed;

[0020] S4: slowly drop the remaining 80% of the aqueous phase into the pretreated emulsion prepared in step S3 in three batches, increase the stirring speed to 600-800 rpm, and continue stirring for 15-20 minutes. After the remaining aqueous phase is completely added, increase the stirring speed to 1000-1500 rpm and stir for 10-15 minutes. After the stirring is completed, homogenize three times. After the treatment is completed, stir at 100-200 rpm and cool to room temperature to obtain an oil suspension.

[0021] Preferably, the concentration of the Bacillus after suspension in step S2 is 10 7 ~10 8 CFU / mL.

[0022] Preferably, the homogenization treatment parameters in step S4 are pressure 100-300 bar, rotation speed 10000-15000 rpm, time 5-10 min, oil droplet refinement 1-10 μm; all treatments in step S4 are maintained at a temperature of 35°C.

[0023] The beneficial effects of the present invention are as follows:

[0024] The present invention introduces a hydrophilic group through an esterification reaction, enhances the amphiphilicity of isopropyl stearate, makes it show better stability in the oil phase, and promotes the uniform dispersion of coconut oil and white oil as a co-emulsifier, ensures the uniform distribution of bacillus in the oil suspension system, avoids local concentration differences, and reduces sedimentation and stratification phenomena; the modified isopropyl stearate forms a stable emulsified layer at the oil / water interface, cooperates with Tween-80, effectively prevents the separation of the oil phase and the water phase, and enhances the uniformity and fluidity of the whole system. The emulsified layer also provides an effective barrier for bacillus to protect it from the influence of the external environment. The medium-chain fatty acids in coconut oil have good emulsifying properties and low viscosity, can effectively improve the fluidity of the system, and provide nutritional support for bacillus at the same time, prolonging its survival time; white oil can reduce the risk of oxidation due to its chemical inertness, and prolong the shelf life of the formula through its low viscosity characteristics, ensuring the stability and fluidity of the system.

[0025] In this system, the hydrophilic part of isopropyl stearate interacts with Bacillus in the aqueous phase, and the hydrophobic part combines with coconut oil and white oil to form a stable emulsion layer; this emulsion layer not only prevents oil-water separation, but also provides physical protection for Bacillus through oil film wrapping, slowing down its inactivation process. The hydroxyl group of modified isopropyl stearate has good hydrophilicity and can form stable intermolecular interactions with the carboxyl group of carboxymethyl-modified hydroxypropyl methylcellulose through hydrogen bonds. This interaction not only improves the adhesion between the two, but also enhances the overall stability of the system, making the molecular arrangement in the aqueous phase environment more orderly, thereby providing a more stable living space for Bacillus. In addition, through the interaction between the hydroxyl group and the carboxyl group of modified isopropyl stearate, the formed complex forms a closed hydrophilic microenvironment in the aqueous phase. This environment reduces the impact of external factors on Bacillus, ensures that its activity is maintained for a longer time, and thus enhances its survival ability. Combined with the nutritional effect of coconut oil and the stability of white oil, Bacillus can obtain a suitable microenvironment in the suspension, thereby prolonging its activity. In order to further enhance the stability of the system, carboxymethyl-grafted hydroxypropyl methylcellulose (HPMC-CM) was introduced into the aqueous phase. Its hydrophilicity and water absorption significantly increased the viscosity of the system. By forming a three-dimensional network structure in the aqueous phase, it played a role in thickening and suspension, preventing Bacillus from settling. In addition, the carboxylmethyl of carboxymethyl-grafted hydroxypropyl methylcellulose carries a negative charge, forming a charge repulsion layer on the surface of the oil droplets, further stabilizing the emulsion and preventing the oil droplets from merging. At the same time, it provides additional water film protection for Bacillus, reducing the impact of environmental changes on its activity. The water absorption of carboxymethyl-grafted hydroxypropyl methylcellulose can also maintain the moisture content in the system, further improving the stability and durability of the suspension. Citric acid, as a chelating agent, effectively chelates metal ions in water to prevent them from reacting adversely with carboxymethyl-modified hydroxypropyl methylcellulose and the negatively charged groups in the cell wall of Bacillus. Citric acid not only maintains the solubility and thickening effect of carboxymethyl-grafted hydroxypropyl methylcellulose, but also ensures the stability and suspension capacity of the entire system by avoiding interference from metal ions.

[0026] In summary, through reasonable formula design, the synergistic effect of modified isopropyl stearate, carboxymethyl-modified hydroxypropyl methylcellulose, coconut oil and white oil significantly improved the survival rate of Bacillus in the oil suspension system, extended the shelf life, and reduced the impact of environmental changes on its activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 It is a graph showing the survival rate test results of the oil suspension prepared in Examples 2 to 3 of the present invention and Comparative Examples 1 to 4;

[0029] Figure 2 It is the oil-water separation index graph of the oil suspension prepared in Examples 2 to 3 of the present invention and Comparative Examples 1 to 4;

[0030] Figure 3 It is a survival rate graph of the high temperature stability test of the oil suspension prepared in Examples 2 to 3 of the present invention and Comparative Examples 1 to 4;

[0031] Figure 4 It is a test chart of ultraviolet resistance of the oil suspension prepared in Examples 2 to 3 of the present invention and Comparative Examples 1 to 4;

[0032] Figure 5 It is a drying stress test diagram of the oil suspension prepared in Examples 2 to 3 of the present invention and Comparative Examples 1 to 4;

[0033] Figure 6 It is a drying moisture change diagram of the oil suspension prepared in Examples 2 to 3 of the present invention and Comparative Examples 1 to 4. DETAILED DESCRIPTION

[0034] The technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. It should be noted that, unless otherwise specified, the raw materials involved in the present invention are purchased through conventional commercial channels, and the Bacillus is Bacillus thuringiensis, purchased from the China General Microbiological Culture Collection Management Center, with a collection number of CGMCC1.15822.

[0035] Embodiment 1: An oil suspension for improving the survival rate of Bacillus, consisting of an oil phase and an aqueous phase, wherein the oil phase comprises the following raw materials in parts by weight: 35 parts of coconut oil, 20 parts of white oil, 10 parts of modified isopropyl stearate, 2 parts of Tween-80 and 0.2 parts of ultraviolet absorber; the aqueous phase comprises the following raw materials in parts by weight: 14 parts of Bacillus, 0.8 parts of grafted modified hydroxypropyl methylcellulose, 0.04 parts of citric acid, 0.05 parts of polydimethylsiloxane, 0.1 parts of leucine, 0.5 parts of glucose and 0.5 parts of trehalose.

[0036] The specific preparation steps of modified isopropyl stearate are as follows:

[0037] A1: Add 10g of isopropyl stearate to 80mL of 50wt% ethanol solution, heat in a water bath to control the temperature at 50℃, stir and dissolve evenly, then slowly add 0.3mL of concentrated sulfuric acid solution, stir and mix evenly, and slowly add 2mL of propylene oxide dropwise during the reaction, heat in a water bath to control the temperature at 50℃, and continue the reaction for 6h;

[0038] A2: After the reaction in step A1 is completed, the reaction solution is cooled to room temperature, 1M NaOH solution is added to adjust the pH of the solution to neutral, the solution is allowed to stand for 10 min, centrifuged at 6000 rpm for 10 min to separate the organic layer, washed three times with distilled water, concentrated to 1 / 4 of the original volume by a rotary evaporator, and vacuum dried at 50°C for 12 h to obtain modified isopropyl stearate;

[0039] The specific preparation steps of the grafted modified hydroxypropyl methylcellulose are as follows:

[0040] B1: Add 10g of hydroxypropyl methylcellulose to 100mL of 50wt% ethanol solution, stir and dissolve evenly at room temperature, slowly add 2g of NaOH, continue stirring for 30min, then slowly add 80mL of 10wt% sodium chloroacetate solution at a rate of 2mL / min, heat in a water bath to control the temperature at 50℃, and stir and react for 6h;

[0041] B2: After the reaction in step B1 is completed, cool to room temperature, adjust the pH to neutral with 1M HCl solution, add 540mL excess ethanol, centrifuge at 5000rpm for 10min to separate the precipitate, wash the precipitate three times with distilled water, and vacuum dry at 50°C for 12h to obtain grafted modified hydroxypropyl methylcellulose;

[0042] The preparation method of the oil suspension for improving the survival rate of Bacillus, the specific preparation steps are as follows:

[0043] S1: Add modified isopropyl stearate to a reactor, then add coconut oil and white oil, heat and control the temperature to 50°C, stir and mix evenly, slowly add Tween-80, stir and disperse evenly, add phenylbenzimidazole sulfonic acid, stir and mix evenly at 200 rpm to obtain an oil phase, and maintain the temperature at 50°C for standby use;

[0044] S2: Suspend Bacillus thuringiensis in sterile water at a concentration of 10 7 CFU / mL, keep the low temperature at 10°C, add grafted modified hydroxypropyl methylcellulose, stir at 300 rpm for 30 min, add citric acid, continue to stir and disperse evenly, then add polydimethylsiloxane and leucine, stir and disperse evenly at 300 rpm, add glucose and trehalose, stir and disperse evenly at 500 rpm to obtain an aqueous phase;

[0045] S3: lowering the temperature of the oil phase prepared in step S1 to 35°C, raising the temperature of the water phase to 20°C, and slowly dropping 20% ​​of the water phase into the oil phase, stirring at 300 rpm for 15 min, controlling the temperature at 35°C during stirring, and obtaining a pretreated emulsion after the stirring is completed;

[0046] S4: The remaining 80% of the aqueous phase was slowly added dropwise to the pretreated emulsion prepared in step S3 in three batches, the stirring speed was increased to 600 rpm, and the stirring was continued for 20 min. After the remaining aqueous phase was completely added, the stirring speed was increased to 1000 rpm, and the stirring was continued for 15 min. After the stirring was completed, the homogenization treatment was performed three times, and the parameters were pressure 100 bar, speed 10000 rpm, time 10 min, and oil droplet refinement 10 μm. The temperature was maintained at 35°C for all treatments. After the treatment was completed, the mixture was stirred at 200 rpm and cooled to room temperature to obtain an oil suspension.

[0047] Embodiment 2: An oil suspension for improving the survival rate of Bacillus, consisting of an oil phase and an aqueous phase, wherein the oil phase comprises the following raw materials in parts by weight: 45 parts of coconut oil, 25 parts of white oil, 15 parts of modified isopropyl stearate, 2 parts of Tween-80 and 0.2 parts of ultraviolet absorber; the aqueous phase comprises the following raw materials in parts by weight: 18 parts of Bacillus, 1 part of grafted modified hydroxypropyl methylcellulose, 0.05 parts of citric acid, 0.05 parts of polydimethylsiloxane, 0.15 parts of leucine, 0.6 parts of glucose and 0.6 parts of trehalose.

[0048] The specific preparation steps of modified isopropyl stearate are as follows:

[0049] A1: Add 10g of isopropyl stearate to 100mL of 50wt% ethanol solution, heat in a water bath to control the temperature at 50℃, stir and dissolve evenly, slowly add 0.5mL of concentrated sulfuric acid solution, stir and mix evenly, and slowly add 2.5mL of propylene oxide dropwise during the reaction, heat in a water bath to control the temperature at 60℃, and continue the reaction for 4h;

[0050] A2: After the reaction in step A1 is completed, the reaction solution is cooled to room temperature, 1M NaOH solution is added to adjust the pH of the solution to neutral, the solution is allowed to stand for 15 minutes, centrifuged at 6000 rpm for 10 minutes to separate the organic layer, washed three times with distilled water, concentrated to 1 / 5 of the original volume by rotary evaporator, and vacuum dried at 50°C for 16 hours to obtain modified isopropyl stearate;

[0051] The specific preparation steps of grafted modified hydroxypropyl methylcellulose are as follows:

[0052] B1: Add 10g of hydroxypropyl methylcellulose to 150mL of 50wt% ethanol solution, stir and dissolve evenly at room temperature, slowly add 2.2g of NaOH, continue stirring for 30min, then slowly add 90mL of 10wt% sodium chloroacetate solution at a rate of 3mL / min, heat in a water bath to control the temperature at 60℃, and stir and react for 5h;

[0053] B2: After the reaction in step B1 is completed, cool to room temperature, adjust the pH to neutral with 1M HCl solution, add 720mL excess ethanol, centrifuge at 5000rpm for 10min to separate the precipitate, wash the precipitate three times with distilled water, and vacuum dry at 50°C for 12h to obtain grafted modified hydroxypropyl methylcellulose;

[0054] The preparation method of the oil suspension for improving the survival rate of Bacillus, the specific preparation steps are as follows:

[0055] S1: Add modified isopropyl stearate to a reactor, then add coconut oil and white oil, heat and control the temperature to 50°C, stir and mix evenly, slowly add Tween-80, stir and disperse evenly, add hydroxyphenylbenzotriazole, stir and mix evenly at 200 rpm to obtain an oil phase, and maintain the temperature at 50°C for standby use;

[0056] S2: Suspend Bacillus thuringiensis in sterile water at a concentration of 10 8 CFU / mL, keep the low temperature at 4°C, add grafted modified hydroxypropyl methylcellulose, stir at 400 rpm for 30 min, add citric acid, continue to stir and disperse evenly, then add polydimethylsiloxane and leucine, stir and disperse evenly at 300 rpm, add glucose and trehalose, stir and disperse evenly at 500 rpm to obtain an aqueous phase;

[0057] S3: lowering the temperature of the oil phase prepared in step S1 to 37°C, raising the temperature of the water phase to 25°C, and slowly dropping 20% ​​of the water phase into the oil phase, stirring at 300 rpm for 15 min, controlling the temperature at 35°C during stirring, and obtaining a pretreated emulsion after the stirring is completed;

[0058] S4: The remaining 80% of the aqueous phase was slowly added dropwise to the pretreated emulsion prepared in step S3 in three batches, the stirring speed was increased to 800 rpm, and the stirring was continued for 15 min. After the remaining aqueous phase was completely added, the stirring speed was increased to 1500 rpm, and the stirring was continued for 10 min. After the stirring was completed, the homogenization treatment was performed three times, and the parameters were pressure 200 bar, speed 12000 rpm, time 8 min, oil droplet refinement 2 μm, and the temperature was maintained at 35°C for all treatments. After the treatment was completed, the mixture was stirred at 100 rpm and cooled to room temperature to obtain an oil suspension.

[0059] Embodiment 3: An oil suspension for improving the survival rate of Bacillus, consisting of an oil phase and an aqueous phase, wherein the oil phase comprises the following raw materials in parts by weight: 40 parts of coconut oil, 22 parts of white oil, 13 parts of modified isopropyl stearate, 2 parts of Tween-80 and 0.3 parts of ultraviolet absorber; the aqueous phase comprises the following raw materials in parts by weight: 16 parts of Bacillus, 1 part of grafted modified hydroxypropyl methylcellulose, 0.06 parts of citric acid, 0.06 parts of polydimethylsiloxane, 0.2 parts of leucine, 0.7 parts of glucose and 0.7 parts of trehalose.

[0060] The specific preparation steps of modified isopropyl stearate are as follows:

[0061] A1: Add 10g of isopropyl stearate to 100mL of 50wt% ethanol solution, heat in a water bath to control the temperature at 50℃, stir and dissolve evenly, then slowly add 0.4mL of concentrated sulfuric acid solution, stir and mix evenly, and slowly add 2.5mL of propylene oxide dropwise during the reaction, heat in a water bath to control the temperature at 70℃, and continue the reaction for 4h;

[0062] A2: After the reaction in step A1 is completed, the reaction solution is cooled to room temperature, 1M NaOH solution is added to adjust the pH of the solution to neutral, the solution is allowed to stand for 20 minutes, centrifuged at 6000 rpm for 10 minutes to separate the organic layer, washed three times with distilled water, concentrated to 1 / 5 of the original volume by rotary evaporator, and vacuum dried at 50°C for 14 hours to obtain modified isopropyl stearate;

[0063] The specific preparation steps of grafted modified hydroxypropyl methylcellulose are as follows:

[0064] B1: Add 10g of hydroxypropyl methylcellulose to 150mL of 50wt% ethanol solution, stir and dissolve evenly at room temperature, slowly add 2.3g of NaOH, continue stirring for 30min, then slowly add 85mL of 10wt% sodium chloroacetate solution at a rate of 4mL / min, heat in a water bath to control the temperature at 60℃, and stir and react for 5h;

[0065] B2: After the reaction in step B1 is completed, cool to room temperature, adjust the pH to neutral with 1M HCl solution, add 705mL excess ethanol, centrifuge at 5000rpm for 10min to separate the precipitate, wash the precipitate three times with distilled water, and vacuum dry at 55°C for 12h to obtain grafted modified hydroxypropyl methylcellulose;

[0066] The preparation method of the oil suspension for improving the survival rate of Bacillus, the specific preparation steps are as follows:

[0067] S1: Add modified isopropyl stearate to a reactor, then add coconut oil and white oil, heat and control the temperature to 55°C, stir and mix evenly, slowly add Tween-80, stir and disperse evenly, add phenylbenzimidazole sulfonic acid, stir and mix evenly at 200 rpm to obtain an oil phase, and maintain the temperature at 55°C for standby use;

[0068] S2: Suspend Bacillus thuringiensis in sterile water at a concentration of 10 8 CFU / mL, keep the low temperature at 7°C, add grafted modified hydroxypropyl methylcellulose, stir at 500 rpm for 25 min, add citric acid, continue to stir and disperse evenly, then add polydimethylsiloxane and leucine, stir and disperse evenly at 300 rpm, add glucose and trehalose, stir and disperse evenly at 500 rpm to obtain an aqueous phase;

[0069] S3: lowering the temperature of the oil phase prepared in step S1 to 35°C, raising the temperature of the water phase to 25°C, and slowly dropping 20% ​​of the water phase into the oil phase, stirring at 300 rpm for 18 minutes, controlling the temperature at 35°C during stirring, and obtaining a pretreated emulsion after the stirring is completed;

[0070] S4: The remaining 80% of the aqueous phase was slowly added dropwise to the pretreated emulsion prepared in step S3 in three batches, the stirring speed was increased to 700 rpm, and the stirring was continued for 18 minutes. After the remaining aqueous phase was completely added, the stirring speed was increased to 1200 rpm, and the stirring was continued for 12 minutes. After the stirring was completed, the homogenization treatment was performed three times, and the parameters were pressure 200 bar, rotation speed 14000 rpm, time 6 minutes, oil droplet refinement 5 μm, and the temperature was maintained at 35°C for all treatments. After the treatment was completed, the mixture was stirred at 150 rpm and cooled to room temperature to obtain an oil suspension.

[0071] Embodiment 4: An oil suspension for improving the survival rate of Bacillus, consisting of an oil phase and an aqueous phase, wherein the oil phase comprises the following raw materials in parts by weight: 45 parts of coconut oil, 25 parts of white oil, 15 parts of modified isopropyl stearate, 3 parts of Tween-80 and 0.3 parts of ultraviolet absorber; the aqueous phase comprises the following raw materials in parts by weight: 18 parts of Bacillus, 1.2 parts of grafted modified hydroxypropyl methylcellulose, 0.06 parts of citric acid, 0.06 parts of polydimethylsiloxane, 0.2 parts of leucine, 0.8 parts of glucose and 0.8 parts of trehalose.

[0072] The specific preparation steps of modified isopropyl stearate are as follows:

[0073] A1: Add 10g of isopropyl stearate to 90mL of 50wt% ethanol solution, heat in a water bath to control the temperature at 50℃, stir and dissolve evenly, then slowly add 0.5mL of concentrated sulfuric acid solution, stir and mix evenly, and slowly add 3mL of propylene oxide dropwise during the reaction, heat in a water bath to control the temperature at 70℃, and continue the reaction for 2h;

[0074] A2: After the reaction in step A1 is completed, the reaction solution is cooled to room temperature, 1M NaOH solution is added to adjust the pH of the solution to neutral, the solution is allowed to stand for 30 minutes, centrifuged at 6000 rpm for 10 minutes to separate the organic layer, washed three times with distilled water, concentrated to 1 / 5 of the original volume by rotary evaporator, and vacuum dried at 50°C for 16 hours to obtain modified isopropyl stearate;

[0075] The specific preparation steps of grafted modified hydroxypropyl methylcellulose are as follows:

[0076] B1: Add 10g of hydroxypropyl methylcellulose to 150mL of 50wt% ethanol solution, stir and dissolve evenly at room temperature, slowly add 2.5g of NaOH, continue stirring for 30min, then slowly add 100mL of 10wt% sodium chloroacetate solution at a rate of 5mL / min, heat in a water bath to control the temperature at 70℃, and stir and react for 3h;

[0077] B2: After the reaction in step B1 is completed, the mixture is cooled to room temperature, the pH is adjusted to neutral using a 1M HCl solution, and then three times the volume of excess ethanol of the original solution is added, and the precipitate is separated by centrifugation. The precipitate is washed three times with distilled water, and vacuum dried at 60°C for 12 hours to obtain grafted modified hydroxypropyl methylcellulose;

[0078] The preparation method of the oil suspension for improving the survival rate of Bacillus, the specific preparation steps are as follows:

[0079] S1: Add modified isopropyl stearate to a reactor, then add coconut oil and white oil, heat and control the temperature to 60°C, stir and mix evenly, slowly add Tween-80, stir and disperse evenly, add hydroxyphenylbenzotriazole, stir and mix evenly at 200 rpm to obtain an oil phase, and maintain the temperature at 60°C for standby use;

[0080] S2: Suspend Bacillus thuringiensis in sterile water at a concentration of 10 8 CFU / mL, keep the low temperature at 4°C, add grafted modified hydroxypropyl methylcellulose, stir at 500 rpm for 20 min, add citric acid, continue to stir and disperse evenly, then add polydimethylsiloxane and leucine, stir and disperse evenly at 300 rpm, add glucose and trehalose, stir and disperse evenly at 500 rpm to obtain an aqueous phase;

[0081] S3: lowering the temperature of the oil phase prepared in step S1 to 40°C, raising the temperature of the water phase to 25°C, and slowly dropping 20% ​​of the water phase into the oil phase, stirring at 300 rpm for 20 minutes, controlling the temperature at 35°C during stirring, and obtaining a pretreated emulsion after the stirring is completed;

[0082] S4: The remaining 80% of the aqueous phase was slowly added dropwise to the pretreated emulsion prepared in step S3 in three batches, the stirring speed was increased to 800 rpm, and the stirring was continued for 15 min. After the remaining aqueous phase was completely added, the stirring speed was increased to 1500 rpm, and the stirring was continued for 10 min. After the stirring was completed, the homogenization treatment was performed three times, and the parameters were pressure 300 bar, rotation speed 15000 rpm, time 5 min, and oil droplet refinement 1 μm. The temperature was maintained at 35°C for all treatments. After the treatment was completed, the mixture was stirred at 200 rpm and cooled to room temperature to obtain an oil suspension.

[0083] Comparative Example 1: The operation of Comparative Example 1 is substantially the same as that of Example 2, except that modified isopropyl stearate is not used in Comparative Example 1.

[0084] Comparative Example 2: The operation of Comparative Example 2 is basically the same as that of Example 2, except that hydroxypropyl methylcellulose is used in Comparative Example 2 to replace the grafted modified hydroxypropyl methylcellulose.

[0085] Comparative Example 3: The operation of Comparative Example 3 is basically the same as that of Example 2, except that soybean oil is used instead of coconut oil in Comparative Example 3.

[0086] Comparative Example 4: The operation of Comparative Example 4 is basically the same as that of Example 2, except that no white oil is added in Comparative Example 4.

[0087] Performance Testing:

[0088] Bacillus survival rate test: 1 mL of the oil suspension prepared in Examples 2 to 3 and Comparative Examples 1 to 4 was diluted 100 times and inoculated onto a sterile plate culture medium. The plate was cultured at 35° C. for 24 h, the number of colonies on the plate was observed, and the initial number of viable Bacillus per milliliter in the sample was calculated, which was recorded as the initial number of bacteria. Then 10 mL of the oil suspension prepared in Examples 2 to 3 and Comparative Examples 1 to 4 was accelerated aged at 42° C. The number of bacteria was sampled and tested on the 14th day. The sample was diluted immediately after being taken out, and the same dilution steps as the initial number of bacteria were repeated to calculate the survival rate / (%) = current number of bacteria / initial number of bacteria × 100%. The results are as follows: Figure 1 shown.

[0089] Depend on Figure 1As a result, the oil suspension prepared by the present invention can effectively maintain a high survival rate of Bacillus. From the results of Comparative Example 1 and Example 2, it can be seen that the lack of modified isopropyl stearate makes it impossible to effectively promote the uniform dispersion of the oil phase and the water phase, the synergistic emulsification effect is affected, the oil-water separation is more likely to occur, the emulsion is unstable, and the Bacillus loses effective physical barrier protection, resulting in a decrease in survival rate; from the results of Comparative Example 2 and Example 2, it can be seen that the grafted modified hydroxypropyl methylcellulose enhances the hydrophilicity and thickening effect by grafting carboxymethyl groups, forms a three-dimensional network structure in the aqueous phase, prevents the oil droplets from merging, and keeps the Bacillus uniformly distributed in the suspension, and after being grafted with hydroxypropyl methylcellulose, the thickening and suspension effects are affected. The results of Comparative Example 3 and Example 2 show that the synergistic effect of the higher viscosity of soybean oil and the lower viscosity of white oil is weakened, resulting in a decrease in the fluidity of the entire system, a deterioration in the dispersibility of the oil phase and the water phase, and an increase in the possibility of oil-water separation and oil droplet merging. This synergistic failure may lead to local Bacillus malnutrition, thereby reducing its survival rate. The results of Comparative Example 4 and Example 2 show that the lack of white oil reduces the fluidity of the entire system, and because white oil has oxidative stability, its absence makes the oil phase (coconut oil) more susceptible to oxidative stress, resulting in a significant decrease in the survival rate of Bacillus.

[0090] Centrifugal stratification test: Take 20 mL of the oil suspension prepared in Examples 2 to 3 and Comparative Examples 1 to 4 and centrifuge at 5000 rpm for 30 min, keep at room temperature, record the stratification, measure the height of the oil layer, emulsion layer, and water layer, repeat the test three times for each sample, take the average value of the results, and calculate the oil-water separation index = separation volume / total sample volume × 100%, where the separation volume refers to the volume of the separated water phase or oil phase. The results are as follows: Figure 2 shown.

[0091] Depend on Figure 2As can be seen from the results, the oil suspension prepared by the present invention is efficient and stable, and has an extremely stable emulsion layer; from the results of Comparative Example 1 and Example 2, it can be seen that the lack of modified isopropyl stearate cannot effectively form a stable emulsion layer at the oil / water interface, resulting in an increased separation trend of the oil phase and the water phase, and a higher separation index; from the results of Comparative Example 2 and Example 2, it can be seen that hydroxypropyl methylcellulose significantly enhances its hydrophilicity and thickening effect by introducing carboxymethyl groups, and forms a charge repulsion layer on the surface of the oil droplets to prevent the oil droplets from merging, while hydroxypropyl methylcellulose does not have such enhanced hydrophilicity and charge repulsion ability, and has weak stability for oil-water emulsions, and the oil-water separation index is significantly increased; from the results of Comparative Example 3 and Example 2, it can be seen that coconut oil contains medium-chain fatty acids and has better emulsification properties, while soybean oil has poor emulsification properties and is difficult to maintain a stable oil / water emulsion layer, which may lead to obvious oil-water separation; from the results of Comparative Example 4 and Example 2, it can be seen that the lack of low viscosity and chemical inertness of white oil may lead to reduced fluidity of the system, and other high-viscosity components are more likely to cause local concentration, resulting in an increase in the oil-water separation index.

[0092] High temperature stability test: 10 mL of the oil suspension prepared in Examples 2 to 3 and Comparative Examples 1 to 4 was placed at 50°C for 24 h and 36 h, and the survival rate of Bacillus before and after exposure was compared. The survival rate / (%) was calculated as current bacterial count / initial bacterial count × 100%. The results are as follows: Figure 3 shown.

[0093] Depend on Figure 3 As can be seen from the results, the oil suspension prepared by the present invention can effectively maintain stability at high temperatures, and the emulsification property is not affected; from the results of Comparative Example 1 and Example 2, it can be seen that due to the weak interfacial stability between the oil phase and the aqueous phase, the emulsion is easy to stratify, so at a high temperature of 50°C, the oil phase and the aqueous phase may gradually separate, resulting in a decrease in the survival rate of Bacillus, especially after 36 hours, a significant decrease in the survival rate; from the results of Comparative Example 2 and Example 2, it can be seen that the thickening and stabilizing effects of hydroxypropyl methylcellulose are not as significant as those of grafted modified hydroxypropyl methylcellulose, especially its charge repulsion ability is weak, and when exposed at 50°C, due to the decrease in emulsion stability, oil droplets are more likely to merge Moreover, the uniformity of the system is weakened, which eventually leads to the instability of the protective barrier of Bacillus, and the survival rate of Bacillus decreases particularly significantly; from the results of Comparative Example 3 and Example 2, it can be seen that due to the high viscosity of soybean oil and its easier oxidation at high temperatures, when exposed at 50°C, the oxidation of soybean oil and the fluidity of the system deteriorate, which may lead to the merging and stratification of oil droplets, and the survival rate of Bacillus decreases significantly; from the results of Comparative Example 4 and Example 2, it can be seen that due to the lack of white oil, the oil phase is more susceptible to oxidation, and the low viscosity characteristics of the system are weakened. Under high temperature conditions, the fluidity of the oil phase is reduced, and the stability of the emulsion layer may deteriorate, resulting in a decrease in the survival rate of Bacillus.

[0094] UV resistance test: Test conditions: UV-C light source with a wavelength of 254nm, the distance between the light source and the oil suspension sample was adjusted to 30cm, and the irradiation intensity was 3mW / cm 2 , irradiation time 1h, 2h, compare the survival rate of Bacillus before and after irradiation, calculate the survival rate / (%) = current bacterial count / initial bacterial count × 100%, the results are as follows Figure 4 shown.

[0095] Depend on Figure 4 As can be seen from the results, the oil suspension prepared by the present invention can effectively resist the influence of ultraviolet rays and can effectively maintain its stability under the influence of ultraviolet rays; from the results of Comparative Example 1 and Example 2, it can be seen that due to the use of unmodified isopropyl stearate, the synergistic effect is significantly affected, the emulsification is poor, the protection ability is poor, and the survival rate is significantly reduced; from the results of Comparative Example 2 and Example 2, it can be seen that due to the use of hydroxypropyl methylcellulose, a stable three-dimensional network and an emulsified layer cannot be effectively synergistically formed, resulting in the sedimentation of Bacillus and the separation of oil and water, and the survival rate is low; from the results of Comparative Example 3 and Example 2, it can be seen that the use of soybean oil leads to high viscosity and poor fluidity, and a stable protective layer cannot be formed, and the survival rate decreases; from the results of Comparative Example 4 and Example 2, it can be seen that the lack of white oil reduces the stability of the system and the antioxidant capacity, resulting in a low survival rate.

[0096] Drying stress test: relative humidity is 20%, temperature is 30℃, exposure is 24h and 48h, and the survival rate of Bacillus before and after irradiation is compared. The survival rate / (%) is calculated as current bacterial count / initial bacterial count×100%. The results are as follows: Figure 5 As shown, a moisture analyzer (Karl Fischer method) was used to measure the moisture content in the sample, and the changes in the moisture content of the sample under a dry environment were compared. The results are shown in Figure 6 shown.

[0097] Depend on Figure 5 and Figure 6 As can be seen from the results, the oil suspension prepared by the present invention can effectively cope with drying stress, and its survival rate and moisture content are not significantly affected; from the results of Comparative Example 1 and Example 2, it can be seen that when the emulsion layer is unstable, moisture is more easily lost, especially under low humidity and high temperature conditions, which will cause the moisture content to decrease rapidly and the Bacillus to be inactivated due to water loss; from the results of Comparative Example 2 and Example 2, it can be seen that the water absorption of the grafted modified hydroxypropyl methylcellulose enables it to retain more moisture in a dry environment, while hydroxypropyl methylcellulose cannot achieve the same effect, resulting in a rapid decrease in the moisture content of the system under dry conditions; from the results of Comparative Example 3 and Example 2, it can be seen that the chemical stability of soybean oil is relatively poor, the viscosity is high, the fluidity is poor, and the emulsification effect is poor, resulting in an unstable emulsion layer; from the results of Comparative Example 4 and Example 2, it can be seen that in the absence of white oil, the overall viscosity of the oil phase is high, resulting in a poor emulsification effect.

[0098] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An oil suspension for improving the survival rate of Bacillus, characterized in that: The oil suspension consists of an oil phase and an aqueous phase. The oil phase comprises the following raw materials in parts by weight: 35-45 parts of coconut oil, 20-25 parts of white oil, 10-15 parts of modified isopropyl stearate, 2-3 parts of Tween-80 and 0.2-0.3 parts of ultraviolet absorber; the aqueous phase comprises the following raw materials in parts by weight: 14-18 parts of bacillus, 0.8-1.2 parts of grafted modified hydroxypropyl methylcellulose, 0.04-0.06 parts of citric acid, 0.05-0.06 parts of polydimethylsiloxane, 0.1-0.2 parts of leucine, 0.5-0.8 parts of glucose and 0.5-0.8 parts of trehalose.

2. An oil suspension concentrate for improving the survival rate of Bacillus according to claim 1, characterized in that: The specific preparation steps of the modified isopropyl stearate are as follows: A1: Add isopropyl stearate to ethanol solution, heat in a water bath to control the temperature, stir and dissolve evenly, then slowly add concentrated sulfuric acid solution, stir and mix evenly, and slowly add propylene oxide dropwise during the reaction, heat in a water bath to control the temperature, and continue the reaction; A2: After the reaction in step A1 is completed, the reaction solution is cooled to room temperature, and NaOH solution is added to adjust the pH of the solution to neutral. The solution is allowed to stand, and the organic layer is separated by centrifugation. The organic layer is washed three times with distilled water, concentrated by a rotary evaporator, and dried in vacuo to obtain modified isopropyl stearate.

3. An oil suspension concentrate for improving the survival rate of bacillus according to claim 2, characterized in that: The ratio of the amount of isopropyl stearate, ethanol solution, concentrated sulfuric acid solution and propylene oxide used in step A1 is 1g:8-10mL:0.03-0.05mL:0.2-0.3mL.

4. An oil suspension concentrate for improving the survival rate of Bacillus according to claim 3, characterized in that: The specific preparation steps of the grafted modified hydroxypropyl methylcellulose are as follows: B1: Add hydroxypropyl methylcellulose to the ethanol solution, stir and dissolve evenly at room temperature, slowly add NaOH, continue to stir and mix, and then slowly drop sodium chloroacetate solution at a rate of 2-5 mL / min, heat in a water bath to control the temperature, and stir to react; B2: After the reaction in step B1 is completed, the mixture is cooled to room temperature, the pH is adjusted to neutral using HCl solution, and then three times the volume of excess ethanol of the original solution is added. The precipitate is separated by centrifugation, and the precipitate is washed three times with distilled water and vacuum dried to obtain grafted modified hydroxypropyl methylcellulose.

5. An oil suspension concentrate for improving the survival rate of Bacillus according to claim 4, characterized in that: The ratio of the amount of hydroxypropyl methylcellulose, ethanol solution, NaOH and sodium chloroacetate solution used in step B1 is 1 g: 10-15 mL: 0.2-0.25 g: 8-10 mL.

6. An oil suspension concentrate for improving the survival rate of Bacillus according to claim 5, characterized in that: The ultraviolet absorber is any one of phenylbenzimidazole sulfonic acid and hydroxyphenylbenzotriazole.

7. A method for preparing an oil suspension concentrate for improving the survival rate of Bacillus according to any one of claims 1 to 6, characterized in that: The specific preparation steps are as follows: S1: Add modified isopropyl stearate to a reactor, then add coconut oil and white oil, heat to control the temperature, stir and mix evenly, slowly add Tween-80, stir and disperse evenly, add ultraviolet absorber, stir and mix evenly to obtain an oil phase, and maintain the temperature for standby use; S2: suspend the Bacillus in sterile water, keep the temperature at 4-10°C, add the grafted modified hydroxypropyl methylcellulose, stir and mix evenly, add citric acid, continue to stir and disperse evenly, then add polydimethylsiloxane and leucine, stir and disperse evenly, add glucose and trehalose, stir and disperse evenly, and obtain an aqueous phase; S3: lowering the temperature of the oil phase prepared in step S1 to 35-40°C, raising the temperature of the water phase to 20-25°C, and slowly dropping 20% ​​of the water phase into the oil phase while continuously stirring. During stirring, the temperature is controlled at 35°C. After the stirring is completed, a pretreated emulsion is obtained; S4: slowly drop the remaining 80% of the aqueous phase into the pretreated emulsion prepared in step S3 in three batches, increase the stirring speed to 600-800 rpm, and continue stirring for 15-20 minutes. After the remaining aqueous phase is completely added, increase the stirring speed to 1000-1500 rpm and stir for 10-15 minutes. After the stirring is completed, homogenize three times. After the treatment is completed, stir and cool to room temperature to obtain an oil suspension.

8. A method for preparing an oil suspension concentrate for improving the survival rate of Bacillus according to claim 7, characterized in that: The concentration of the Bacillus after suspension in step S2 is 10 7 ~10 8 CFU / mL.

9. A method for preparing an oil suspension concentrate for improving the survival rate of Bacillus according to claim 8, characterized in that: The homogenization treatment parameters in step S4 are pressure 100-300 bar, rotation speed 10000-15000 rpm, time 5-10 min, oil droplet refinement 1-10 μm; all treatments in step S4 are maintained at a temperature of 35°C.

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