Complex microbial inoculant, effervescent granules and application

Abstract

The invention provides a complex microbial inoculant, effervescent granules and application, and belongs to the technical field of microorganisms. The invention provides a complex microbial inoculant. The complex microbial inoculant comprises bacillus halotolerant SSF1 and bacillus licheniformis LU1. The complex microbial inoculant has relatively high enzyme activity of endo-glucosidase and relatively good cellulose degradation capability. The complex microbial inoculant is used as an active component and can be combined with a disintegrating agent, a lubricating agent, an adhesive and the like to prepare effervescent granules. The obtained effervescent granules are short in disintegration time and large in foaming amount, the pH value of the effervescent granules is close to the pH value of pure water after the effervescent granules are completely disintegrated, the viable count of the complex microbial inoculant can be efficiently kept, and the complex microbial inoculant can maintain high activity. The result of the embodiment shows that when the effervescent granules of the complex microbial inoculant are applied to wheat planting, the growth of wheat can be remarkably promoted, the germination number of wheat can be increased, and the overground part length, the underground part length, the fresh weight root-crown ratio and the dry weight root-crown ratio of wheat can be improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a compound bacterial agent, effervescent granules, and their applications. Background Technology

[0002] Effervescent granules are tablets that release a large amount of carbon dioxide upon contact with water, made by adding binders, lubricants, fillers, and other additives to a suitable acid or alkali source as disintegrants. Effervescent granules offer advantages such as being dust-free, easy to apply, and highly efficient in application. They also provide uniform pesticide dispersion, low phytotoxicity to crops, and are less prone to leakage during transportation and storage, aligning with the development trend of high efficiency, safety, and ease of use.

[0003] Microbial inoculants have significant advantages in promoting crop growth and degrading straw. However, their activity is often limited by environmental conditions when applied directly to straw or crops. Developing suitable formulations of microbial inoculants can overcome these limitations to some extent, offering advantages in maintaining microbial activity or enhancing their effectiveness. Currently, there are relatively few technologies available for preparing effervescent granules of microbial inoculants. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a compound microbial agent that can be used as an active ingredient to prepare effervescent granules. The effervescent granules can maintain the high activity of the microbial agent, and the resulting effervescent granules have good effects and can significantly promote crop growth.

[0005] The objective of this invention is achieved through the following technical solution: This invention provides a compound microbial agent comprising halophilic Bacillus SSF1 and Bacillus licheniformis LU1; the preservation number of the halophilic Bacillus SSF1 is CGMCC No. 34189; and the preservation number of the Bacillus licheniformis LU1 is CCTCC No: M 20231025.

[0006] Preferably, the mass ratio of halophilic Bacillus SSF1 powder to Bacillus licheniformis LU1 powder in the compound bacterial agent is 1:(1.5~3); the viable count of the halophilic Bacillus SSF1 powder is 4×10⁻⁶. 13 ~5×10 13 The viable count of the Bacillus licheniformis LU1 bacterial powder was 8 × 10⁻⁶. 13 ~9.5×10 13 .

[0007] This invention provides a method for preparing the compound microbial agent described in the above-mentioned technical solution, comprising: A compound bacterial agent was prepared by mixing salt-tolerant Bacillus SSF1 bacterial powder and Bacillus licheniformis LU1 bacterial powder.

[0008] Preferably, the preparation method of the halophilic Bacillus SSF1 bacterial powder and Bacillus licheniformis LU1 bacterial powder is as follows: the halophilic Bacillus SSF1 bacterial suspension and the Bacillus licheniformis LU1 bacterial suspension are respectively mixed with a freeze-drying protectant and then freeze-dried to obtain halophilic Bacillus SSF1 bacterial powder and Bacillus licheniformis LU1 bacterial powder; the freeze-drying protectant includes skim milk powder, fructooligosaccharides and monosodium glutamate.

[0009] This invention provides effervescent granules, comprising the compound microbial agent described in the above technical solution or the compound microbial agent prepared by the preparation method described in the above technical solution.

[0010] Preferably, the effervescent granules include a disintegrant; the disintegrant includes citric acid and tartaric acid; the mass ratio of citric acid to tartaric acid is (1~1.25):(1~1.25); and the amount of disintegrant added to the effervescent granules is 55wt.%~65wt.%.

[0011] Preferably, the effervescent granules include a lubricant; the lubricant includes polyethylene glycol 6000; and the amount of lubricant added to the effervescent granules is 1 wt.% to 5 wt.%.

[0012] Preferably, the effervescent granules include a binder; the binder includes polyvinylpyrrolidone; and the amount of binder added to the effervescent granules is 1 wt.% to 5 wt.%.

[0013] Preferably, the amount of compound microbial agent added to the effervescent granules is 10wt.%~20wt.%.

[0014] This invention provides the application of the effervescent granules described in the above technical solution in promoting wheat growth.

[0015] The beneficial effects of this invention are: This invention provides a compound microbial agent comprising *Bacillus halophilus* SSF1 and *Bacillus licheniformis* LU1; the preservation number of *Bacillus halophilus* SSF1 is CGMCC No. 34189; and the preservation number of *Bacillus licheniformis* LU1 is CCTCC No. M 20231025. The compound microbial agent exhibits high endoglucosidase activity and good cellulose degradation ability. Using this compound microbial agent as the active ingredient, it can be combined with disintegrants, lubricants, and binders to prepare effervescent granules. The prepared effervescent granules have a short disintegration time, a large foaming volume, and a pH value close to that of pure water after complete disintegration, effectively maintaining the viable bacterial count of the compound microbial agent and ensuring its high activity. The results of the embodiments of this invention demonstrate that using the effervescent granules of the compound microbial agent in wheat cultivation can significantly promote wheat growth, increase the number of germinating wheat plants, and improve the aboveground length, underground length, fresh weight root-to-shoot ratio, and dry weight root-to-shoot ratio of wheat.

[0016] Biological Preservation Instructions Salt-resistant Bacillus SSF1, classified and named as follows: Bacillus halotolerans It was deposited on April 14, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34189.

[0017] Bacillus licheniformis LU1, classified and named as follows: Bacillus licheniformis It was deposited on June 15, 2023 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC No: M20231025. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Example 1: Glucose standard curve; Figure 2 The images show the morphological observation, Congo red staining, and Gram staining identification results of strain B1. Figure 3 The effervescent granules prepared in Example 3; Figure 4 This is a graph showing the number of germinations in each treatment group in Example 4; Figure 5The aboveground growth status of wheat in each treatment group in Example 4 is shown in the figure. Figure 6 The image shows the growth status of the whole wheat plant in each treatment group of Example 4. Detailed Implementation

[0020] This invention provides a compound microbial agent comprising halophilic Bacillus SSF1 and Bacillus licheniformis LU1; the preservation number of the halophilic Bacillus SSF1 is CGMCC No. 34189; and the preservation number of the Bacillus licheniformis LU1 is CCTCC No. M. 20231025.

[0021] The salt-tolerant Bacillus SSF1 provided by this invention was isolated from soil before spring sowing in Qingshuihe County, Hohhot City, Inner Mongolia.

[0022] As an optional embodiment of the present invention, the viable count ratio of Bacillus halophilus SSF1 powder to Bacillus licheniformis LU1 powder in the compound bacterial agent can be 1:1.5~1:3, or 1:2; the viable count of the Bacillus halophilus SSF1 powder can be 4×10⁻⁶. 13 ~5×10 13 It can also be 4.4 × 10 13 Or 4.5×10 13 The viable count of the Bacillus licheniformis LU1 bacterial powder can be 8 × 10⁻⁶. 13 ~10×10 13 It can also be 8.8×10 13 Or 9.0×10 13 The compound microbial agent provided by this invention has high endoglucosidase activity and good cellulose degradation ability. Using this compound microbial agent as the active ingredient, it can be combined with disintegrants, lubricants, and binders to prepare effervescent granules. The prepared effervescent granules can efficiently maintain the viable bacterial count of the compound microbial agent, thus maintaining its high activity. The results of the embodiments of this invention show that using the effervescent granules of the compound microbial agent in wheat cultivation can significantly promote wheat growth, increase the number of germinating wheat plants, and improve the aboveground length, underground length, fresh weight root-shoot ratio, and dry weight root-shoot ratio of wheat.

[0023] This invention provides a method for preparing the compound microbial agent described in the above-mentioned technical solution, comprising: A compound bacterial agent was prepared by mixing salt-tolerant Bacillus SSF1 bacterial powder and Bacillus licheniformis LU1 bacterial powder.

[0024] This invention does not impose any particular limitation on the preparation method of the halophilic Bacillus SSF1 and Bacillus licheniformis LU1 bacterial powders; any conventional method for preparing bacterial powders in the art can be used. As an optional embodiment of this invention, the preparation method of the halophilic Bacillus SSF1 and Bacillus licheniformis LU1 bacterial powders can be as follows: mixing the halophilic Bacillus SSF1 bacterial suspension and the Bacillus licheniformis LU1 bacterial suspension with a freeze-drying protectant respectively, and then freeze-drying to obtain the halophilic Bacillus SSF1 and Bacillus licheniformis LU1 bacterial powders. This invention does not impose any particular limitation on the preparation method of the halophilic Bacillus SSF1 and Bacillus licheniformis LU1 bacterial suspensions; any conventional method in the art can be used.

[0025] As an optional embodiment of the present invention, the preparation method of the halophilic Bacillus SSF1 bacterial suspension can be as follows: culturing the halophilic Bacillus SSF1 to the stationary phase to obtain a halophilic Bacillus SSF1 bacterial solution; separating and resuspending the bacterial cells from the halophilic Bacillus SSF1 bacterial solution to obtain a halophilic Bacillus SSF1 bacterial suspension. The present invention does not specifically limit the culturing method of the halophilic Bacillus SSF1; conventional culturing methods in the art can be used. After obtaining the halophilic Bacillus SSF1 bacterial solution, the present invention preferably separates the bacterial cells from the halophilic Bacillus SSF1 bacterial solution by centrifugation; the centrifugation speed can be 3500 r / min; the centrifugation time can be 20 min. After centrifugation to obtain the bacterial cells, the present invention preferably washes the bacterial cells. The present invention preferably uses 0.9 wt.% physiological saline for washing; the washing can be performed twice. After washing, the wet bacterial cells are collected and resuspended; the method for collecting the wet bacterial cells includes centrifugation; the centrifugation speed can be 3500 r / min; the centrifugation time can be 20 min. In this invention, the resuspension is preferably performed using physiological saline; the mass-to-volume ratio of wet bacterial cells to physiological saline during resuspension can be 1 g: 2 mL. After resuspension, a suspension of halophilic Bacillus SSF1 cells is obtained.

[0026] As an optional embodiment of the present invention, the preparation method of the *Bacillus licheniformis* LU1 bacterial suspension can be as follows: culturing *Bacillus licheniformis* LU1 to the stationary phase to obtain *Bacillus licheniformis* LU1 bacterial solution; separating the bacterial cells from the *Bacillus licheniformis* LU1 bacterial solution and resuspending them to obtain the *Bacillus licheniformis* LU1 bacterial suspension. The present invention does not specifically limit the culturing method of *Bacillus licheniformis* LU1; conventional culturing methods in the art can be used. After obtaining the *Bacillus licheniformis* LU1 bacterial solution, the present invention preferably separates the bacterial cells from the *Bacillus licheniformis* LU1 bacterial solution by centrifugation; the centrifugation speed can be 3500 r / min; the centrifugation time can be 20 min. After centrifugation to obtain the bacterial cells, the present invention preferably washes the bacterial cells. The present invention preferably uses 0.9 wt.% physiological saline for washing; the washing can be performed twice. After washing, the wet bacterial cells are collected and resuspended; the method for collecting the wet bacterial cells includes centrifugation; the centrifugation speed can be 3500 r / min; the centrifugation time can be 20 min. In this invention, the resuspension is preferably performed using physiological saline; the mass-to-volume ratio of wet bacterial cells to physiological saline during resuspension can be 1 g: 2 mL. After resuspension, a Bacillus licheniformis LU1 bacterial suspension is obtained.

[0027] In an optional embodiment of the present invention, the lyophilization protectant comprises skim milk powder, fructooligosaccharides, and monosodium glutamate. In an optional embodiment of the present invention, the mass fraction of skim milk powder in the lyophilization protectant can be 10-14 wt.%, or 12 wt.%; the mass fraction of fructooligosaccharides in the lyophilization protectant can be 10-14 wt.%, or 12 wt.%; and the mass fraction of monosodium glutamate in the lyophilization protectant can be 5-7 wt.%, or 6 wt.%. The lyophilization protectant can use water as a solvent.

[0028] In this invention, the halophilic Bacillus SSF1 bacterial suspension is preferably mixed with a lyophilization protectant; the volume ratio of the halophilic Bacillus SSF1 bacterial suspension to the lyophilization protectant can be 1:1. After mixing, the mixture is lyophilized. This invention does not specifically limit the lyophilization method; any conventional lyophilization method in the art can be used. This invention obtains halophilic Bacillus SSF1 bacterial powder through lyophilization.

[0029] In this invention, the *Bacillus licheniformis* LU1 bacterial suspension is preferably mixed with a freeze-drying protectant; the volume ratio of the *Bacillus licheniformis* LU1 bacterial suspension to the freeze-drying protectant can be 1:1. After mixing, the mixture is freeze-dried. This invention does not specifically limit the freeze-drying method; any conventional freeze-drying method in the art can be used. This invention obtains *Bacillus licheniformis* LU1 bacterial powder through freeze-drying.

[0030] After obtaining halophilic Bacillus SSF1 powder and Bacillus licheniformis LU1 powder, this invention mixes the halophilic Bacillus SSF1 powder and Bacillus licheniformis LU1 powder to obtain a composite microbial agent. As an optional embodiment of this invention, the ratio of viable bacteria counts when mixing the halophilic Bacillus SSF1 powder and Bacillus licheniformis LU1 powder can be 1:2. This invention does not specifically limit the mixing method; any conventional mixing method in the art can be used. After mixing, this invention obtains a composite microbial agent.

[0031] This invention provides effervescent granules, comprising the composite microbial agent described in the above-described technical solution or the composite microbial agent prepared by the preparation method described in the above-described technical solution. As an optional embodiment of this invention, the disintegrant of the effervescent granules comprises citric acid and tartaric acid; the mass ratio of citric acid to tartaric acid is (1~1.25):(1~1.25), or it can be 1:1.25, 1:1, or 1.25:1; the amount of disintegrant added to the effervescent granules can be 55wt.%~65wt.%, or it can be 55wt.%, 60wt.%, or 65wt.%. As an optional embodiment of this invention, the lubricant of the effervescent granules comprises polyethylene glycol 6000; the amount of lubricant added to the effervescent granules can be 1wt.%~5wt.%, or it can be 1wt.%, 2wt.%, 3wt.%, 4wt.%, or 5wt.%. As an optional embodiment of the present invention, the binder of the effervescent granules can be polyvinylpyrrolidone; the amount of binder added to the effervescent granules can be 1wt.%~5wt.%, or 1wt.%, 2wt.%, 3wt.%, 4wt.%, or 5wt.%. As an optional embodiment of the present invention, the amount of compound microbial agent added to the effervescent granules can be 10wt.%~20wt.%, or 10wt.%, 11wt.%, 12wt.%, 13wt.%, 14wt.%, 15wt.%, 16wt.%, 17wt.%, 18wt.%, 19wt.%, or 20wt.%.

[0032] The effervescent granules provided by this invention have a short disintegration time, a large foaming volume, and a pH value close to that of pure water after complete disintegration, which enables microbial protection to have high activity.

[0033] The present invention relates to the application of effervescent granules in promoting wheat growth. The results of the embodiments of the present invention demonstrate that applying the effervescent granules during wheat seed germination significantly promotes wheat growth, increases the number of germinated wheat seeds, and improves the aboveground length, underground length, fresh weight root-to-shoot ratio, and dry weight root-to-shoot ratio of wheat.

[0034] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0035] The reagents used in the following technical solutions and their sources are as follows: Citric acid, Tianjin New Technology Industrial Park Kemao Chemical Reagent Co., Ltd.; Tartaric acid, Shanghai Maclean Biochemical Technology Co., Ltd.; Sodium bicarbonate, Fuchen Chemical Reagent Co., Ltd.; Sodium carbonate, Sinopharm Chemical Reagent Co., Ltd.; Calcium carbonate, Tianjin Xinbote Chemical Co., Ltd.; Polyethylene glycol 6000, Sinopharm Chemical Reagent Co., Ltd.; Magnesium stearate, Tianjin Xinbote Chemical Co., Ltd.; Talc, Shangqiu Liangfeng Sanitary Products Co., Ltd.; Porous starch, Xi'an Zelang Biotechnology Co., Ltd.; Sodium carboxymethyl cellulose, Shandong Top Bioengineering Co., Ltd.; Polyvinylpyrrolidone, Fuchen Chemical Reagent Co., Ltd.; 75% ethanol, Shandong Zhuojian Medical Technology Co., Ltd.; LB broth, Beijing Aoboxing Biotechnology Co., Ltd.; Agar, Beijing Aoboxing Biotechnology Co., Ltd.; Corn starch, Shandong Keyuan Biochemical Co., Ltd.; Sodium chloride, Tianjin Xinbote Chemical Co., Ltd.; Skim milk powder, Nanjing Baidakang Biotechnology Co., Ltd.; Monosodium glutamate, Lotus Health Industry Group Co., Ltd.; Fructooligosaccharides, Shanghai Yuanye Biotechnology Co., Ltd.

[0036] Preparation of test culture media: All solutions and culture media used were prepared with distilled water.

[0037] LB liquid medium (2 L): 40 g LB broth.

[0038] LB solid medium (2 L): Add 30 g of agar to the LB liquid medium.

[0039] Preparation of other solutions: Skim milk powder (100 mL): Accurately weigh 12 g of skim milk powder and dissolve it in 80 mL of distilled water. Make up the volume to 100 mL and sterilize at 108℃ for 10 min. Store at room temperature.

[0040] Fructooligosaccharides (100 mL): Accurately weigh 12 g of fructooligosaccharides, dissolve them in 80 mL of distilled water, bring the volume up to 100 mL, sterilize at 115℃ for 15 min, and store at room temperature.

[0041] Monosodium glutamate (100 mL): Accurately weigh 6 g of sodium glutamate, dissolve it in 80 mL of distilled water, bring the volume up to 100 mL, sterilize at 115℃ for 15 min, and store at room temperature.

[0042] 0.9% physiological saline (1 L): Accurately weigh 9 g of sodium chloride and dissolve it in 80 mL of distilled water. Make up to 1000 mL and sterilize at 121℃ for 15 min. Store at room temperature.

[0043] Example 1 Test strain The two strains of halophilic Bacillus SSF1 (CGMCC No. 34189) and Bacillus licheniformis LU1 (CCTCC NO: M20231025) used in the experiment were provided by the Institute of Biotechnology, Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences.

[0044] I. Screening and Classification Identification Process of Salt-Tolerant Bacillus SSF1 1. Screening process for halophilic Bacillus SSF1: Sample Collection: The soil samples used were from farmland in Qingshuihe County, Hohhot City, Inner Mongolia (111°0.68′E, 39°0.92′N) where straw was plowed and returned to the field. Corn was planted in this area in both 2019 and 2020, and straw was plowed and returned to the field after the autumn harvest. Soil samples were collected before sowing in the spring of 2021. Farmland soil with a straw degradation rate ≥50% was selected as the sample for this study. A five-point sampling method was used, where the topsoil was lightly scraped to collect the topsoil from 5-10 cm below the surface, mixed thoroughly, and stored in sterile bags at 4°C for later use.

[0045] Isolation of cellulose-degrading bacteria: 5g of collected soil sample was mixed into an Erlenmeyer flask containing 45mL of enrichment medium and cultured with shaking at 30℃ and 170rpm for 2 days. In a clean bench, 1mL of the enriched bacterial culture was transferred to a 9mL physiological saline test tube and cultured according to a 10... -5 ~10 -9 Dilute the sample using five different dilution gradients and mix thoroughly by shaking. Take 200 μL of each gradient suspension and spread it onto sodium carboxymethyl cellulose (CMC-Na), microcrystalline cellulose, and starch solid medium, repeating the process three times. Incubate at 30°C for 24-48 hours, or until colonies appear on the medium.

[0046] Initial screening of lignocellulose-degrading bacteria: The hydrolysis ratio (HCR) of the strains directly reflects the effectiveness of microbial degradation of lignocellulose. The hydrolysis ratio was calculated from the diameter of the degradation zone (D) and the colony diameter (d). A higher ratio indicates better degradation. Generally, a hydrolysis ratio (HCR) ≥ 2 is considered to have a better cellulose degradation effect. First, based on sodium carboxymethyl cellulose (CMC-Na) medium, 18 candidate strains were obtained by Congo red staining and comparison of strain hydrolysis ratio values. Second, based on microcrystalline cellulose (MCC) medium and starch medium, 9 candidate strains were further screened by Congo red and iodine staining and comparison of strain hydrolysis ratio values, and they were numbered SSF1~SSF9. The main results are shown in Table 1.

[0047] Table 1. Hydrolysis zone size of SSF1-SSF9 strains

[0048] Note: Different letters indicate that different strains have significantly different hydrolysis rates on the same culture medium (P < 0.05).

[0049] Secondary screening of lignocellulose-degrading bacteria Cellulase activity is an important indicator of the enzyme production capacity of cellulose-degrading bacteria. Based on the cellulase activity of each strain, the cellulase production capacity of each strain can be detected to obtain candidate strains with high cellulose degradation capacity, thus preparing for subsequent experiments.

[0050] A glucose standard curve can be used to accurately determine the glucose content in a sample. A glucose standard curve is plotted with glucose content on the x-axis and absorbance at 540 nm on the y-axis. The regression equation for this standard curve, obtained through linear fitting, is: y = 1.6267x + 0.0071 R². 2 = 0.9982, correlation coefficient greater than 0.995, the standard curve fits well and can reflect the relationship between absorbance and glucose content well. Figure 1 This curve can be used as a standard curve for measuring cellulase activity, and then the size of cellulase activity can be calculated.

[0051] Assay for cellulase activity The test strain was cultured on LB medium at 30℃ and 170 rpm for 2 days to obtain the strain culture medium. The supernatant in the strain culture medium was separated and used as crude enzyme solution for subsequent cellulase activity determination.

[0052] Determination of total cellulase activity Filter paper enzyme activity (FPA) was determined. 0.1 g of filter paper strip was added to a 25 mL stoppered colorimetric tube. The filter paper strip was then moistened with 2.00 mL of citrate-disodium hydrogen phosphate buffer (pH = 4.8). 1.00 mL of crude enzyme solution (1.00 mL of inactivated crude enzyme solution was added to the blank control) and incubated at 50 °C for 30 min. 3 mL of DNS reagent was added, and the mixture was boiled in a water bath for 10 min. The mixture was then cooled to a final volume of 20 mL and the absorbance was measured at 540 nm. Each tube was repeated three times, and the average value was taken as the final result.

[0053] Assay for EG or Cx enzyme (endo-β-1,4-glucosidase) activity The activity of carboxymethyl cellulose (CMC) enzyme was determined using a method that combines the following steps: 1 mL of crude enzyme solution (or 1 mL of inactivated crude enzyme solution for the blank control) and 2 mL of 1% CMC-Na solution (pH = 4.8, citrate-disodium hydrogen phosphate buffer) were added to a 25 mL stoppered colorimetric tube. The tube was incubated at 50 °C for 30 min, then 3.00 mL of DNS reagent was added. After mixing, the tube was boiled in a water bath for 10 min, cooled with cold water, and the volume was adjusted to 20.00 mL. The absorbance was measured at 540 nm. Each tube was repeated three times, and the average value was taken as the final result.

[0054] Determination of CBH or C1 enzyme (exo-β-1,4-glucosidase) activity The microcrystalline cellulose (MCC) enzyme activity assay was performed. 1 mL of crude enzyme solution (1 mL of inactivated crude enzyme solution for the blank control) and 2 mL of 1% CMC-Na solution (pH = 4.8 citrate-disodium hydrogen phosphate buffer) were added to a 25 mL stoppered colorimetric tube. The tube was incubated at 50 °C for 30 min, then 3.00 mL of DNS reagent was added. After mixing, the tube was boiled in a water bath for 10 min, cooled with cold water, and brought to a final volume of 20.00 mL with deionized water. The absorbance was measured at 540 nm. Each tube was repeated three times, and the average value was taken as the final result.

[0055] β-1,4-glucosidase activity assay Add 1.00 mL of crude enzyme solution (1.00 mL of inactivated crude enzyme solution for the blank group) and 2.00 mL of 1% salicin solution (pH = 4.8 citrate-disodium hydrogen phosphate buffer) to a 25 mL stoppered colorimetric tube. Incubate at 50 °C for 30 min, add 3.00 mL of DNS reagent, mix well, boil in a water bath for 10 min, cool with cold water, and bring the volume to 20 mL. Mix well and measure the absorbance at 540 nm. Repeat 3 times for each tube, and take the average value of the final result.

[0056] Endoglucosidase, exoglucosidase, and β-glucosidase are key enzyme systems for cellulose degradation. Filter paper enzyme can, to some extent, reflect the total enzyme activity of this complex enzyme system of three cellulases. The cellulase activities of nine isolated strains were analyzed using endoglucosidase, exoglucosidase, β-glucosidase, and filter paper enzyme. The results are shown in Table 2.

[0057] Table 2. Cellulase activity of isolated strains

[0058] Note: Different letters indicate that different strains have significantly different hydrolysis rates on the same culture medium (P < 0.05).

[0059] The cellulase production characteristics of the nine candidate strains showed significant differences (P < 0.05). The filter paper enzyme activity ranged from 29.58 to 67.86 U / mL, with strain SSF1 exhibiting the highest activity at 67.86 U / mL. Endoglucosidase activity ranged from 17.11 to 54.39 U / mL, with strain SSF6 showing the highest activity at 54.39 U / mL, followed by strain SSF4 at 52.64 U / mL. Exonuclease activity ranged from 14.86 to 78.59 U / mL, with strain SSF6 exhibiting the highest activity, followed by strain SSF1 at 70.94 U / mL. β-glucosidase activity ranged from 17.50 to 58.96 U / mL, with strain SSF1 showing the highest β-glucosidase activity. Based on the activities of total cellulase, endoglucosidase, exoglucosidase, and β-glucosidase produced by candidate strains, strains with high cellulose degradation ability were screened. Among the nine candidate strains, SSF6 showed the highest overall enzyme activity and had a high potential for cellulose degradation, while strain SSF1 had a slightly lower cellulose production ability than SSF6. Strain SSF1 will be used in subsequent applications.

[0060] 2. SSF1 classification and identification process: The whole genome DNA of strain SSF1 was extracted using a DNA extraction kit. The DNA was then amplified by PCR using universal 16S primers (27F and 1492R) and sent to a sequencing company for sequencing. The obtained bacterial nucleotide sequence (strain 16S rDNA) was entered into the NCBI database for BLAST alignment. Reference strain sequences with a similarity >97% were selected for genus identification.

[0061] The 16S rDNA nucleotide sequence of strain SSF1 is shown in SEQ ID NO.1, specifically:

[0062] The results showed that strain SSF1 and Bacillus halotolerans With a sequence similarity of 99%, belonging to the genus Bacillus, strain SSF1 was identified as a salt-tolerant Bacillus. Bacillus halotolerans ).

[0063] II. Screening and Classification Identification Process of Bacillus licheniformis LU1 1. Screening process for strain LU1 Sample Collection: The soil samples used were from farmland in Qingshuihe County, Hohhot City, Inner Mongolia (111°0.68′E, 39°0.92′N) where straw was plowed and returned to the field. Corn was planted in this area in both 2019 and 2020, and straw was plowed and returned to the field after the autumn harvest. Soil samples were collected before sowing in the spring of 2021. Farmland soil with a straw degradation rate ≥50% was selected as the sample for this study. A five-point sampling method was used, where the topsoil was lightly scraped to collect the topsoil from 5-10 cm below the surface, mixed thoroughly, and stored in sterile bags at 4°C for later use.

[0064] 2. Isolation of cellulose-degrading bacteria: 5g of collected soil sample was mixed into an Erlenmeyer flask containing 45mL of enrichment medium and cultured with shaking at 30℃ and 170rpm for 2 days. In a clean bench, 1mL of the enriched bacterial culture was transferred to a 9mL physiological saline test tube and cultured according to a 10... -5 ~10 -9 Dilute the sample using five different dilution gradients and mix thoroughly by shaking. Spread 200 μL of each gradient suspension onto sodium carboxymethyl cellulose (CMC-Na) solid medium, repeating the process three times. Incubate at 30°C for 24–48 h, or until colonies appear on the medium.

[0065] 3. Initial Screening of Lignocellulose-Degrading Bacteria: The hydrolysis ratio (HCR) of the strains directly reflects the effectiveness of microbial degradation of lignocellulose. The hydrolysis ratio is calculated from the diameter of the degradation zone (D) and the colony diameter (d). A higher ratio indicates better degradation. Generally, a hydrolysis ratio (HCR) ≥ 2 is considered to indicate better cellulose degradation. Based on sodium carboxymethyl cellulose (CMC-Na) medium, six candidate strains were obtained by Congo red staining and comparison of strain hydrolysis ratios, and were numbered B1 to B6. The main results are shown in Table 3.

[0066] Table 3. Hydrolysis zone size of strains B1-B6

[0067] Note: Different letters indicate that different strains have significantly different hydrolysis rates on the same culture medium (P < 0.05).

[0068] Morphological observation, Congo red staining, and Gram staining identification results of strain B1 are as follows: Figure 2 As shown.

[0069] 4. Secondary screening of lignocellulose-degrading bacteria Cellulase activity is an important indicator of the enzyme production capacity of cellulose-degrading bacteria. Based on the cellulase activity of each strain, the cellulase production capacity of each strain can be detected to obtain candidate strains with high cellulose degradation capacity, thus preparing for subsequent experiments.

[0070] A glucose standard curve can be used to accurately determine the glucose content in a sample. A glucose standard curve is plotted with glucose content on the x-axis and absorbance at 540 nm on the y-axis. The regression equation for this standard curve, obtained through linear fitting, is: y = 1.6267x + 0.0071 R². 2 = 0.9982, correlation coefficient greater than 0.995, the standard curve fits well and can reflect the relationship between absorbance and glucose content well. Figure 1 This curve can be used as a standard curve for measuring cellulase activity, and then the size of cellulase activity can be calculated.

[0071] Assay for cellulase activity The test strain was cultured on LB medium at 30℃ and 170 rpm for 2 days to obtain the strain culture medium. The supernatant in the strain culture medium was separated and used as crude enzyme solution for subsequent cellulase activity determination.

[0072] For the determination of sodium carboxymethyl cellulose (CCMC) enzyme activity, 0.5 mL of crude enzyme solution was mixed with an equal volume of 1% CCMC substrate solution in a test tube. A test tube inactivated before adding the substrate was used as a blank control. The mixture was placed in a 50°C constant temperature water bath for 30 minutes to allow the enzymatic reaction to occur. Immediately after the reaction, 1.5 mL of DNS reagent was added to terminate the reaction and initiate the color development process. All test tubes were heated in a boiling water bath for 5-10 minutes to allow the reducing sugar to fully react with the DNS reagent to generate a brownish-red product, and then cooled to room temperature. The absorbance of the solution was measured at 540 nm using a spectrophotometer. The amount of reducing sugar produced was calculated based on the glucose standard curve. Finally, the enzyme activity was calculated with 1 μmol of reducing sugar produced per minute as one unit of enzyme activity.

[0073] The activity of EG or Cx enzymes (endo-β-1,4-glucosidase) was determined using the carboxymethyl cellulose (CMC) enzyme activity assay. 1 mL of crude enzyme solution (1 mL of inactivated crude enzyme solution for the blank control) and 2 mL of 1% CMC-Na solution (citrate-disodium hydrogen phosphate buffer, pH = 4.8) were added to a 25 mL stoppered colorimetric tube. The tube was incubated at 50 °C for 30 min, then 3.00 mL of DNS reagent was added. After mixing, the tube was boiled in a water bath for 10 min, cooled with cold water, and brought to a final volume of 20.00 mL. The absorbance was measured at 540 nm. Each tube was repeated three times, and the average value was taken as the final result.

[0074] The activity of CBH or C1 enzymes (exo-β-1,4-glucosidase) was determined using the microcrystalline cellulose (MCC) enzyme activity assay. 1 mL of crude enzyme solution (1 mL of inactivated crude enzyme solution for the blank group) and 2 mL of 1% CMC-Na solution (citrate-disodium hydrogen phosphate buffer, pH = 4.8) were added to a 25 mL stoppered colorimetric tube. The tube was incubated at 50 °C for 30 min, then 3.00 mL of DNS reagent was added. After mixing, the tube was boiled in a boiling water bath for 10 min, cooled with cold water, and brought to a final volume of 20.00 mL with deionized water. The absorbance was measured at 540 nm. Each tube was repeated three times, and the final result was the average value.

[0075] β-1,4-glucosidase activity assay Add 1.00 mL of crude enzyme solution (1.00 mL of inactivated crude enzyme solution for the blank group) and 2.00 mL of 1% salicin solution (citrate-disodium hydrogen phosphate buffer, pH = 4.8) to a 25 mL stoppered colorimetric tube. Incubate at 50 °C for 30 min, add 3.00 mL of DNS reagent, mix well, boil in a water bath for 10 min, cool with cold water, and bring the volume to 20 mL. Mix well and measure the absorbance at 540 nm. Repeat three times for each tube, and take the average value of the final results. Carboxymethyl cellulose sodium enzyme, endoglucosidase, exoglucosidase, and β-glucosidase are key enzyme systems for cellulose degradation. The cellulase activities of six isolated strains were analyzed using endoglucosidase, exoglucosidase, β-glucosidase, and carboxymethyl cellulose sodium enzyme, and the results are shown in Table 4.

[0076] Table 4 Enzyme activities of four enzymes in strains B1-B6

[0077] Note: Different letters indicate that different strains have significantly different hydrolysis rates on the same culture medium (P < 0.05).

[0078] The cellulase production characteristics of the six candidate strains showed significant differences (P < 0.05). Among them, B1 showed better overall performance, so B1 was selected for the preparation of effervescent tablets, and B1 was renamed LU1 during preservation.

[0079] 2. The whole genome DNA of strain LU1 was extracted using a DNA extraction kit. Then, the strain DNA was amplified by PCR using universal 16S primers (27F and 1492R), and sent to a sequencing company for sequencing. The obtained bacterial nucleotide sequence (strain 16S rDNA) was entered into the NCBI database for BLAST alignment. Reference strain sequences with a similarity >97% were selected for genus identification.

[0080] The 16S rDNA nucleotide sequence of strain LU1 is shown in SEQ ID NO.2, specifically as follows:

[0081] The results showed that strain LU1 and Bacillus licheniformis With a sequence similarity of 99%, belonging to the genus Bacillus, strain LU1 was identified as Bacillus licheniformis. Bacillus licheniformis ).

[0082] Example 2: Preparation of Compound Microbial Powder To improve the overall degradation efficiency and stability of the composite bacterial strain and to ensure that there are no antagonistic interactions among the selected strains, a strain compatibility study was conducted. The growth of each strain on the plates was observed. Bacillus licheniformis LU1 and B. halotolerans There is no growth inhibition among SSF1 cells, and they can grow well, therefore, they were selected. Bacillus licheniformis LUl and B. halotolerans SSF1 was used to construct a composite bacterial system.

[0083] bacterial strain construction and enzyme production capacity detection Strains SSF1 and LU1 were cultured overnight on LB liquid medium at 170 rpm and 30°C until OD500 was reached. 600 ≥1, isolate the bacterial cells in the culture medium, resuspend them in sterile water, and obtain SSF1 seed culture and LU1 seed culture respectively.

[0084] SSF1 seed culture and LU1 seed culture were mixed at viable cell ratios of 1:1, 1:2 and 1:3, respectively, and then cultured in LB liquid medium at 170 rpm and 30℃ for 12 h to obtain a composite bacterial culture. The activity of endoglucosidase in the composite bacterial culture was detected, and the results are shown in Table 5.

[0085] Table 5. Endoglucosidase activity in the culture medium of the mixed bacterial strain

[0086] Using cellulase activity as the criterion, a highly efficient cellulase-producing compound bacterial strain was screened by measuring the CMCase activity of the constructed bacterial strains. Three different addition ratios of the compound bacterial strain D were designed, with the enzyme activities arranged from highest to lowest as D2>D1>D3. Strain D2 exhibited the highest CMCase activity, at 248.21 U / mL. The highly efficient compound bacterial strain constructed in this experiment consisted of strain SSF1 and strain LU1 at a viable count ratio of 1:2 for subsequent experiments. Different strains could coexist synergistically, providing a basis for their combined biodegradation of lignocellulose.

[0087] Example 3: Preparation of Compound Microbial Powder The freeze-drying protectant used contains 10 wt.% skim milk powder, 10 wt.% fructooligosaccharides and 5 wt.% monosodium glutamate.

[0088] 1. Salt-tolerant Bacillus SSF1 was cultured in LB medium at 30°C and 70 rpm; Bacillus licheniformis LU1 was cultured in LB medium at 30°C and 170 rpm. Two Bacillus cultures that had reached the stable phase were centrifuged at 3500 r / min for 20 min, the supernatant was discarded, and the cultures were washed twice with 0.9 wt.% physiological saline. After centrifugation at 3500 r / min for 20 min, 150 g of wet cells of each of halophilic Bacillus SSF1 and Bacillus licheniformis LU1 were collected. The cells were then resuspended in 300 mL of physiological saline. An equal volume of 300 mL of the bacterial suspension was mixed with a freeze-drying protectant and pre-frozen at -20℃ for 12 h. Finally, the pre-frozen cells were freeze-dried for 36 h to obtain Bacillus powder. Using the above method, halophilic Bacillus SSF1 powder and Bacillus licheniformis LU1 powder were obtained, with the viable count of the halophilic Bacillus SSF1 powder being 9 × 10⁻⁶. 13 CFU / g; The viable count of Bacillus licheniformis LU1 bacterial powder was 1.32 × 10⁻⁶ CFU / g. 15 CFU / g.

[0089] 2. According to the results of previous laboratory experiments, when the ratio of halophilic Bacillus SSF1 to Bacillus licheniformis LU1 is 1:2, the activity of endoglucosidase is the highest and the cellulose degradation ability is the strongest.

[0090] 3. Mix SSF1 and LU1 evenly at a ratio of 1:2 for live bacteria count to prepare compound bacterial powder.

[0091] Example 4: Preparation of Effervescent Granules 1. Determination of the type and proportion of disintegrants Citric acid and sodium bicarbonate were selected as the acid source and alkali source, respectively. The ratio of the two was screened, with three ratios selected: 1:1, 1:1.25, and 1.25:1. The disintegration time and foaming amount of different ratios were measured, and the results are shown in Table 3.

[0092] The specific experimental method was as follows: Citric acid and sodium bicarbonate were mixed at mass ratios of 1:1, 1:1.25, and 1.25:1, respectively, to obtain three groups of disintegrant mixtures with different proportions, which were used as disintegrant 1, disintegrant 2, and disintegrant 3 for subsequent experiments. The binder in the effervescent granules was 3% polyvinylpyrrolidone; the filler was corn starch; the lubricant was 3% polyethylene glycol 6000; and the disintegrant was 60%. Effervescent granules 1-3 all used this formula (the percentages are mass fractions), and the mass of the effervescent tablet was 0.6 g.

[0093] The raw materials used to prepare effervescent granules 1 are: 0.09 g of the compound bacterial powder of Example 3, 0.36 g of disintegrant 1, 0.018 g of binder, 0.114 g of filler and 0.018 g of lubricant.

[0094] The raw materials used to prepare effervescent granules 2 are: 0.09 g of the compound bacterial powder of Example 3, 0.36 g of disintegrant 2, 0.018 g of binder, 0.114 g of filler and 0.018 g of lubricant.

[0095] The raw materials used to prepare effervescent granules 3 are: 0.09 g of the compound bacterial powder of Example 3, 0.36 g of disintegrant 3, 0.018 g of binder, 0.114 g of filler and 0.018 g of lubricant.

[0096] The preparation method of effervescent granules is as follows: Tableting is performed using direct compression technology. The acid and alkali sources are weighed according to their ratio, and then mixed thoroughly using a mixer to obtain a disintegrant. The disintegrant, binder, and filler are sieved (0.25mm screen size) and then mixed with the compound bacterial powder until homogeneous. Finally, a lubricant is added, and after all raw materials are mixed thoroughly, the mixture is directly compressed into tablets (1cm diameter, 8mm thickness) using a 25-hole tablet press.

[0097] After the effervescent granules were prepared, the disintegration time and foaming amount of the effervescent granules were tested, and the results are shown in Table 6.

[0098] Table 6. Disintegration time and foaming amount of effervescent granules prepared with different acid and carbon source ratios.

[0099] Note: Different letters represent significant differences in disintegration time and foaming amount at the 0.05 level.

[0100] The experimental results showed that, after screening, the effervescent granules exhibited the shortest disintegration time when the mass ratio of citric acid to sodium bicarbonate was 1.25:1, which was significantly shorter (p < 0.05) than the mass ratios of 1:1 and 1:1.25. Simultaneously, the foaming amount at this ratio was also significantly higher (p < 0.05) than the other two ratios, thus meeting the selection criteria.

[0101] 2. Determination of the amount of disintegrant added Based on the optimal acid and carbon source ratio in step 1, effervescent granules were prepared with disintegrant addition levels of 55%, 60%, and 65%. The preparation of the effervescent granules was observed, and indicators such as disintegration time and foaming volume were measured to further determine the optimal disintegrant addition level. Citric acid and sodium bicarbonate were mixed at a mass ratio of 1.25:1 to obtain a disintegrant mixture, which was used as the disintegrant for addition tests. The binder was 3% polyvinylpyrrolidone, the filler was corn starch, and the lubricant was 3% polyethylene glycol 6000. The mass of the effervescent tablet was 0.6 g.

[0102] The specific experimental method is as follows: The raw materials used to prepare effervescent granules 1 are: 0.09 g of the compound bacterial powder of Example 3, 0.33 g (55%) of disintegrant, 0.018 g of binder, 0.144 g of filler and 0.018 g of lubricant.

[0103] The raw materials used to prepare effervescent granules 2 are: 0.09 g of the compound bacterial powder from Example 3, 0.36 g (60%) of disintegrant, 0.018 g of binder, 0.114 g of filler and 0.018 g of lubricant.

[0104] The raw materials used to prepare effervescent granules 3 are: 0.09 g of the compound bacterial powder of Example 3, 0.39 g (65%) of disintegrant, 0.018 g of binder, 0.084 g of filler and 0.018 g of lubricant.

[0105] The preparation method of effervescent granules is the same as described in 1 above.

[0106] After the effervescent granules were prepared, the disintegration phenomenon and foaming amount of the effervescent granules were tested, and the results are shown in Table 7.

[0107] Table 7 Determination of the amount of disintegrant added

[0108] Note: Different letters indicate significant differences in disintegration time at the 0.05 level.

[0109] The experimental results show that when the disintegrant content is 60%, the effervescent granules disintegrate smoothly and completely, and the disintegration time is significantly shorter (p<0.05) than that of the two ratios of 55% and 65%. Moreover, the solution after disintegration is clear and has a large amount of foaming, which meets the selection criteria.

[0110] 3. Determining the type of lubricant The lubricants used in the experiment were talc, magnesium stearate, and polyethylene glycol 6000. The optimal lubricant was determined by observing the appearance, disintegration time, and disintegration phenomena of the effervescent granules. The disintegrant was a mixture of citric acid and sodium bicarbonate at a mass ratio of 1.25:1. The binder was polyvinylpyrrolidone; the filler was corn starch; and all lubricants were added at a rate of 0.018 g (3%). The mass of each effervescent tablet was 0.6 g.

[0111] The specific experimental method is as follows: The raw materials used to prepare effervescent granules 1 are: 0.09 g of the compound bacterial powder of Example 3, 0.36 g (60%) of disintegrant, 0.018 g of binder, 0.114 g of filler and 0.018 g of talc.

[0112] The raw materials used to prepare effervescent granules 2 are: 0.09 g of the compound bacterial powder from Example 3, 0.36 g (60%) of disintegrant, 0.018 g of binder, 0.114 g of filler and 0.018 g of magnesium stearate.

[0113] The raw materials used to prepare effervescent granules 3 are: 0.09 g of the compound bacterial powder of Example 3, 0.36 g (60%) of disintegrant, 0.018 g of binder, 0.114 g of filler and 0.018 g of polyethylene glycol 6000.

[0114] The preparation method of effervescent granules is the same as described in 1 above.

[0115] After the effervescent granules were prepared, their appearance, disintegration time, disintegration phenomenon and other indicators were observed. The results are shown in Table 8.

[0116] Table 8 Determination of Lubricant Type

[0117] Note: Different letters indicate significant differences in disintegration time at the 0.05 level.

[0118] The experimental results showed that the effervescent granules prepared from the three raw materials all had smooth surfaces. However, when polyethylene glycol 6000 was used as a lubricant, the disintegration time was the shortest, and significantly shorter (p < 0.05) than when talc and magnesium stearate were used as lubricants. Although the disintegration time of talc as a lubricant was also significantly longer than that of magnesium stearate, it was not chosen due to its poor disintegration performance, incomplete disintegration, low foaming volume, and turbid solution after disintegration. Therefore, polyethylene glycol 6000 was selected as the lubricant for the effervescent granules in this experiment.

[0119] 4. Determining the amount of lubricant to be added Polyethylene glycol 6000, selected in step 3, was used as a lubricant. Effervescent granules were prepared with lubricant addition amounts of 1%, 3%, and 5%. The optimal lubricant dosage was further determined by observing the appearance of the effervescent granules and measuring indicators such as disintegration time and foaming amount. Citric acid and sodium bicarbonate were mixed at a mass ratio of 1.25:1 to obtain a disintegrant mixture, which was used as the disintegrant. The raw materials for the effervescent granules were the same as those in step 3 above.

[0120] The specific experimental method is as follows: The raw materials used to prepare effervescent granules 1 are: 0.09 g of the compound bacterial powder of Example 3, 0.36 g (60%) of disintegrant, 0.018 g of binder, 0.006 g (1%) of polyethylene glycol 6000 and filler, with the filler added to make the total amount of raw materials per effervescent granule 0.6 g.

[0121] The raw materials used to prepare effervescent granules 2 are: 0.09 g of the compound bacterial powder from Example 3, 0.36 g (60%) of disintegrant, 0.018 g of binder, 0.114 g of filler and 0.018 g (3%) of polyethylene glycol 6000.

[0122] The raw materials used to prepare effervescent granules 3 are: 0.09 g of the compound bacterial powder of Example 3, 0.36 g (60%) of disintegrant, 0.018 g of binder, 0.03 g (5%) of polyethylene glycol 6000 and filler. The amount of filler added is up to 0.6 g of the total raw materials per effervescent granule.

[0123] The preparation method of effervescent granules is the same as described in 1 above.

[0124] After the effervescent granules were prepared, the appearance of the effervescent granules and the disintegration time and foaming amount were observed. The results are shown in Table 9.

[0125] Table 9 Determination of Lubricant Dosage

[0126] Note: Different letters indicate significant differences in disintegration time at the 0.05 level.

[0127] The experimental results show that when the dosage of polyethylene glycol 6000 is 3%, the effervescent granules have the shortest disintegration time, which is significantly shorter (p < 0.05) than when the dosage is 1% and 5%. The granules disintegrate completely in water and produce a large amount of foam. Although the disintegration time is also significantly shorter when the dosage is 1% than when it is 5% (p < 0.05), its disintegration performance is poor, with incomplete disintegration and low foaming. Therefore, the dosage of polyethylene glycol 6000 is 3%.

[0128] 5. Determining the type of adhesive Porous starch, carboxymethyl cellulose, and polyvinylpyrrolidone were selected as binders. The optimal binder was determined by measuring the appearance, disintegration time, and effervescence properties of the effervescent granules. The disintegrant was a mixture of citric acid and sodium bicarbonate at a mass ratio of 1.25:1. Polyethylene glycol 6000 was used as a lubricant. Corn starch was used as a filler. The mass of each effervescent tablet was 0.6 g.

[0129] The specific experimental method is as follows: The raw materials used to prepare effervescent granules 1 are: 0.09 g of the compound bacterial powder of Example 3, 0.36 g (60%) of disintegrant, 0.018 g of porous starch, 0.114 g of filler and 0.018 g (3%) of lubricant.

[0130] The raw materials used to prepare effervescent granules 2 are: 0.09 g of the compound bacterial powder from Example 3, 0.36 g (60%) of disintegrant, 0.018 g of carboxymethyl cellulose, 0.114 g of filler and 0.018 g (3%) of lubricant.

[0131] The raw materials used to prepare effervescent granules 3 are: 0.09 g of the compound bacterial powder of Example 3, 0.36 g (60%) of disintegrant, 0.018 g of polyvinylpyrrolidone, 0.114 g of filler and 0.018 g (3%) of lubricant.

[0132] The preparation method of effervescent granules is the same as described in 1 above.

[0133] After the effervescent granules were prepared, their appearance, disintegration time, and effervescent properties were measured, and the results are shown in Table 10.

[0134] Table 10 Determination of Adhesive Type

[0135] Note: Different letters indicate significant differences in disintegration time at the 0.05 level.

[0136] The experimental results show that when polyvinylpyrrolidone is used as a binder to prepare effervescent granules, the surface is smooth and there is no adhesion. The disintegration time is the shortest and significantly shorter than that of porous starch and sodium carboxymethyl cellulose (p < 0.05). Polyvinylpyrrolidone has good disintegration performance, complete disintegration, and high foaming volume. Therefore, polyvinylpyrrolidone is selected as the best binder.

[0137] 6. Determining the amount of adhesive to be added After determining the type of adhesive, three dosages of 1%, 3%, and 5% were selected. The optimal dosage was further determined by measuring indicators such as disintegration time and foaming amount. The disintegrant was a mixture of citric acid and sodium bicarbonate at a mass ratio of 1.25:1. Polyethylene glycol 6000 was used as a lubricant. Polyvinylpyrrolidone was used as the adhesive. Corn starch was used as the filler. The effervescent tablet weighed 0.6 g.

[0138] The specific experimental method is as follows: The raw materials used to prepare effervescent granules 1 are: 0.09 g of the compound bacterial powder of Example 3, 0.36 g (60%) of disintegrant, 0.006 g (1%) of binder, 0.018 g (3%) of lubricant and filler, and the amount of filler added is up to 0.6 g of the total raw materials per effervescent granule.

[0139] The raw materials used to prepare effervescent granules 2 are: 0.09 g of the compound bacterial powder from Example 3, 0.36 g (60%) of disintegrant, 0.018 g (3%) of binder, 0.114 g of filler and 0.018 g (3%) of lubricant.

[0140] The raw materials used to prepare effervescent granules 3 are: 0.09 g of the compound bacterial powder of Example 3, 0.36 g (60%) of disintegrant, 0.03 g (5%) of binder, 0.018 g (3%) of lubricant and filler, with the filler added to make the total amount of raw materials per effervescent granule 0.6 g.

[0141] The preparation method of effervescent granules is the same as described in 1 above.

[0142] After the effervescent granules were prepared, their appearance, disintegration time, and effervescent properties were measured. The results are shown in Table 11.

[0143] Table 11 Determination of Adhesive Dosage

[0144] Note: Different letters represent significant differences in disintegration time and foaming amount at the 0.05 level.

[0145] The experimental results show that when the mass fraction of polyvinylpyrrolidone is 3%, the effervescent granules have the shortest disintegration time and the highest foaming amount, both of which are significantly better than (p < 0.05) when the mass fraction is 1% and 3%. Although the disintegration time of polyvinylpyrrolidone at 1% is significantly different from that at 5%, the foaming amount is less, and there is no difference at the 0.05 level compared with the 5% dosage. Therefore, this experiment selected 3% as the optimal addition amount of polyvinylpyrrolidone.

[0146] 7. Determining the Dosage of Microbial Powder Addition The prepared compound bacterial powder was added at three dosages: 10%, 15%, and 20%. The optimal dosage was further determined by measuring indicators such as disintegration time and foaming amount. The disintegrant was a mixture of citric acid and sodium bicarbonate at a mass ratio of 1.25:1. Polyethylene glycol 6000 was used as a lubricant. Polyvinylpyrrolidone was used as a binder. Corn starch was used as a filler. The effervescent tablet weighed 0.6 g.

[0147] The specific experimental method is as follows: The raw materials used to prepare effervescent granules 1 are: 0.06 g (10%) of the compound bacterial powder of Example 3, 0.36 g (60%) of the disintegrant, 0.018 g (3%) of the binder, 0.018 g (3%) of the lubricant and the filler. The amount of filler added is up to 0.6 g of the total raw materials per effervescent granule.

[0148] The raw materials used to prepare effervescent granules 2 are: 0.09 g (15%) of the compound bacterial powder of Example 3, 0.36 g (60%) of the disintegrant, 0.018 g (3%) of the binder, 0.018 g (3%) of the lubricant and the filler. The amount of filler added is up to 0.6 g of the total raw materials per effervescent granule.

[0149] The raw materials used to prepare effervescent granules 3 are: 0.12 g (20%) of the compound bacterial powder of Example 3, 0.36 g (60%) of the disintegrant, 0.018 g (3%) of the binder, 0.018 g (3%) of the lubricant and the filler. The amount of filler added is up to 0.6 g of the total raw materials per effervescent granule.

[0150] The preparation method of effervescent granules is the same as described in 1 above.

[0151] After the effervescent granules were prepared, the disintegration time, effervescent properties and foaming amount were measured, and the results are shown in Table 12.

[0152] Table 12 Determination of Microbial Powder Addition Amount

[0153] Note: Different letters represent significant differences in disintegration time and foaming amount at the 0.05 level.

[0154] The experimental results show that when the amount of bacterial powder added is 15%, the disintegration time is the shortest, and compared with the other two addition amounts, the complete disintegration time is significantly shortened (p < 0.05), and the foaming volume is significantly greater than that of the addition amounts of 10% and 15%. Although the disintegration time at 10% is also significantly shorter than that at 20% (p < 0.05), the disintegration is not smooth or complete. Furthermore, when the amount of bacterial powder added is 15%, the pH value of the solution after complete disintegration is close to that of pure water. Therefore, 15% was selected as the optimal amount of bacterial powder added. Under this condition, the effervescent granules prepared are as follows: Figure 3 As shown.

[0155] 8. Optimal dilution factor selection for counting viable bacteria in effervescent granules. The disintegrant is a mixture of citric acid and sodium bicarbonate in a mass ratio of 1.25:1. Polyethylene glycol 6000 is used as a lubricant. Polyvinylpyrrolidone is used as a binder. The filler is corn starch.

[0156] The raw materials used to prepare effervescent granules 1 were: 0.09 g (15%) of the compound bacterial powder from Example 3, 0.36 g (60%) of the disintegrant, 0.018 g (3%) of the binder, 0.018 g (3%) of the lubricant, and a filler. The filler was added to a total of 0.6 g of raw materials per effervescent granule.

[0157] The preparation method of effervescent granules is the same as described in 1 above.

[0158] Dilute 1 g of effervescent granules in 50 mL of sterile saline to obtain the stock solution. Take 1 mL of the stock solution and add it to 9 mL of sterile saline, mix well, and obtain the final solution (10 mL). -1 Diluent, repeat the above steps to prepare 10 solutions sequentially. -2 10 -3 For different gradient bacterial solutions, the pipette tip needs to be changed each time to avoid cross-contamination between different gradients. Spread the diluted bacterial solutions of each gradient onto LB medium, incubate upside down in an incubator at 30°C, and after 24 h, select the dilutions with colony counts between 30 and 300 on the plate for counting, and calculate the viable count of the effervescent granules using the following formula.

[0159] viable count = average colony count × dilution factor × (1 / coating volume).

[0160] The unit for coating volume is mL.

[0161] The growth of each colony on LB plates is shown in Table 13.

[0162] Table 13 Colony growth status

[0163] Based on the colony growth on LB plates, this experiment selected a dilution concentration of 10. -9 The viable count was performed on the plate, and the viable count of the effervescent granules was calculated to be 1.97 × 10⁻⁶ according to the viable count formula. 13 CFU / g.

[0164] Example 5: Wheat pot experiment verification This experiment consisted of four groups: a control group (CK), a group receiving effervescent granules with 10 live bacteria counts, and a group receiving 10 live bacteria counts. 8 CFU / g, 10 9CFU / g, 10 10 The experimental group treated with CFU / g was tested to assess the growth-promoting effect of effervescent granules on wheat by measuring indicators such as wheat germination rate, aboveground length, underground length, fresh weight root-to-shoot ratio, and dry weight root-to-shoot ratio.

[0165] 1. In this experiment, seeds of uniform size and plump grains were selected as experimental subjects. Before planting, the seeds were soaked in a 75% ethanol aqueous solution for 1 minute and then rinsed with deionized water 5 to 7 times.

[0166] 2. Planting Add potting soil to 2 / 3 of the pot's height, then evenly place 20 wheat seeds into the pot. Mark the planting height on the pot and continue adding potting soil until it is full.

[0167] In Experiment 1 (Group I), effervescent granules were dissolved in 120 L (one effervescent tablet) of water to obtain an effervescent granule solution. The viable bacteria count in the effervescent granule solution was 10. 8 CFU / g. Pour the effervescent granule solution into the flowerpot, ensuring it is thoroughly soaked but not overflowing.

[0168] In Experiment 2 (Group II), effervescent granules were dissolved in 12 L (one effervescent tablet) of water to obtain an effervescent granule solution. The viable bacteria count in the effervescent granule solution was 10. 9 CFU / g. Pour the effervescent granule solution into the flowerpot, ensuring it is thoroughly soaked but not overflowing.

[0169] In Experiment 3 (Group III), effervescent granules were dissolved in 1.2 L (one effervescent tablet) of water to obtain an effervescent granule solution. The viable bacteria count in the effervescent granule solution was 10. 10 CFU / g. Pour the effervescent granule solution into the flowerpot, ensuring it is thoroughly soaked but not overflowing.

[0170] Experiments 1 through 3 were conducted in 6 parallel experiments for each treatment. After the first application of effervescent granule solution, the effervescent granule solution was applied once every 2 days.

[0171] The control group was irrigated with an equal amount of sterile water instead of effervescent granule solution.

[0172] 3. Experimental Data Recording On the day of harvesting, measure the plant height, number of germinated plants, above-ground fresh weight, and below-ground fresh weight of the wheat, and calculate the fresh weight-to-shoot ratio. Pack the above-ground and below-ground parts of the wheat into kraft paper bags and dry them at 85℃ to constant weight. Measure the dry weight of the above-ground and below-ground parts separately, and calculate the dry weight-to-shoot ratio.

[0173] Wheat seedlings planted for two weeks were selected for various index measurements.

[0174] 4. Results (1) The number of germinations in each treatment group is as follows: Figure 4 As shown.

[0175] The results showed that the number of germinations in all three experimental groups was greater than that in the control group, and the difference was significant at the 0.05 level. The number of germinations in experimental group 2 was also significantly different from that in the control group and the other two treatment groups at the 0.05 level, but there was no difference between experimental group 1 and experimental group 3.

[0176] (2) The aboveground length, underground length, fresh weight root-to-shoot ratio, and dry weight root-to-shoot ratio of each treatment group are shown in Table 14. The wheat growth status diagrams for each treatment group are shown below. Figures 5-6 As shown. Figure 5 Figures showing the aboveground growth status of wheat in each treatment group; Figure 6 The diagram shows the overall growth status of wheat plants in each treatment group. Figures 5-6 ck in the figure represents the control group, 10 8 The wheat growth status of Experiment 1 group was 10. 9 The wheat growth status of experimental group 2, 10 10 The growth status of wheat in the three experimental groups.

[0177] Table 14 Statistical Table of Wheat Indicators

[0178] Note: Different letters represent significant differences in aboveground length, underground length, fresh weight root-to-shoot ratio, and dry weight root-to-shoot ratio at the 0.05 level.

[0179] Analysis of the experimental results showed that the aboveground length, underground length, fresh weight root-to-shoot ratio, and dry weight root-to-shoot ratio of the three experimental groups were significantly different from those of the control group at the 0.05 level. This indicates that the three concentrations of Bacillus effervescent granules all have the ability to promote wheat growth, and the experimental group 2 was significantly different from the other three groups at the 0.05 level.

[0180] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A compound microbial agent, characterized in that, The compound microbial agent includes halophilic Bacillus SSF1 and Bacillus licheniformis LU1; the preservation number of halophilic Bacillus SSF1 is CGMCC No. 34189; the preservation number of Bacillus licheniformis LU1 is CCTCC No: M 20231025.

2. The compound microbial agent according to claim 1, characterized in that, The viable cell ratio of Bacillus halophilus SSF1 powder to Bacillus licheniformis LU1 powder in the compound microbial agent is 1:(1.5~3); the viable cell count of the Bacillus halophilus SSF1 powder is 4×10⁻⁶. 13 ~5×10 13 CFU / g; The viable count of the Bacillus licheniformis LU1 powder is 8 × 10⁻⁶. 13 ~10×10 13 CFU / g.

3. A method for preparing the compound microbial agent according to claim 1 or 2, characterized in that, include: A compound bacterial agent was prepared by mixing salt-tolerant Bacillus SSF1 bacterial powder and Bacillus licheniformis LU1 bacterial powder.

4. The preparation method according to claim 3, characterized in that, The preparation method of the halophilic Bacillus SSF1 bacterial powder and Bacillus licheniformis LU1 bacterial powder is as follows: the halophilic Bacillus SSF1 bacterial suspension and the Bacillus licheniformis LU1 bacterial suspension are respectively mixed with a freeze-drying protectant and then freeze-dried to obtain halophilic Bacillus SSF1 bacterial powder and Bacillus licheniformis LU1 bacterial powder; the freeze-drying protectant includes skim milk powder, fructooligosaccharides and monosodium glutamate.

5. An effervescent granule, characterized in that, The compound microbial agent includes the compound microbial agent described in claim 1 or 2, or the compound microbial agent prepared by the preparation method described in claim 3 or 4.

6. The effervescent granules according to claim 5, characterized in that, The effervescent granules include a disintegrant; the disintegrant includes citric acid and tartaric acid; the mass ratio of citric acid to tartaric acid is (1~1.25):(1~1.25); the amount of disintegrant added to the effervescent granules is 55wt.%~65wt.%.

7. The effervescent granules according to claim 5, characterized in that, The effervescent granules include a lubricant; the lubricant includes polyethylene glycol 6000; the amount of lubricant added to the effervescent granules is 1 wt.% to 5 wt.%.

8. The effervescent granules according to claim 5, characterized in that, The effervescent granules include a binder; the binder includes polyvinylpyrrolidone; the amount of binder added to the effervescent granules is 1 wt.% to 5 wt.%.

9. The effervescent granules according to claim 5, characterized in that, The amount of compound microbial agent added to the effervescent granules is 10wt.%~20wt.%.

10. The use of the effervescent granules according to any one of claims 5 to 9 in promoting wheat growth.

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