A lipopeptide-producing bacillus subtilis bacterial agent, a preparation method and application thereof

Abstract

The application discloses a kind of lipopeptide-producing bacillus subtilis inoculant and its preparation method and application, belong to microbial inoculant preparation technical field.The method is realized long-term stable maintenance of lipopeptide-producing bacillus subtilis and its bacteriostatic activity by trehalose, chitosan and mannitol ternary protection system, combined with calcium alginate three-dimensional network embedding technology.Specifically comprising: trehalose, chitosan and mannitol are added to lipopeptide-producing bacillus subtilis fermentation broth, to obtain protection liquid;Sodium alginate, mature flour and rice bran powder are mixed to prepare carrier;Protection liquid is mixed with carrier and stirred, calcium chloride solution is sprayed to make sodium alginate crosslinking form calcium alginate three-dimensional network structure, and is crushed to obtain.The application is embedded and fixed by the egg box structure of calcium alginate, effectively prevents oxidative degradation and activity loss, so that the survival rate of viable count is maintained at more than 75% after 90 days of normal temperature storage, the application process is simple, low in cost, and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculant preparation technology, specifically to a lipopeptide-producing Bacillus subtilis inoculant, its preparation method, and its application. Background Technology

[0002] Bacillus subtilis ( Bacillus subtilis As an important probiotic and biocontrol strain, Bacillus subtilis can produce a variety of antibacterial substances, exhibiting significant inhibitory effects against Staphylococcus aureus, Escherichia coli, and Salmonella pathogens. These antibacterial substances are the core active ingredients in Bacillus subtilis's antibacterial activity, and their stability directly determines the product's antibacterial efficacy and application value. However, these antibacterial substances are highly susceptible to degradation and inactivation during storage due to light, oxidation, and temperature. Furthermore, a decrease in the number of viable bacteria also leads to a reduction in the subsequent production of antibacterial substances, severely restricting the industrial application of Bacillus subtilis inoculants.

[0003] In existing technologies, the preparation methods for microbial agents include freeze-drying, spray drying, and the addition of protective agents. Among these, freeze-drying can cause mechanical damage to cell membranes due to ice crystal formation during the freezing process, and antibacterial substances are also at risk of oxidation under prolonged vacuum conditions. Spray drying, with its instantaneous processing at inlet air temperatures of 180℃~220℃, results in a loss of more than 50% of viable bacteria, and the antibacterial substances undergo thermal degradation and oxidation at high temperatures, leading to a loss of 60%~70% of antibacterial activity, which affects product quality. The addition of protective agents typically involves adding single protective agents such as glycerol, trehalose, or skim milk powder, which has limited protective effects. After 30 days of storage at room temperature, the number of viable bacteria decreases by more than 50%, and the antibacterial activity decreases by more than 60%.

[0004] Sodium alginate is a natural polysaccharide extracted from brown algae, composed of β-D-mannuronic acid (M unit) and α-L-guluronic acid (G unit). Sodium alginate can combine with calcium ions to form calcium alginate gel, in which the G unit binds with the calcium ions... 2+ A characteristic egg-box structure is formed, constructing a stable three-dimensional network system. This three-dimensional network has good biocompatibility and encapsulation protection functions, and has been widely used in cell fixation and drug sustained release. However, existing calcium alginate encapsulation technologies mainly use drop-addition or extrusion methods to prepare gel spheres, which are complex processes with long production cycles (24 to 48 hours) and low production efficiency. Furthermore, when the calcium ion concentration is too high, it is easy to form complexes with antibacterial substances and precipitate, resulting in a significant decrease in antibacterial activity, with a loss of more than 40% of antibacterial activity. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a lipopeptide-producing Bacillus subtilis inoculant, its preparation method, and its application.

[0006] The first aspect of this invention provides a method for preparing a lipopeptide-producing Bacillus subtilis inoculant, comprising the following steps: The protective system was combined with lipopeptide-producing Bacillus subtilis ( Bacillus subtilis The fermentation broth of NMG-GLJ001 was mixed at a mass ratio of 1:2.5~3.5 to obtain a protective solution. The protective system consisted of trehalose, chitosan, and mannitol at a mass ratio of 1:1:0.4~0.6. The lipopeptide-producing Bacillus subtilis NMG-GLJ001 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 11, 2026, with accession number CGMCC NO.37891. The NMG-GLJ001 on the accession certificate is the lipopeptide-producing Bacillus subtilis NMG-GLJ001 of this invention, classified and named as Bacillus subtilis. Bacillus subtilis The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Sodium alginate, cooked flour, and rice bran powder were mixed in a mass ratio of 2~4:8~12:100 to obtain a carrier mixture; The protective liquid and the carrier mixture are mixed to obtain a wet material. A soluble calcium salt solution is sprayed into the wet material to carry out a cross-linking reaction, so that sodium alginate and calcium ions cross-link to form a three-dimensional calcium alginate network structure. After drying at 45℃~55℃ for 5.5h~6h, the lipopeptide-producing Bacillus subtilis inoculant is obtained. The mass percentage of the soluble calcium salt solution is 1%~5%, and the spraying amount of the soluble calcium salt solution is 3%~8% of the mass of the wet material.

[0007] This invention employs a calcium alginate eggshell structure to construct a three-dimensional network embedding system. By controlling the mass percentage of the soluble calcium salt solution to 1%–5% and the spraying amount to 3%–8% of the wet material mass, a three-dimensional network is obtained that embeds and fixes bacteria and antibacterial substances without complexing or precipitating with the antibacterial substances, thus ensuring antibacterial activity. This invention uses a spraying method to spray calcium ion solution onto the wet material for calcium ion cross-linking. This allows for precise control of the calcium ion addition, ensuring both effective embedding and antibacterial effects. Furthermore, the entire preparation process is simple and quick, requiring only 8–12 minutes.

[0008] In another preferred embodiment, the viable count of the Bacillus subtilis NMG-GLJ001 fermentation broth is 5 × 10⁻⁶. 8 ~2×10 9 CFU / mL.

[0009] In another preferred embodiment, the trehalose is D-(+)-trehalose dihydrate with a purity ≥98%. Trehalose is a non-reducing disaccharide with a unique "water substitution" function. During the drying process, it can replace water molecules to form hydrogen bonds with biological macromolecules such as proteins, lipids, and peptides, maintaining their natural conformation and preventing denaturation and inactivation. Trehalose can also form a glassy matrix, encapsulating live bacteria and antibacterial substances within it, isolating oxygen, and preventing oxidative degradation.

[0010] The chitosan has a degree of deacetylation of 80%–95% and a molecular weight of 10–100 kDa. As a product of chitin deacetylation, chitosan carries a positive charge and can bind to negatively charged phospholipids on bacterial cell membranes through electrostatic interactions, forming a protective layer and enhancing cell membrane stability. Simultaneously, chitosan possesses antioxidant properties, scavenging free radicals and protecting antibacterial substances from oxidation. Furthermore, chitosan and sodium alginate (negatively charged) can form a polyelectrolyte complex, enhancing the encapsulation effect.

[0011] The mannitol is D-mannitol with a purity of ≥98%. As a polyol-based permeation protectant, mannitol can maintain the osmotic pressure balance inside and outside the cell and prevent cell dehydration damage during the drying process. At the same time, mannitol has free radical scavenging ability and can work synergistically with chitosan to protect antibacterial substances from oxidative degradation.

[0012] In another preferred embodiment, the soluble calcium salt is made of calcium chloride.

[0013] In another preferred embodiment, the cooked flour is wheat flour that has been cooked at 120°C to 140°C for 5 to 10 minutes; The viscosity of the sodium alginate is 200 mPa·s to 800 mPa·s.

[0014] In another preferred embodiment, the protective system is mixed with the lipopeptide-producing Bacillus subtilis NMG-GLJ001 at 15°C to 30°C and stirred at 80 rpm to 150 rpm for 8 min to 15 min.

[0015] In another preferred embodiment, the crosslinking reaction takes 5 to 15 minutes.

[0016] The second aspect of this invention provides a lipopeptide-producing Bacillus subtilis inoculant prepared by the aforementioned method, characterized in that the viable count of the lipopeptide-producing Bacillus subtilis inoculant is 7 × 10⁻⁶. 8 CFU / g ~9×10 8 CFU / g.

[0017] The third aspect of the present invention provides the application of the aforementioned lipopeptide-producing Bacillus subtilis agent in the preparation of antibacterial agents, wherein the antibacterial agents are used to inhibit Staphylococcus aureus, Escherichia coli and Salmonella.

[0018] The fourth aspect of this invention provides the application of the aforementioned Bacillus subtilis inoculant in the preparation of antibacterial feed additives.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention uses a calcium alginate egg carton structure to construct a three-dimensional network embedding system, and adopts a spraying method for calcium ion cross-linking. The process is simple and time-saving, requiring only 8 to 12 minutes. The amount of soluble calcium salt solution with a mass percentage of 1% to 5% is precisely controlled at 3% to 8% of the wet material mass to avoid excessive calcium ions complexing with antibacterial substances. The three-dimensional network formed by the cross-linking of sodium alginate and calcium ions embeds and fixes the bacteria and antibacterial substances, forming a physical barrier: (1) blocking oxygen: the network pore size is <100nm, which effectively reduces the diffusion of oxygen into the interior and reduces the oxidation rate of antibacterial substances; (2) fixing active ingredients: the antibacterial substances are embedded in the network to prevent loss and degradation; (3) buffering pH changes: calcium alginate has a certain buffering capacity to maintain the stability of the microenvironment; (4) mechanical protection: the three-dimensional network provides mechanical support to protect the spores and cell structures from damage during drying and storage. The trehalose-chitosan-mannitol ternary protection system in this invention exhibits synergistic effects, with the three components working together to maintain a 75%–80% viable bacterial count retention rate over 90 days. The product's antibacterial activity is significantly superior to the 50%–65% antibacterial activity of single or two-component protection systems. This invention employs a low-temperature drying method of 45℃–55℃, far lower than the high-temperature method of spray drying (180℃–220℃), thus avoiding thermal degradation of the antibacterial substances and thermal shrinkage and deformation of the calcium alginate network. Experiments have demonstrated that after drying at 50℃ for 6 hours, the calcium alginate network structure remains intact, and the product, diluted 4000 times, exhibits a 15mm inhibition zone against Staphylococcus aureus. In contrast, after spray drying (180℃ air intake), the product, diluted 1000 times, only exhibits an 8mm–9mm inhibition zone, indicating a significant loss of antibacterial activity.

[0020] This invention uses a spraying method for calcium ion crosslinking, eliminating the need for traditional calcium alginate encapsulation methods such as dropwise addition or extrusion to prepare gel spheres. It also eliminates the need for high-cost equipment such as freeze drying or spray drying. It can be achieved using only a conventional mixer + spray device + oven or fluidized bed. The drying cost per ton of product is only RMB 1,800 to RMB 2,200, and the equipment investment is less than RMB 800,000, which is significantly lower than freeze drying or spray drying.

[0021] The present invention demonstrates through experiments that the Bacillus subtilis inoculant obtained by the method of the present invention, after being diluted 4000 times, exhibits an inhibition zone of up to 15 mm against Staphylococcus aureus, after being diluted 2000 times, an inhibition zone of up to 12 mm against Escherichia coli K88, and after being diluted 1000 times, an inhibition zone of up to 12 mm against Salmonella. This is significantly superior to the control group without calcium alginate cross-linking (the inhibition zone was only 10 mm to 11 mm after being diluted 500 times).

[0022] The Bacillus subtilis inoculant of this invention is a pale yellow to light brown powder with a characteristic aroma of rice bran powder. It has good flowability, making it easy to transport and add. 8 CFU / g ~9×10 8 CFU / g. The Bacillus subtilis inoculant, after a 4000-fold dilution, showed an inhibition zone of 15 mm against Staphylococcus aureus; after a 2000-fold dilution, it showed an inhibition zone of 12 mm against Escherichia coli K88; and after a 1000-fold dilution, it showed an inhibition zone of 12 mm against Salmonella. This indicates that the Bacillus subtilis inoculant of this invention has a significant inhibitory effect on both Gram-positive cocci and Gram-negative bacilli. Attached Figure Description

[0023] Figure 1 The figure shows the antibacterial effect of different dilutions on different pathogen indicator bacteria in Example 1 of the present invention; 25923 in the figure represents Staphylococcus aureus ATCC 25923; K88 represents Escherichia coli K88; 1344 represents Salmonella CVCC1344, and the same applies below; the numbers in the figure represent the dilution factor, and the same applies below.

[0024] Figure 2 This is a diagram showing the antibacterial effect of different dilutions on different pathogenic indicator bacteria in Comparative Example 1 of the present invention.

[0025] Figure 3 This is a diagram showing the antibacterial effect of different dilutions on different pathogenic indicator bacteria in Comparative Example 2 of this invention.

[0026] Figure 4 This is a diagram showing the antibacterial effect of different dilutions on different pathogenic indicator bacteria in Comparative Example 3 of this invention.

[0027] Figure 5 This is a diagram showing the antibacterial effect of different dilutions on different pathogenic indicator bacteria in Comparative Example 4 of this invention.

[0028] Figure 6 This is a diagram showing the antibacterial effect of different dilutions on different pathogenic indicator bacteria in Comparative Example 5 of this invention. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0030] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Chinese patent CN108497850A discloses a protective agent for Bacillus subtilis preparations, using a combination of skim milk powder and sucrose. However, its shelf life at room temperature is only 60 days, and its antibacterial activity decays rapidly. CN109517758A discloses a sodium alginate encapsulation method for preparing Bacillus subtilis particles, but it uses a traditional calcium ion cross-linking method to prepare gel spheres, which is complex, has a long production cycle (24-48 hours), and excessive calcium ions complex with the antibacterial substances, resulting in a loss of more than 40% of antibacterial activity. Therefore, developing a simple, low-cost method for preparing Bacillus subtilis preparations that can effectively protect the activity of antibacterial substances has significant theoretical and practical value.

[0032] Based on this, the present invention utilizes a ternary protection system of trehalose, chitosan, and mannitol, combined with calcium alginate three-dimensional network encapsulation technology, to achieve long-term stable maintenance of the antibacterial activity of lipopeptide-producing Bacillus subtilis. The specific preparation method is as follows:

[0033] The protective system was mixed with the fermentation broth of Bacillus subtilis NMG-GLJ001 at a mass ratio of 1:2.5~3.5 to obtain the protective solution; the protective system was composed of trehalose, chitosan and mannitol at a mass ratio of 1:1:0.4~0.6. Sodium alginate, cooked flour, and rice bran powder were mixed in a mass ratio of 2~4:8~12:100 to obtain a carrier mixture; The protective liquid and the carrier mixture are mixed to obtain a wet material. Calcium ions are added to the wet material to carry out a cross-linking reaction, so that sodium alginate and calcium ions cross-link to form a three-dimensional calcium alginate network structure. The mixture is then dried at 45℃~55℃ for 5.5h~6h and pulverized to obtain the lipopeptide-producing Bacillus subtilis inoculant. The amount of calcium ions added is 3%~8% of the mass of the wet material.

[0034] The following is a detailed description of a Bacillus subtilis inoculant, its preparation method, and its application.

[0035] The specific method for preparing the lipopeptide-producing Bacillus subtilis NMG-GLJ001 fermentation broth used in the examples is as follows: Lipopeptide-producing Bacillus subtilis NMG-GLJ001 was inoculated into seed culture medium (6 g / L peptone, 3 g / L yeast extract, 6 g / L NaCl, pH 7.0) and cultured at 37℃ and 210 rpm for 12 hours. Then, 3% of the inoculum was transferred to fermentation medium (30 g / L corn starch, 20 g / L soybean meal, 5 g / L yeast extract, 10 g / L corn steep liquor, 2 g / L K₂HPO₄, 0.5 g / L MgSO₄·7H₂O, pH 7.0) and fermented at 37℃ and 200 rpm for 16 hours, yielding a viable count of 11.5 × 10⁻⁶ cells / mL. 9 Fermentation broth of Bacillus subtilis NMG-GLJ001 with CFU / mL lipopeptide production.

[0036] The lipopeptide-producing Bacillus subtilis NMG-GLJ001 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 11, 2026, with accession number CGMCC NO.37891. The NMG-GLJ001 on the accession certificate is the same lipopeptide-producing Bacillus subtilis NMG-GLJ001 used in this invention, and its classification and nomenclature number is Bacillus subtilis. Bacillus subtilis The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The lipopeptide-producing Bacillus subtilis NMG-GLJ001 can produce surfactantin, iturin, and fengycin, lipopeptide antibacterial substances, which have significant inhibitory effects on Staphylococcus aureus, Escherichia coli, and Salmonella pathogens.

[0037] Method for determining viable cell count: Plate count method, LB medium, incubated at 37°C for 24 hours.

[0038] Antibacterial activity assay method: Referring to GB / T 38483-2020 "Determination of Antibacterial Activity of Secondary Metabolites of Microbial Antibiotics - Inhibition Zone Method", and combining the characteristics of the lipopeptide-producing Bacillus subtilis agent of this invention, the Oxford cup method (agar diffusion method) was used to determine the antibacterial activity. The specific steps are as follows: (1) Reagents and materials Main culture medium (prepared with primary water, refer to Appendix of GB / T 38483-2020): MHB liquid culture medium (200mL): Weigh 4.8g of MHB culture medium, add 200mL of primary water and stir to dissolve, dispense into Erlenmeyer flasks, and autoclave at 121℃ for 15min.

[0039] LB solid medium (200mL): Weigh 2g sodium chloride, 2g peptone, 1g yeast extract, and 4g agar powder, add 200mL of primary water and stir to dissolve. Sterilize at 121℃ for 15min.

[0040] Plain agar medium (200mL): Weigh 4g of agar powder, add 200mL of primary water and stir to dissolve. Sterilize at 121℃ for 15min. 0.85% physiological saline: Weigh 8.5g of sodium chloride, add primary water to a final volume of 1000mL, sterilize at 121℃ for 20min, and use as a sample dilution solution.

[0041] Oxford cups: Sterilize at 121℃ for 20 minutes before use.

[0042] (2) Activation of indicator bacteria and preparation of bacterial suspension Pick a single colony from the slant and inoculate it into 15 mL of MHB liquid medium (100 mL Erlenmeyer flask). Incubate at 37°C with shaking at 200 rpm for 14 h until the late logarithmic growth phase. Control the OD of the bacterial culture. 600 The value is between 1.8 and 2.0, and the concentration of the working bacterial suspension is 1×10⁻⁶. 8 CFU / mL, store at 4℃ for later use.

[0043] (3) Sample preparation Referring to GB / T 38483-2020 standard, and considering the characteristics of the lipopeptide-producing Bacillus subtilis agent of this invention, the following processing method was adopted: 1.0 g of the lipopeptide-producing Bacillus subtilis agent was accurately weighed and added to 9.0 mL of sterile 0.85% physiological saline. The mixture was extracted at 37°C and 200 rpm for 30 min by shaking to fully dissolve the lipopeptide-based antibacterial substances, yielding the stock solution (i.e., a 10-fold dilution of the agent). Using a 10-fold serial dilution method, 100-fold, 500-fold, 1000-fold, 2000-fold, and 4000-fold dilutions were prepared sequentially to obtain sample solutions of different dilution ratios. These solutions were stored at 4°C and used on the same day for future reference.

[0044] (4) Preparation of double-layer detection plate Lower layer of sterile agar substrate: Place sterile agar medium in a 60℃ water bath for insulation, pour 10mL into the bottom of a 90mm diameter sterile culture dish, cover the bottom of the dish, and let it solidify horizontally.

[0045] Oxford cup placement: Using sterile forceps, place 4 Oxford cups symmetrically on the solidified plain agar plate (spaced ≥25mm apart, ≥15mm from the edge of the plate), and gently press them to ensure they fit tightly against the agar surface without gaps.

[0046] Upper layer of bacterial agar: Cool the sterilized LB solid medium to 50°C; take a 50mL sterile centrifuge tube, add 40mL of LB medium and 400μL of the working bacterial suspension prepared in (2) above according to the 1% addition principle, and gently invert to mix; slowly pour 20mL / plate onto the plain agar plate with Oxford cups placed on it, ensuring that the position of the Oxford cups is not displaced, allow it to solidify horizontally, and then use sterile forceps to vertically pull out the Oxford cups, leaving regular circular channels to make a double-layer plate for antibacterial detection. The prepared detection plates can be stored in a 4°C refrigerator for later use, and it is recommended to use them on the same day.

[0047] (5) Adding samples Label the bottom of each test plate with the sample name, dilution factor, indicator bacteria name, and date. Accurately pipette 100 μL of sample solution at different dilutions and slowly add it to the wells along the wall of an Oxford cup (hold the pipette steady with your left hand while adding the sample to prevent liquid from overflowing or dripping onto the plate surface). Use 0.85% sterile saline as a negative control.

[0048] (6) Cultivation After adding the sample, place the plate upright in a 36±1℃ incubator for 24 hours. Keep the plate stable when moving it to prevent liquid from overflowing and affecting the results.

[0049] (7) Measurement of inhibition zone Immediately after the culture is completed, photograph the test plate for archiving; use vernier calipers to measure the diameter along the inhibition zone.

[0050] Example 1 A method for preparing a lipopeptide-producing Bacillus subtilis inoculum includes the following steps: Step 1: Take 5 parts of trehalose (D-(+)-trehalose dihydrate, purity 99%), 5 parts of chitosan (degree of deacetylation 90%, molecular weight 50kDa), and 0.5 parts of mannitol (D-type, purity 99%) and add them to 300 parts of Bacillus subtilis NMG-GLJ001 fermentation broth. Stir at 100 rpm for 10 minutes at 20℃ to obtain a protective solution.

[0051] Step 2: Mix 3 parts sodium alginate (viscosity 600 mPa·s), 10 parts cooked flour (wheat flour cooked at 130℃ for 8 minutes) and 100 parts rice bran powder, and stir at 40 rpm for 8 minutes to obtain the carrier mixture.

[0052] Step 3: Slowly add the protective solution to the carrier mixture and stir at 60 rpm for 20 minutes.

[0053] Step 4: Prepare a 2.5% calcium chloride solution by mass, spray it evenly at a rate of 5% of the wet material weight, and let it stand for 10 minutes to crosslink and form a three-dimensional network structure of calcium alginate.

[0054] Step 5: Spread the cross-linked material evenly on a drying tray with a layer thickness of 2.5cm, and dry it in a 50℃ oven for 6 hours until the moisture content is 10%.

[0055] Step 6: Grind through a 30-mesh sieve, then seal and store in an aluminum foil composite bag filled with nitrogen.

[0056] The performance of the lipopeptide-producing Bacillus subtilis inoculant prepared above was tested: Appearance: Pale yellow powder with the unique aroma of rice bran powder and a slight seaweed scent.

[0057] viable bacteria count: 7.5 × 10⁻⁶ 8 CFU / g.

[0058] Moisture content: 10%.

[0059] Particle size: 20 mesh~40 mesh.

[0060] The inhibition zone diameter against Staphylococcus aureus ATCC 25923 after a 4000-fold dilution was 15 mm; the inhibition zone diameter against Escherichia coli K88 after a 2000-fold dilution was 12 mm; and the inhibition zone diameter against Salmonella CVCC 1344 after a 1000-fold dilution was 12 mm. Figure 1 As shown in Table 1, the stability test (stored at room temperature 25℃) was conducted.

[0061] Table 1 Stability Results As can be seen from the results in Table 1, the viable count of the lipopeptide-producing Bacillus subtilis agent prepared in this invention was 77% after 90 days of storage at room temperature. After dilution of 4000 times, the inhibition zone against Staphylococcus aureus was still 14 mm, and after dilution of 1000 times, the inhibition zones against Escherichia coli K88 and Salmonella were both 12 mm. This indicates that the three-dimensional network encapsulation of calcium alginate combined with the trehalose-chitosan-mannitol ternary protection system can effectively maintain the viable count and broad-spectrum antibacterial activity of lipopeptide-producing Bacillus subtilis NMG-GLJ001.

[0062] Comparative Example 1 The difference from Example 1 is that there is no protective agent and no cross-linking, that is, no trehalose, chitosan and mannitol are added, and no calcium alginate cross-linking is performed. 300 parts of the fermentation broth of Bacillus subtilis NMG-GLJ001 producing lipopeptide is directly mixed with the carrier mixture (3 parts sodium alginate, 10 parts cooked flour and 100 parts rice bran powder) and dried. Other conditions are the same as in Example 1.

[0063] The performance of the lipopeptide-producing Bacillus subtilis inoculant prepared above was tested: Appearance: Pale yellow powder.

[0064] viable count: 6.8 × 10⁻⁶ 8 CFU / g.

[0065] Moisture content: 10%.

[0066] The inhibition zone diameter against Staphylococcus aureus ATCC 25923 after a 1000-fold dilution was 14 mm; the inhibition zone diameter against Escherichia coli K88 after a 500-fold dilution was 12 mm; and the inhibition zone diameter against Salmonella CVCC 1344 after a 500-fold dilution was 12 mm. Figure 2 As shown in Table 2, the results of the stability test (stored at room temperature of 25℃) are as follows.

[0067] Table 2 Stability Results The results showed that without the protectant and without calcium alginate cross-linking, the viable bacterial count was only 38% after 90 days, and the antibacterial activity was severely lost. The highest dilution factor against Staphylococcus aureus decreased from 1000× to 500×, indicating that both the protectant system and the three-dimensional network embedding of calcium alginate were crucial for maintaining activity.

[0068] Comparative Example 2 The difference from Example 1 is that it is a two-component protective agent + cross-linking, that is, only 5 parts of trehalose and 0.5 parts of mannitol are added (chitosan is not added). Other conditions are the same as in Example 1.

[0069] The performance of the lipopeptide-producing Bacillus subtilis inoculant prepared above was tested: Appearance: Pale yellow powder viable bacteria count: 7.0 × 10⁻⁶ 8 CFU / g.

[0070] Moisture content: 10%.

[0071] The inhibition zone diameter against Staphylococcus aureus ATCC 25923 after a 2000-fold dilution was 14 mm; the inhibition zone diameter against Escherichia coli K88 after a 1000-fold dilution was 12 mm; and the inhibition zone diameter against Salmonella CVCC 1344 after a 500-fold dilution was 12 mm. Figure 3 As shown in Table 3, the results of the stability test (stored at room temperature of 25℃) are as follows.

[0072] Table 3 Stability Results The results showed that although the two-component protection system (lacking chitosan) had calcium alginate cross-linking, the viable bacterial retention rate was only 54% after 90 days, indicating a significant loss of antibacterial activity. The absence of chitosan led to decreased cell membrane stability, and the lack of the polyelectrolyte complex formed by chitosan and sodium alginate weakened the encapsulation effect.

[0073] Comparative Example 3 The difference from Example 1 is that the ternary protective agent is added without cross-linking, that is, 5 parts of trehalose, 5 parts of chitosan and 0.5 parts of mannitol are added, but calcium alginate cross-linking is not performed (calcium chloride solution is not sprayed), and other conditions are the same as in Example 1.

[0074] The performance of the lipopeptide-producing Bacillus subtilis inoculant prepared above was tested: Appearance: Pale yellow powder.

[0075] viable bacteria count: 7.3 × 10⁻⁶ 8 CFU / g.

[0076] Moisture content: 10%.

[0077] The inhibition zone diameter against Staphylococcus aureus ATCC 25923 after a 2000-fold dilution was 14 mm; the inhibition zone diameter against Escherichia coli K88 after a 1000-fold dilution was 12 mm; and the inhibition zone diameter against Salmonella CVCC 1344 after a 1000-fold dilution was 12 mm. Figure 4 As shown in Table 4, the results of the stability test (stored at room temperature of 25℃) are as follows.

[0078] Table 4 Stability Results The results showed that although the ternary protection system was effective, without the three-dimensional network encapsulation of calcium alginate, the viable bacterial retention rate was only 62% after 90 days, and the antibacterial activity was also significantly lower than in Example 1. The lack of the physical barrier and oxygen-barrier protection provided by the calcium alginate eggshell structure led to oxidative degradation of the antibacterial substances during storage, resulting in accelerated loss of activity.

[0079] Comparative Example 4 The difference from Example 1 is the addition of a ternary protective agent and excess Ca. 2+ Crosslinking involves adding 5 parts trehalose, 5 parts chitosan, and 0.5 parts mannitol, but using an excess of calcium chloride solution for crosslinking (using an 8% calcium chloride solution by mass, with a spraying amount of 15% of the wet material weight), with other conditions the same as in Example 1.

[0080] The performance of the lipopeptide-producing Bacillus subtilis inoculant prepared above was tested: Appearance: Pale yellow powder.

[0081] viable count: 6.5 × 10⁻⁶ 8 CFU / g.

[0082] Moisture content: 10%.

[0083] The inhibition zone diameter against Staphylococcus aureus ATCC 25923 after a 1000-fold dilution was 11 mm; the inhibition zone diameter against Escherichia coli K88 after a 500-fold dilution was 10 mm; and the inhibition zone diameter against Salmonella CVCC 1344 after a 500-fold dilution was 10 mm. Figure 5 As shown in Table 5, the stability test (stored at room temperature 25℃) yielded the following results.

[0084] Table 5 Stability Results The results showed that excess Ca 2+ (The 15% dosage, significantly higher than the 5% in Example 1,) complexed and precipitated with lipopeptide antibacterial substances, resulting in a loss of approximately 26% of the initial antibacterial activity (compared to Example 1, the inhibition zone for Staphylococcus aureus decreased from 15 mm to 11 mm). Although the viable cell count retention rate was still acceptable (68%), the antibacterial activity was severely impaired, indicating that Ca... 2+ Dosage must be precisely controlled; excessive use can be harmful.

[0085] Comparative Example 5 The difference from Example 1 is that a ternary protective agent + crosslinking + high-temperature drying is used, that is, 5 parts of trehalose, 5 parts of chitosan and 0.5 parts of mannitol are added, and a 2.5% calcium chloride solution is sprayed for crosslinking (5% of the wet material weight), but a spray drying method is used (inlet air temperature 180℃, atomization pressure 0.2MPa), and other steps are the same as in Example 1.

[0086] The performance of the lipopeptide-producing Bacillus subtilis inoculant prepared above was tested: Appearance: Light yellow powder.

[0087] viable bacteria count: 3.5 × 10⁻⁶ 8 CFU / g (53% initial viable count loss).

[0088] Moisture content: 4%.

[0089] The inhibition zone diameter against Staphylococcus aureus ATCC 25923 after a 1000-fold dilution was 10 mm; the inhibition zone diameter against Escherichia coli K88 after a 500-fold dilution was 8 mm; and the inhibition zone diameter against Salmonella CVCC 1344 after a 500-fold dilution was 8 mm. Figure 6 As shown in the figure. Stability test (stored at room temperature of 25℃), the results are shown in Table 6.

[0090] Table 6 Stability Results The results showed that high-temperature (180℃) spray drying caused severe losses: (1) the initial viable count was only 3.5 × 10⁻⁶. 8(1) The CFU / g was reduced by 53% compared to Example 1; (2) The initial antibacterial activity was reduced by about 40% (the Staphylococcus aureus inhibition zone decreased from 15 mm to 9 mm); (3) The viable bacteria retention rate after 90 days was only 40%, which was much lower than the 77% in Example 1. High temperature caused: ① thermal damage to spores and vegetative cells; ② thermal degradation and oxidation of lipopeptide antibacterial substances; ③ thermal shrinkage and deformation of the calcium alginate network at high temperature, resulting in increased pore size and decreased protective effect.

[0091] By comparing the above five comparative systems, the following results can be obtained: (1) The necessity of synergy in the ternary protection system Comparative Example 2 (two-component) vs Example 1 (ternary): 90-day viability retention rate 54% vs 77%, Staphylococcus aureus inhibition zone 1000×-11mm vs 4000×-14mm, indicating that the lack of chitosan leads to a significant decrease in cell membrane stability and embedding effect.

[0092] (2) The key role of calcium alginate cross-linking Comparative Example 3 (without crosslinking) vs Example 1 (with crosslinking): The initial antibacterial activities were similar, but after 90 days, the inhibition zone of Staphylococcus aureus was 2000×-12mm vs 4000×-14mm, and the stability of the antibacterial substance was significantly different, proving that the oxygen barrier protection function of the three-dimensional network of the egg carton is crucial.

[0093] (3) Ca² + Precise control of dosage Comparative Example 4 (Excess Ca²) + ) vs Example 1 (appropriate amount of Ca²) + The initial antibacterial activity decreased by 26% (1000×-11mm vs 4000×-15mm), indicating excessive Ca²⁺. + It can form complexes with lipopeptides and precipitate, and must be precisely controlled within the range of 3% to 8% of the wet material weight.

[0094] (4) The protective effect of low-temperature drying Comparative Example 5 (180°C spray drying) vs Example 1 (50°C oven drying): initial viable cell loss 53%, initial antibacterial activity loss 40%, 90-day retention rate 40% vs 77%, demonstrating the necessity of low-temperature drying for the protection of heat-sensitive lipopeptides and spore activity.

[0095] (5) Serious consequences of lack of protective measures Comparative Example 1 (no protectant + no cross-linking): The viability rate after 90 days was only 38%, and the antibacterial activity was almost completely lost (only a 10 mm inhibition zone could be detected at 500× dilution), proving that a complete protection technology solution must be adopted.

[0096] Comprehensive data show that the lipopeptide-producing Bacillus subtilis inoculant prepared by the method of this invention maintained a viable count of 77% after 90 days of storage at room temperature. Even after a 4000-fold dilution, it still exhibited a 14mm inhibition zone against Staphylococcus aureus, which is significantly superior to all comparative examples (viable counts of 38%–68%, with the highest dilutions ranging from 500 to 2000 times). This fully demonstrates the synergistic advantages of the trehalose-chitosan-mannitol ternary protection system and the calcium alginate three-dimensional network encapsulation technology.

[0097] Application Example: Broiler Feed Additives The lipopeptide-producing Bacillus subtilis agent prepared in Example 1 was added to the basal diet of broilers for a feeding trial to study the effects of different doses of the lipopeptide-producing Bacillus subtilis agent on the growth performance, serum biochemical indicators and intestinal morphology of broilers, and to explore the optimal dose for broiler farming.

[0098] Experimental design: 180 AA broiler chickens of similar weight at 1 day old were selected and randomly divided into 3 groups of 60 chickens each, with 10 replicates per group and 6 chickens per replicate.

[0099] The control group was fed a corn-soybean meal basal diet.

[0100] Experimental group 1 was supplemented with 250 mg / kg of Bacillus subtilis lipopeptide inoculant (live count 1.0 × 10⁻⁶). 9 In experimental group 2, 500 mg / kg of Bacillus subtilis lipopeptide inoculant (2.0 × 10⁻⁶ CFU / kg feed) was added to the basal diet. 9 CFU / kg feed).

[0101] The lipopeptide-producing Bacillus subtilis inoculant is a pale yellow powder with a viable count of 4.0 × 10⁻⁶ cells. 9 The CFU / g of this lipopeptide-producing Bacillus subtilis showed good inhibitory effects against common intestinal pathogens such as Escherichia coli and Salmonella. The basal diet was formulated according to the "Broiler Feeding Standard" (NY / T 33-2004) and the NRC (1994) broiler nutritional requirements, divided into two stages: 1-21 days old (early stage) and 22-42 days old (late stage). The experimental period was 42 days.

[0102] Feeding and Management: The experiment was conducted in a standardized broiler house. The house temperature was controlled at 33-35℃ for 1-3 day old chickens, and then decreased by 2-3℃ each week until it was kept constant at around 21℃. The relative humidity was maintained at 60%-70%. 24-hour continuous lighting was used. During the experiment, the broilers had free access to feed and water. They were vaccinated according to the routine immunization program, and daily hygiene and disinfection were carried out.

[0103] Measurement indicators and methods: (1) Growth performance: On the morning of the 1st, 21st and 42nd day of the experiment, the weight of each replicate broiler was weighed on an empty stomach. During the experiment, the feed consumption was recorded on a replicate basis, and the average daily weight gain (ADG) and feed conversion ratio (F / G) were calculated. (2) Serum biochemical indicators: On the 42nd day of the experiment, one broiler close to the average weight was randomly selected from each replicate, and 10 mL of blood was collected from the wing vein. The serum was separated by centrifugation at 3500 r / min for 15 min. The contents of total protein (TP), albumin (ALB), blood urea nitrogen (BUN), total cholesterol (TC), triglycerides (TG) and glucose (Glu) in the serum were measured using a fully automated biochemical analyzer. (3) Immune indicators: The thymus and bursa of Fabricius were separated, and the surface fascia and fat were removed before weighing. The immune organ index (fresh weight of organs / live weight before slaughter) was calculated. The contents of immunoglobulins IgA, IgG and IgM in the serum were measured by enzyme-linked immunosorbent assay (ELISA). (4) Intestinal tissue morphology: The middle segment of the jejunum was separated into approximately 2 cm sections, rinsed with physiological saline, and fixed in 4% paraformaldehyde fixative for at least 24 hours. Paraffin sections (5 μm) were routinely prepared and stained with hematoxylin and eosin (HE). The villus height and crypt depth were measured using Image-Pro Plus 7.0 image analysis software. Ten complete villus-crypt structures were randomly selected from each sample for measurement, and the villus height to crypt depth ratio (V / C) was calculated. (5) Statistical analysis of data: The experimental data were analyzed using SPSS 26.0 statistical software for one-way ANOVA. When the differences were significant, Duncan's method was used for multiple comparisons. The experimental results are expressed as mean ± standard deviation. P < 0.05 was considered significant, and P < 0.01 was considered extremely significant. The results are shown in Table 7.

[0104] Table 7. Effects of different doses of Bacillus subtilis inoculant on broiler growth performance. Note: Different letters above the numbers indicate a significant difference at the p=0.05 level, while the presence of the same letter or the absence of a letter indicates no significant difference at the p=0.05 level.

[0105] As shown in Table 7, compared with the control group, the addition of 250 mg / kg and 500 mg / kg of Bacillus subtilis lipopeptide to the diet significantly increased the final body weight and average daily weight gain of broilers (P<0.01) and significantly reduced the feed conversion ratio (P<0.01). The 500 mg / kg group showed the best results: from day 1 to 42, the average daily weight gain increased by 21.6% compared to the control group (48.95 g / d vs 59.53 g / d), and the feed conversion ratio decreased by 30.9% (2.59 vs 1.79). The body weight at day 42 reached 2548.94 g, an increase of 21.2% compared to the control group. The 250 mg / kg group showed a 10.0% increase in average daily weight gain and a 16.2% decrease in feed conversion ratio from day 1 to 42, with effects between the control and 500 mg / kg groups, indicating a clear dose-response relationship.

[0106] Table 8. Effects of adding Bacillus subtilis, a lipopeptide-producing bacterium, to the serum biochemical indicators of broilers. As shown in Table 8, the addition of Bacillus subtilis lipopeptide-producing agent significantly increased serum glucose, urea nitrogen, total protein, and albumin levels (P<0.01), indicating that the Bacillus subtilis lipopeptide-producing agent improved the body's nutrient metabolism and protein synthesis capacity. In the 500 mg / kg group, serum total protein increased by 31.2% (29.45 g / L vs 38.65 g / L), albumin increased by 40.3% (14.65 g / L vs 20.55 g / L), and glucose increased by 22.7% (12.10 mmol / L vs 14.85 mmol / L) compared to the control group. The improvement in each indicator in the 250 mg / kg group was between that of the control group and the 500 mg / kg group.

[0107] Table 9. Effects of adding Bacillus subtilis, a lipopeptide-producing bacterium, to the organ index of broilers. Note: Different letters above the numbers indicate different meanings. p A statistically significant difference is indicated at the 0.05 level; the presence or absence of identical letters indicates a statistically significant difference. p There was no significant difference at the 0.05 level.

[0108] As shown in Table 9, the addition of lipopeptide-producing Bacillus subtilis significantly increased the thymus index and serum immunoglobulin levels (P<0.01), indicating that the agent enhanced the body's immune function. In the 500 mg / kg group, serum IgA increased by 94.1% (0.34 mg / L vs 0.66 mg / L), IgG by 62.8% (4.73 g / L vs 7.70 g / L), IgM by 63.4% (1.83 g / L vs 2.99 g / L), and the thymus index by 72.7% (0.11% vs 0.19%) compared to the control group. The increase in immunoglobulins was smaller in the 250 mg / kg group, with IgA increasing by only 8.8%, which was not significantly different from the control group. However, the 500 mg / kg group showed a significant increase, indicating a dose-threshold effect in the immune enhancement.

[0109] Table 10 Effects of dietary supplementation with lipopeptide-producing Bacillus subtilis inoculum on intestinal morphology in broilers. Note: Different letters above the numbers indicate different meanings. p A statistically significant difference is indicated at the 0.05 level; the presence or absence of identical letters indicates a statistically significant difference. p There was no significant difference at the 0.05 level.

[0110] As shown in Table 10, the jejunal villus height in the 500 mg / kg group was significantly increased by 16.5% compared to the control group (1244.16 μm vs 1449.29 μm, P < 0.01), the crypt depth was significantly decreased by 3.8% (175.97 μm vs 169.24 μm, P < 0.01), and the V / C ratio was increased by 21.0% (7.08 vs 8.57, P < 0.01). The increased villus height, decreased crypt depth, and increased V / C ratio indicate an increased intestinal absorptive area, enhanced nutrient absorption capacity, and improved intestinal health. The villus height and V / C ratio in the 250 mg / kg group did not differ significantly from the control group, suggesting that the improvement in intestinal morphology also exhibits a dose-threshold effect.

[0111] The results of the experiment showed that adding lipopeptide-producing Bacillus subtilis to the broiler diet could significantly improve growth performance, enhance immune function, and optimize intestinal tissue structure, and there was a clear dose-response relationship. (1) Mechanism of growth performance improvement: The average daily weight gain of the 500mg / kg group increased by 21.6% and the feed conversion ratio decreased by 30.9% in 1 day and 42 days. The main mechanisms included: ① Lipopeptide antibacterial substances produced by Bacillus subtilis inhibited the growth of intestinal pathogens (Escherichia coli, Salmonella) and maintained the balance of intestinal microecology; ② Improved intestinal morphology and structure, villus height increased by 16.5%, V / C ratio increased by 21.0%, and enhanced the ability to absorb nutrients; ③ Promoted protein metabolism, serum total protein increased by 31.2%, and albumin increased by 40.3%. (2) Immunostimulation effect: Serum IgA, IgG, and IgM increased by 94.1%, 62.8%, and 63.4% respectively in the 500mg / kg group, and the thymus index increased by 72.7%. Bacillus subtilis, as a probiotic, enhances the body's disease resistance by stimulating the intestinal immune system and promoting the secretion of immunoglobulins. (3) Determination of the optimal dosage: Considering growth performance, immune indicators and intestinal health indicators, 500 mg / kg (2.0 × 10⁻⁶ live bacteria) 9 The optimal dosage was (CFU / kg feed). At this dosage, all indicators reached their optimal levels and were significantly better than the 250 mg / kg group. Although the 250 mg / kg group improved growth performance, it did not show significant effects in immune enhancement and intestinal morphology optimization, indicating that the lower dosage did not reach the threshold for full efficacy.

[0112] Economic benefit analysis (taking the 500mg / kg group as an example): Based on the experimental results, the economic benefits of the 500mg / kg dosage group were analyzed. (1) Increased weight gain: The average daily weight gain of the experimental group increased by 10.58g / d. Based on the 42-day experimental period, the weight gain of each broiler increased by an additional 445g (10.58g / d × 42 days). (2) Feed saving: The feed conversion ratio decreased from 2.59 to 1.79, a reduction of 30.9%. Based on the total weight gain of approximately 2.5kg per broiler over 42 days, the control group consumed 6.475kg of feed, while the experimental group consumed 4.475kg of feed, saving approximately 1.0kg / bird of feed. (3) Cost accounting: The number of live bacteria in the microbial agent was 4.0 × 10 9CFU / g, adding 500mg / kg requires 0.5kg of microbial agent / ton of feed. Each broiler consumes about 4.5kg of feed in 42 days, and the amount of microbial agent used is about 2.25g / bird. Calculated at a microbial agent price of 5 yuan / kg, the cost of microbial agent is 0.011 yuan / g × 2.25g = 0.025 yuan / bird. Considering the processing loss factor, it is calculated at 0.26 yuan / bird. (4) Profit calculation: Weight gain profit = 0.445kg × 10 yuan / kg (broiler price) = 4.45 yuan / bird; Feed saving profit = 1.0kg × 3 yuan / kg (feed price) = 3.0 yuan / bird; Net profit = 4.45 + 3.0 - 0.26 = 7.19 yuan / bird. (5) Input-output ratio: 1:27.7 (input 0.26 yuan, output 7.19 yuan). Economic benefit analysis shows that adding 500 mg / kg Bacillus subtilis NMG to broiler feed has significant economic benefits. The cost increase per broiler is only 0.26 yuan, but the overall benefit from increased weight gain and feed savings reaches 7.19 yuan, resulting in a high input-output ratio of 1:27.7. Based on a farm with 10,000 broilers, each batch can generate an additional net profit of approximately 71,900 yuan, demonstrating significant economic benefits.

[0113] Conclusions: (1) Adding Bacillus subtilis NMG to broiler diets can significantly improve growth performance, improve feed conversion ratio, enhance immune function, and optimize intestinal tissue structure. (2) Based on comprehensive growth performance, serum biochemical indicators, immune indicators, and intestinal morphology indicators, the optimal addition dose was determined to be 500 mg / kg (2.0 × 10⁻⁶ viable bacteria). 9 (CFU / kg feed). At this dose, the average daily weight gain increased by 21.6% from day 1 to day 42, the feed conversion ratio decreased by 30.9%, serum IgA increased by 94.1%, intestinal villus height increased by 16.5%, and V / C ratio increased by 21.0%. (3) Economic benefit analysis showed that the input-output ratio of adding 500mg / kg Bacillus subtilis NMG was 1:27.7, and each broiler chicken could increase net income by 7.19 yuan, which has good application prospects and promotion value.

[0114] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a lipopeptide-producing Bacillus subtilis inoculant, characterized in that, Includes the following steps: The protective system was combined with lipopeptide-producing Bacillus subtilis ( Bacillus subtilis The fermentation broth of NMG-GLJ001 was mixed at a mass ratio of 1:2.5~3.5 to obtain a protective solution; the protective system consisted of trehalose, chitosan and mannitol at a mass ratio of 1:1:0.4~0.6; Bacillus subtilis NMG-GLJ001, a lipopeptide-producing bacterium, was deposited at the China General Microbiological Culture Collection Center on March 11, 2026, with accession number CGMCC NO.37891; Sodium alginate, cooked flour, and rice bran powder were mixed in a mass ratio of 2~4:8~12:100 to obtain a carrier mixture; The protective liquid and the carrier mixture are mixed to obtain a wet material. A soluble calcium salt solution is sprayed into the wet material to carry out a cross-linking reaction, so that sodium alginate and calcium ions cross-link to form a three-dimensional calcium alginate network structure. After drying at 45℃~55℃ for 5.5h~6h, the lipopeptide-producing Bacillus subtilis inoculant is obtained. The mass percentage of the soluble calcium salt solution is 1%~5%, and the spraying amount of the soluble calcium salt solution is 3%~8% of the mass of the wet material.

2. The method for preparing the lipopeptide-producing Bacillus subtilis inoculant according to claim 1, characterized in that, The viable cell count of the Bacillus subtilis NMG-GLJ001 fermentation broth containing lipopeptides was 5 × 10⁻⁶. 8 CFU / mL ~2×10 9 CFU / mL.

3. The method for preparing the lipopeptide-producing Bacillus subtilis inoculant according to claim 1, characterized in that, The trehalose is D-(+)-trehalose dihydrate with a purity ≥98%; The degree of deacetylation of the chitosan is 80%~95%, and the molecular weight is 10kDa~100kDa; The mannitol is D-mannitol with a purity of ≥98%.

4. The method for preparing the lipopeptide-producing Bacillus subtilis inoculant according to claim 1, characterized in that, The reagent used for the soluble calcium salt is calcium chloride.

5. The method for preparing the lipopeptide-producing Bacillus subtilis inoculant according to claim 1, characterized in that, The cooked flour is wheat flour that has been cooked at 120℃~140℃ for 5min~10min; The viscosity of the sodium alginate is 200 mPa·s to 800 mPa·s.

6. The method for preparing the lipopeptide-producing Bacillus subtilis inoculant according to claim 1, characterized in that, The protective system was mixed with the fermentation broth of the lipopeptide-producing Bacillus subtilis NMG-GLJ001 at 15℃~30℃ and stirred at 80rpm~150rpm for 8min~15min.

7. The method for preparing the lipopeptide-producing Bacillus subtilis inoculant according to claim 1, characterized in that, The cross-linking reaction takes 5 to 15 minutes.

8. A lipopeptide-producing Bacillus subtilis inoculant prepared by the preparation method according to any one of claims 1 to 7, characterized in that, The viable count of the lipopeptide-producing Bacillus subtilis agent is 7 × 10⁻⁶. 8 CFU / g ~9×10 8 CFU / g.

9. The application of the lipopeptide-producing Bacillus subtilis agent according to claim 8 in the preparation of an antibacterial agent, characterized in that, The antibacterial agent is used to inhibit Staphylococcus aureus, Escherichia coli and Salmonella.

10. The application of the lipopeptide-producing Bacillus subtilis agent according to claim 8 in the preparation of antibacterial feed additives.

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