A method for preparing microbial preparation by low-temperature step-by-step flocculation

Abstract

The invention discloses a method for preparing a microbial preparation by low-temperature step-by-step flocculation, belongs to the technical field of microbial preparations, and is characterized in that the preparation method of the microbial preparation comprises: taking a fermentation liquid of Bacillus amyloliquefaciens, adding calcium chloride with a mass ratio of (0.02-0.2)%, adding resin, stirring and adsorbing for 1-2 hours; fishing out the resin, adding the treated liquid to a sodium hydrogen phosphate solution, adding calcium chloride with a mass ratio of 25-30%, stirring for 1-2 hours, filtering and separating to obtain a flocculent and a clear liquid, adding diatomaceous earth and resin to the flocculent to obtain a flocculent, naturally air-drying at low temperature, and crushing to obtain a microbial preparation. The beneficial effects of the invention are: the invention creatively uses calcium chloride as a flocculant, and while preparing a microbial preparation containing Bacillus amyloliquefaciens by step-by-step flocculation, it also greatly improves the yield of 2-3-butanediol.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial preparations, and more specifically relates to a method for preparing a microbial preparation by low-temperature step-by-step flocculation. Background Art

[0002] 2-3-Butanediol is an important platform compound, widely used in chemical, food, medicine and materials fields. It is currently mainly derived from petroleum raw materials. However, with the shortage of petroleum resources, it is particularly necessary to develop a process for biorefining 2-3-butanediol using renewable biomass as raw materials.

[0003] Currently, most strains used to ferment and produce 2,3-butanediol, including Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae and Serratia marcescens, are mesophilic bacteria, and the fermentation temperature is generally 37°C or below. Due to the low fermentation temperature, it is easy to be contaminated by miscellaneous bacteria, and high requirements are placed on fermentation equipment and production operations.

[0004] At the same time, these strains are conditional pathogens and are not suitable for industrial applications. The thermophilic strains reported to produce 2,3-butanediol have weak xylose utilization ability and low optical purity of the product 2,3-butanediol. Xylose is the second most abundant monosaccharide in nature, mainly present in the form of polymers such as hemicellulose in straw raw materials, and is an important non-grain biomass resource. At the same time, inulin raw material is also a widely distributed non-grain biomass raw material, which has been used to produce ethanol, lactic acid and other chemicals in recent years. There are many research reports, but the production of 2,3-butanediol by this raw material is less studied and the production level is low. Therefore, it is necessary to develop a biologically safe strain that can efficiently utilize biomass raw materials such as xylose and inulin to produce 2,3-butanediol.

[0005] Shandong University has conducted a comprehensive study on the production of 2-3-butanediol and obtained multiple strains of Klebsiella, Enterobacter, Serratia, and Bacillus that produce 2-3-butanediol, especially Bacillus amyloliquefaciens LR., which has a better conversion rate to 2,3-butanediol.

[0006] Bacillus amyloliquefaciens belongs to the genus Bacillus. During the fermentation process, Bacillus amyloliquefaciens can produce antibacterial substances, antibacterial proteins, and lipopeptide antibiotics, especially having antagonistic effects on other pathogenic bacteria.

[0007] At present, 2,3-butanediol is prepared by separating the bacteria from the fermentation broth of Bacillus amyloliquefaciens through membrane filtration or flocculation sedimentation. However, the excretion of the separated bacteria causes environmental pollution.

[0008] Therefore, it is a new idea to use the fermentation broth of Bacillus amyloliquefaciens to prepare microbial preparations through flocculation, so as to obtain bacterial protein feed with antagonistic effect on other pathogenic bacteria. Summary of the invention

[0009] In order to solve the above problems and overcome the shortcomings of the prior art, the present invention provides a method for preparing a microbial preparation by low-temperature step-by-step flocculation, which can effectively solve the problem of preparing 2,3-butanediol after separating the bacteria from the fermentation broth of Bacillus amyloliquefaciens by membrane filtration or flocculation sedimentation, and the problem of environmental pollution caused by the excretion of the separated bacteria.

[0010] The specific technical solution of the present invention to solve the above technical problems is: a method for preparing a microbial preparation by low-temperature step-by-step flocculation, characterized in that the method for preparing the microbial preparation comprises:

[0011] (1) Take the fermentation liquid of Bacillus amyloliquefaciens, add calcium chloride at a mass ratio of 0.02-0.2%, and stir thoroughly for 1-2 hours; add resin at a mass ratio of 10-15%, and stir and adsorb for 1-2 hours; use a grate to remove the resin to obtain the treated liquid for standby use;

[0012] (2) Adding the treatment liquid to a 10-25% sodium hydrogen phosphate solution as a reaction aid, wherein the amount of sodium hydrogen phosphate added is 4-6% of the mass of the treatment liquid, and stirring for 1-2 hours;

[0013] (3) Add 25-30% calcium chloride by mass, stir for 1-2 hours, and filter to separate the floccules and clear liquid.

[0014] (4) Add 2-3% diatomaceous earth and the resin obtained in step (1) to the flocs according to the mass ratio of the fermentation liquid of Bacillus amyloliquefaciens, stir for 1-2 hours to obtain flocs, naturally air-dry at low temperature, and grind to obtain a microbial preparation.

[0015] Furthermore, the Bacillus amyloliquefaciens fermentation broth is obtained by fermenting Bacillus amyloliquefaciens, and the content of 2-3-butanediol in the Bacillus amyloliquefaciens fermentation broth is 36-42 g / L.

[0016] Furthermore, the Bacillus amyloliquefaciens fermentation broth is obtained by fermenting Bacillus amyloliquefaciens, and the fermentation conditions are as follows: in the fermentation medium, the seed inoculation amount is 5%, the rotation speed is 500 rpm, the ventilation volume is 4 L / min, the initial glucose concentration is 80 g / L, the initial pH=7.0, the fermentation temperature is 42°C, and the fermentation is carried out for 48 hours.

[0017] Furthermore, the fermentation medium comprises, in parts by mass, 14.7 parts of corn steep liquor dry powder, 5.8 parts of yeast powder, 1 part of ammonium citrate, 6.5 parts of anhydrous sodium acetate, 0.5 parts of MgSO4, 2 parts of K2HPO4, and 80 parts of glucose.

[0018] Furthermore, the resin model is D3520.

[0019] Furthermore, the stirring frequency of step (1) is 0.85-1.00 Hz, the stirring frequency of step (2) is 1.35-1.5 Hz, the stirring frequency of step (3) is 20-30 Hz, and the stirring frequency of step (4) is 10-15 Hz.

[0020] The beneficial effects of the present invention are:

[0021] (1) The present invention creatively uses calcium chloride as a flocculant to prepare a microbial preparation containing Bacillus amyloliquefaciens by step-by-step flocculation, while also greatly improving the yield of 2-3-butanediol.

[0022] (2) The present invention firstly adsorbs the small molecular substances in the clear liquid through the resin, and then cleverly separates the resin from the system, and then flocculates it through high concentration of calcium chloride. It can not only retain 2-3-butanediol in the clear liquid to a great extent, but also effectively avoid the destruction of the resin by high concentration of calcium chloride, which causes the adsorbed substances to re-enter the clear liquid. The destroyed resin releases the active ingredients, which is beneficial to the activity of the microbial preparation in the later stage. Moreover, the porous particle state of the resin can also prevent the microbial preparation from getting damp and hardening.

[0023] (3) Calcium chloride not only has a flocculation effect, but also cooperates with disodium hydrogen phosphate to produce a common ion effect and a salting-out effect in the later stage, which can remove 2-3-butanediol from the system, especially from the water in the flocculent body, thereby improving the yield and allowing the 2-3-butanediol part of the clear liquid to separate from the clear liquid, which is beneficial to the later distillation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Attached Figure 1 It is a product diagram of the present invention;

[0025] Attached Figure 2 It is a state diagram of the filter cake of the plate and frame filtration of the present invention;

[0026] Attached Figure 3 This is a product diagram of Comparative Example 2 of the present invention; in the accompanying drawings: DETAILED DESCRIPTION

[0027] In the description of the present invention, specific details are only for a full understanding of the embodiments of the present invention, but those skilled in the art should know that the implementation of the present invention is not limited to these details. In addition, well-known structures and functions are not described or shown in detail to avoid blurring the key points of the embodiments of the present invention. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0028] Specific implementation of the present invention:

[0029] In order to better understand the present invention, a specific embodiment is used for illustration. It is worth emphasizing that the effect of this embodiment is not substantially different from the various embodiments within the protection scope of the present invention, including the respective reagents and the content ratio of the reagents, and all of them can achieve the effects described in the present invention and solve the above-mentioned problems. Other combinations are not described here.

[0030] The Bacillus amyloliquefaciens used in the following examples is from the Microbiology Laboratory of Shandong University and belongs to the genus Bacillus. The Bacillus amyloliquefaciens of the present invention is cited by reference. The high-yield 2,3-butanediol property of Bacillus amyloliquefaciens does not belong to the protection scope of the present invention. The present invention only borrows the fermentation broth of the strain to study the influence of the low-temperature step-by-step flocculation process on the fermentation broth.

[0031] The amyloliquefaciens fermentation liquid is obtained by fermenting Bacillus amyloliquefaciens, and the fermentation conditions are in a fermentation medium, and the fermentation medium is in parts by mass: 14.7 parts of corn syrup dry powder, 5.8 parts of yeast powder, 1 part of ammonium citrate, 6.5 parts of anhydrous sodium acetate, 0.5 parts of MgSO4, 2 parts of K2HPO4, and 80 parts of glucose;

[0032] The seed inoculation amount was 5%, the rotation speed was 500 rpm, the ventilation volume was 4 L / min, the initial glucose concentration was 80 g / L, the initial pH was 7.0, the fermentation temperature was 42°C, and the fermentation time was 48 hours.

[0033] Among them, the content of 2-3-butanediol in the fermentation broth of Bacillus amyloliquefaciens is 36-42g / L.

[0034] The actual total content of 2-3-butanediol in 1L of Bacillus amyloliquefaciens fermentation broth was 41.6 g. Example 1

[0035] (1) Take the fermentation liquid of Bacillus amyloliquefaciens, add 0.02% calcium chloride by mass, and stir it thoroughly at 0.85Hz for 1h; add 10% resin by mass, stir and adsorb for 1h; use a grate to remove the resin and obtain the treated liquid for standby use;

[0036] (2) Adding a 10% sodium hydrogen phosphate solution as a reaction aid to the treatment liquid, the amount of sodium hydrogen phosphate added being 4% of the mass of the treatment liquid, and stirring at 1.35 Hz for 1 hour;

[0037] (3) Add 25% calcium chloride by mass to the fermentation broth, stir at 20 Hz for 1 hour, and filter to separate the flocs and clear liquid.

[0038] (4) Add 2% diatomaceous earth and the resin D3520 obtained in step (1) to the flocs according to the mass ratio of the fermentation liquid of Bacillus amyloliquefaciens, stir at 10 Hz for 1-2 hours to obtain flocs, air-dry at low temperature, and grind to obtain a microbial preparation. Example 2

[0039] (1) Take the fermentation liquid of Bacillus amyloliquefaciens, add 0.2% calcium chloride by mass, and stir it at 1.00Hz for 2 hours; add 15% resin by mass, stir and adsorb for 2 hours; use a grate to remove the resin and obtain the treated liquid for standby use;

[0040] (2) Adding the treatment liquid with a 25% sodium hydrogen phosphate solution as a reaction aid, the amount of sodium hydrogen phosphate added is 6% of the mass of the treatment liquid, and stirring at 1.5 Hz for 2 hours;

[0041] (3) Add 30% calcium chloride by mass to the fermentation broth, stir at 30 Hz for 2 hours, and filter to separate the flocs and clear liquid.

[0042] (4) Add 3% diatomaceous earth and the resin D3520 obtained in step (1) to the flocs according to the mass ratio of the fermentation liquid of Bacillus amyloliquefaciens, stir at 15 Hz for 2 hours to obtain flocs, dry them naturally at low temperature, and grind them to obtain a microbial preparation. Example 3

[0043] (1) Take the fermentation liquid of Bacillus amyloliquefaciens, add 0.1% calcium chloride by mass, and stir it thoroughly at 1Hz for 1.5 hours; add 12% resin by mass, stir and adsorb for 1.5 hours; use a grate to remove the resin and obtain the treated liquid for standby use;

[0044] (2) Adding a 20% sodium hydrogen phosphate solution as a reaction aid to the treatment liquid, the amount of sodium hydrogen phosphate added being 5% of the mass of the treatment liquid, and stirring at 1.4 Hz for 1.5 h;

[0045] (3) Add 28% calcium chloride by mass to the fermentation broth, stir at 25 Hz for 1.5 hours, and filter to separate the flocs and clear liquid.

[0046] (4) Add 2.5% diatomaceous earth and the resin D3520 obtained in step (1) to the flocs according to the mass ratio of the fermentation liquid of Bacillus amyloliquefaciens, stir at 13 Hz for 1-2 hours to obtain flocs, dry naturally at low temperature, and grind to obtain a microbial preparation.

[0047] In order to more intuitively demonstrate the process advantages of the present invention, the present invention adopts a low-temperature step-by-step flocculation process method and the same process adopts an equivalent replacement method for comparison.

[0048] Comparative Example 1:

[0049] The preparation method is the same as that of Example 3, except that: in the preparation process of this comparative example, a single flocculation method is adopted; specifically:

[0050] (1) Take the fermentation liquid of Bacillus amyloliquefaciens, add 12% resin by mass, stir and adsorb for 1.5 hours; use a grate to remove the resin to obtain the treated liquid for standby use;

[0051] (2) Adding a 20% sodium hydrogen phosphate solution as a reaction aid to the treatment liquid, the amount of sodium hydrogen phosphate added being 5% of the mass of the treatment liquid, and stirring at 1.4 Hz for 1.5 h;

[0052] (3) Add 28.2% calcium chloride by mass, stir at 25 Hz for 1.5 hours, and filter to separate the floccules and clear liquid.

[0053] (4) Add 2.5% diatomaceous earth and the resin D3520 obtained in step (1) to the flocs according to the mass ratio of the fermentation liquid of Bacillus amyloliquefaciens, stir at 13 Hz for 1-2 hours to obtain flocs, dry naturally at low temperature, and grind to obtain a microbial preparation.

[0054] Comparative Example 2:

[0055] The preparation method is the same as that of Example 3, except that no resin is added during the preparation of this comparative example;

[0056] Comparative Example 3:

[0057] The preparation method is the same as that of Example 3, except that: in the preparation process of this comparative example, the resin is not fished out by a grate;

[0058] Comparative Example 4:

[0059] The preparation method is the same as that of Example 3, except that in the preparation process of this comparative example, the reaction auxiliary agent disodium hydrogen phosphate is replaced by sodium chloride;

[0060] Comparative Example 5:

[0061] The preparation method is the same as that of Example 3, except that in the preparation process of this comparative example, the flocculant calcium chloride is replaced with aluminum sulfate;

[0062] Comparative Example 6:

[0063] The preparation method is the same as that of Example 3, except that in the preparation process of this comparative example, the flocculant calcium chloride is replaced with polyacrylamide;

[0064] Detection method:

[0065] Wherein, the detection method of the content of 2-3-butanediol in the fermentation broth is:

[0066] After the fermentation broth was centrifuged, the fermentation broth test solution was obtained. The gas chromatography method was used. The instrument was Shimadzu GC-2014C gas chromatograph. The internal standard method was used to quantitatively determine butanediol. The fermentation broth test solution was mixed with ethyl acetate, an extractant with internal standard isoamyl alcohol, in a ratio of 1:1, and then shaken for 20 seconds. After centrifugation at 12,000 rpm, the upper organic phase was taken for gas chromatograph detection. The specific analysis conditions were: the injector and ion flame detector were maintained at 260°C, the column temperature was maintained at 80°C, the sample measurement time was 3 minutes, and the injection volume was 1 μL.

[0067] Wherein, the detection method of the content of 2-3-butanediol in the clear solution is:

[0068] After the clear liquid was centrifuged, the clear liquid test liquid was obtained, and the gas chromatography method was used. The instrument was Shimadzu GC-2014C gas chromatograph, and the internal standard method was used to quantitatively determine butanediol. The clear liquid test liquid was mixed with ethyl acetate, an extractant with internal standard isoamyl alcohol, in a ratio of 1:1, and then shaken for 20 seconds. After centrifugation at 12000rpm, the upper organic phase was taken for gas chromatography detection. The specific analysis conditions were: the injector and ion flame detector were maintained at 260°C, the column temperature was maintained at 80°C, the sample measurement time was 3 minutes, and the injection volume was 1μL.

[0069] The total amount of 2-3-butanediol in the fermentation broth and the total amount of 2-3-butanediol in the clear liquid were calculated based on the determined 2-3-butanediol content.

[0070] Table 1: Detection indexes of the content of butanediol in different embodiments and comparative examples 2-3

[0071]

[0072] From the data analysis in Table 1, we can see that:

[0073] (1) In Examples 1-3, the total amount of 2-3-butanediol in the clear liquid was lower than that in the fermentation broth. This may be because 2-3-butanediol is soluble in water, and the flocs obtained by filtration and separation after flocculation still contain a certain amount of water, which dissolves part of the 2-3-butanediol, resulting in a decrease in the total amount of 2-3-butanediol.

[0074] The total amount of 2-3-butanediol in the clear solution of Examples 1-3 was significantly increased compared to the total amount of 2-3-butanediol in the clear solution of Comparative Example 1.

[0075] (2) Comparative Example 1 and Example 3 differ in whether or not step-by-step flocculation is used.

[0076] In Comparative Example 1, the 2-3-butanediol content in the clear solution was 32.3 g, which was lower than the 2-3-butanediol content in the clear solution of Example 3, which was 39.2 g.

[0077] This is due to the effect of step-by-step flocculation. The principle may be that the main effect of low-concentration calcium chloride on the cell membrane is to cause changes in membrane potential through calcium ion influx, thereby affecting the function and state of the cell; calcium ions in calcium chloride can enter the cell through calcium channels, causing changes in membrane potential, leading to cell excitation and the generation of action potentials. The above mechanism causes the release of 2-3-butanediol in the cell to the outside of the cell, increasing the overall 2-3-butanediol content;

[0078] However, since high concentration of calcium chloride was directly added in Comparative Example 1, the protein flocculated, which in turn reduced the content of 2-3-butanediol. In addition, the flocculent still contained a certain amount of water, which dissolved part of the 2-3-butanediol, resulting in the lowest 2-3-butanediol content in the clear liquid of this group.

[0079] (2) Comparison of Comparative Examples 2-3 and Example 3:

[0080] In Comparative Example 2, no resin was added, while in Comparative Example 3, resin was added but not removed in advance. From the perspective of the 2-3-butanediol content in the clear liquid, there was little difference from Example 3. However, from the perspective of filtration effect and product purity, the impurity content in Comparative Example 2-3 was relatively high and extremely difficult to filter. This may be because the water-soluble small molecule protein was not easy to settle under the action of the flocculant and entered the clear liquid, which not only brought a bottleneck to the filtration, but also brought more impurities to the subsequent distillation and purification, and also caused some useful active ingredients to be unable to enter the microbial preparation, resulting in a certain amount of waste;

[0081] However, although the resin was added in Comparative Example 3, the effect of Comparative Example 2 was no different, the impurity content was high, and it was extremely difficult to filter. This may be because the resin was destroyed by the high concentration of calcium chloride, causing the adsorbed substances to re-enter the clear liquid.

[0082] The present invention firstly adsorbs the small molecular substances in the clear liquid by resin, and then cleverly separates the resin from the system, and then flocculates by high concentration of calcium chloride, which can not only retain 2-3-butanediol in the clear liquid to a great extent, but also effectively avoids the destruction of the resin by high concentration of calcium chloride, causing the adsorbed substances to re-enter the clear liquid.

[0083] It is worth emphasizing that the resin added later is destroyed in the high concentration of calcium chloride. Since it is separated from the clear liquid system at this time, it will not cause water-soluble proteins and polysaccharides to enter the clear liquid and bring bottlenecks to the filtration. More importantly, this destroyed resin will release the active ingredients, which is beneficial to the activity of the subsequent microbial preparations. Moreover, the porous particle state of the resin can also prevent the microbial preparations from getting damp and hardening.

[0084] (3) Comparison of Example 4 and Example 3:

[0085] In Comparative Example 4, the reaction aid is sodium chloride, which is a sodium salt relative to disodium hydrogen phosphate. The disodium hydrogen phosphate and calcium chloride in Example 3 work together to remove 2-3-butanediol from the system, especially from the floccules, through the common ion effect and the salting-out effect. In this way, the 2-3-butanediol in the clear liquid is separated from the clear liquid, which is beneficial to the subsequent distillation.

[0086] From the data in Table 1, there is little difference between Comparative Example 4 and Example 3 because the detection method of the clear liquid 2-3-butanediol is to measure the total amount by organic solvent extraction method. From the appearance, salting out separation occurs after the clear liquid is refined for several hours.

[0087] The salting-out separation effect of Comparative Example 4 is not good, which may be due to the fact that calcium chloride and sodium chloride produce common ion competition, resulting in poor solubility of sodium chloride, and then the total dissolved ions of the whole system are reduced, and the common ion effect and salting-out effect are poor.

[0088] (4) Comparison of Examples 5-6 with Example 3:

[0089] From the data in Table 1, the content of 2-3-butanediol in the clear solution is lower than that in Example 3, and the overall flocculation effect is not much different. The reason may be that:

[0090] First, although aluminum sulfate and polyacrylamide have flocculation effects, they do not have the effect of step-by-step flocculation because they do not have the effect of low-concentration calcium ions. The principle is that the main effect of low-concentration calcium chloride on the cell membrane is to cause changes in membrane potential through calcium ion influx, thereby affecting the function and state of the cell; the calcium ions in calcium chloride can enter the cell through calcium channels, causing changes in membrane potential, leading to cell excitation and the generation of action potentials. The above mechanism causes the 2-3-butanediol in the cell to be released outside the cell, increasing the overall 2-3-butanediol content;

[0091] Its calcium dichloride not only acts as a flocculation, but also cooperates with disodium hydrogen phosphate to produce common ion effect and salting-out effect in the later stage, which can remove 2-3-butanediol from the system, especially from the flocculent, thereby increasing the yield and also allowing the 2-3-butanediol in the clear liquid to separate from the clear liquid, which is beneficial to the later distillation.

[0092] In summary:

[0093] (1) The present invention creatively uses calcium chloride as a flocculant to prepare a microbial preparation containing Bacillus amyloliquefaciens by step-by-step flocculation, while also greatly improving the yield of 2-3-butanediol.

[0094] (2) The present invention firstly adsorbs the small molecular substances in the clear liquid through the resin, and then cleverly separates the resin from the system, and then flocculates it through high concentration of calcium chloride. It can not only retain 2-3-butanediol in the clear liquid to a great extent, but also effectively avoid the destruction of the resin by high concentration of calcium chloride, which causes the adsorbed substances to re-enter the clear liquid. The destroyed resin releases the active ingredients, which is beneficial to the activity of the microbial preparation in the later stage. Moreover, the porous particle state of the resin can also prevent the microbial preparation from getting damp and hardening.

[0095] (3) Calcium chloride not only has a flocculation effect, but also cooperates with disodium hydrogen phosphate to produce common ion effect and salting-out effect in the later stage, which can remove 2-3-butanediol from the system, especially from the water of the flocculent body, improve the yield, and make the 2-3-butanediol part of the clear liquid separate from the clear liquid, which is beneficial to the later distillation.

Claims

1. A method for preparing a microbial preparation by low-temperature stepwise flocculation, characterized in that The preparation method of the microbial preparation comprises: (1) Take the fermentation liquid of Bacillus amyloliquefaciens, add calcium chloride at a mass ratio of 0.02-0.2%, and stir thoroughly for 1-2 hours; add resin at a mass ratio of 10-15%, and stir and adsorb for 1-2 hours; use a grate to remove the resin to obtain the treated liquid for standby use; (2) adding a reaction aid to the treatment liquid, wherein the reaction aid is a 10-25% disodium hydrogen phosphate solution, and the amount of disodium hydrogen phosphate added is 4-6% of the mass of the treatment liquid, and stirring for 1-2 hours; (3) Add 25-30% calcium chloride to the fermentation broth, stir for 1-2 hours, and filter to separate the flocs and clear liquid. (4) Add 2-3% diatomaceous earth and the resin obtained in step (1) to the flocs according to the mass ratio of the fermentation liquid of Bacillus amyloliquefaciens, stir for 1-2 hours to obtain flocs, naturally air-dry at low temperature, and grind to obtain a microbial preparation.

2. The method for preparing a microbial preparation by low-temperature stepwise flocculation according to claim 1, characterized in that The Bacillus amyloliquefaciens fermentation liquid is obtained by fermenting Bacillus amyloliquefaciens, and the content of 2-3-butanediol in the Bacillus amyloliquefaciens fermentation liquid is 36-42 g / L.

3. The method for preparing a microbial preparation by low-temperature stepwise flocculation according to claim 2, characterized in that The Bacillus amyloliquefaciens fermentation liquid is obtained by fermenting Bacillus amyloliquefaciens, and the fermentation conditions are as follows: in a fermentation medium, a seed inoculation amount of 5%, a rotation speed of 500 rpm, a ventilation volume of 4 L / min, an initial glucose concentration of 80 g / L, an initial pH of 7.0, a fermentation temperature of 42° C., and a fermentation time of 48 hours.

4. The method for preparing a microbial preparation by low-temperature stepwise flocculation according to claim 3, characterized in that The fermentation medium comprises, in parts by mass, 14.7 parts of corn steep liquor dry powder, 5.8 parts of yeast powder, 1 part of ammonium citrate, 6.5 parts of anhydrous sodium acetate, 0.5 parts of MgSO4, 2 parts of K2HPO4, and 80 parts of glucose.

5. The method for preparing a microbial preparation by low-temperature stepwise flocculation according to claim 1, characterized in that The resin model is D3520.

6. The method for preparing a microbial preparation by low-temperature stepwise flocculation according to claim 1, characterized in that The stirring frequency of step (1) is 0.85-1.00 Hz, the stirring frequency of step (2) is 1.35-1.5 Hz, the stirring frequency of step (3) is 20-30 Hz, and the stirring frequency of step (4) is 10-15 Hz.

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