A compound microbial feed additive, a preparation method and application thereof

Abstract

This invention discloses a composite microbial feed additive, its preparation method, and its application, belonging to the field of microbial materials technology. The additive is prepared by fermenting a mixture of *Priscilla megaterium* CGMCC1.10466, *Bacillus laterosporus* CGMCC1.2827, and *Bacillus licheniformis* CGMCC28445 at a volume ratio of (2-4):(1-3):5), followed by mixing with a composite carrier containing glucomannan and xylooligosaccharides and then drying. This invention utilizes the synergistic effect of the three microorganisms to efficiently degrade ammonia nitrogen, inhibit urease activity, significantly improve the intestinal health of broilers, reduce ammonia emissions in the breeding environment, and enhance growth performance. It is suitable for feeding white-feathered or yellow-feathered broilers throughout their entire growth cycle or in stages.

Description

Technical Field

[0001] This invention belongs to the field of microbial materials technology, specifically relating to a composite microbial feed additive, its preparation method, and its application. Background Technology

[0002] The large-scale and intensive development of broiler farming has placed higher demands on the nutritional value and functionality of feed. Gut health is a core factor determining the nutrient absorption efficiency, growth performance, and farming benefits of broilers. However, the accumulation of ammonia in chicken houses during the farming process not only pollutes the environment but also induces respiratory diseases in broilers, reduces their immunity, and even directly affects the gut microecological balance, forming a vicious cycle of "environmental deterioration - gut damage - growth retardation," which seriously restricts the green and sustainable development of the broiler farming industry.

[0003] Microbial feed additives, due to their green, safe, and residue-free characteristics, are gradually replacing traditional chemical additives and becoming the core means of regulating the intestinal health of broilers and improving the breeding environment.

[0004] For example, Chinese patent application CN201510649272.9 provides a microbial feed additive containing Bacillus coagulans FM603 or its fermentation culture. The Bacillus coagulans FM603 fermentation culture contains bacteriocins, which have antibacterial activity against Gram-positive pathogens such as Listeria monocytogenes, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Clostridium perfringens, and Clostridium perfringens. This bacteriocin has a molecular weight of 4276.45 Da and a partial amino acid sequence of Ala-Gly-His-Dhb-Phe-Val-Dhb-Gly-Pro. It is relatively stable under heat, acid, pepsin, or trypsin treatment, but is easily degraded and inactivated by streptomycin. This feed additive can improve the egg production rate of laying hens, reduce the feed conversion ratio, and improve egg quality; increase feed intake and daily weight gain in piglets, and reduce the feed conversion ratio; and increase daily weight gain and serum lysozyme content in broilers, while reducing the feed conversion ratio and mortality rate.

[0005] However, while existing technologies can improve daily weight gain and reduce mortality in broilers, they only focus on intestinal antibacterial activity and nutrient absorption, without addressing the improvement of ammonia emissions in the farming environment. Furthermore, single-strain microbial additives have functional limitations and poor intestinal colonization stability, making it difficult to meet the comprehensive needs of modern broiler farming. Therefore, developing a compound microbial feed additive with a reasonable strain ratio, stable processing, and sufficient live bacteria count, which can simultaneously promote broiler intestinal health and reduce ammonia emissions, has become an urgent technical problem to be solved in the current broiler farming industry. Summary of the Invention

[0006] This invention addresses the technical bottlenecks of existing compound microbial feed additives, such as limited functionality, poor stability of preparation processes, and inability to simultaneously regulate broiler gut health and reduce ammonia emissions in the breeding environment. It provides a compound microbial feed additive, its preparation method, and its application.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A compound microbial feed additive, made from Priestella megaterium ( Priestia megatherium ), Bacillus retroflexus ( Brevibacillus laterosporus ), Bacillus licheniformis ( Bacillus licheniformis The mixture was fermented at a volume ratio of (2-4):(1-3):5, and then mixed with a carrier at a mass ratio of 1:(5-8) and dried to obtain the final product.

[0008] Furthermore, the strain number of *Priscilla megaterium* is CGMCC1.10466, purchased from the China General Microbiological Culture Collection Center, with an original deposit date of March 18, 2010; the strain number of *Bacillus laterosporus* is CGMCC1.2827, purchased from the China General Microbiological Culture Collection Center, with an original deposit date of August 28, 2001. Both *Priscilla megaterium* and *Bacillus laterosporus* can be purchased through the collection center and do not require biological preservation. The *Bacillus licheniformis* (… Bacillus licheniformis The accession number for this *Bacillus licheniformis* is CGMCC28445. It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on September 12, 2023. This *Bacillus licheniformis* was donated by another party.

[0009] Furthermore, the carrier is a composite carrier of wheat bran, corn cob powder, rice bran, glucomannan, and xylooligosaccharide. Wheat bran, corn cob powder, and rice bran are mixed in a mass ratio of 1:2:1 as the base material, and the base material is mixed with glucomannan and xylooligosaccharide in a mass ratio of 8:(0.8-1.2):(0.5-0.8).

[0010] Glucomannan and xylooligosaccharides are added as functional additives. Glucomannan can enhance the cell encapsulation rate and gastric acid tolerance, while xylooligosaccharides specifically promote the colonization of beneficial bacteria and inhibit the proliferation of ammonia-producing putrefactive bacteria. The two work synergistically to improve the survival rate and functional expression efficiency of the compound bacterial agent in the intestine.

[0011] A method for preparing a compound microbial feed additive includes the following steps: S1. Activation of bacterial strains: Inoculate *Priestella megaterium*, *Bacillus laterosporus*, and *Bacillus licheniformis* into sterilized LB liquid medium and culture with shaking at 25-30℃ and 150-200 rpm for 18-24 hours to obtain activated seed solutions of each strain. S2. Seed culture expansion: The activated seed culture obtained in step S1 is transferred to the seed culture medium at an inoculation rate of 5%-10% by volume. The culture is shaken and cultured for 12-16 hours at a temperature of 25-30℃ and a rotation speed of 150-200 rpm to obtain the secondary seed culture of each strain. S3. Mixed Fermentation: The secondary seed cultures of *Priscilla megaterium*, *Bacillus laterosporus*, and *Bacillus licheniformis* obtained in step S2 are mixed at a volume ratio of (2-4):(1-3):5. This mixture is then inoculated into the fermentation medium at an inoculum volume of 5%-8% of the total volume. Fermentation is carried out at a temperature of 32-35℃, a rotation speed of 180-220 rpm, and an aeration rate of 1:0.5-1.0 vvm until the total viable cell count is ≥2.0 × 10⁻⁶. 9 CFU / mL was used to obtain a composite fermentation broth; S4. Carrier pretreatment: Mix wheat bran, corn cob powder, and rice bran evenly in a mass ratio of 1:2:1, and pulverize them through an 80-100 mesh sieve to obtain the base material; add glucomannan and xylooligosaccharide in the above base material in a mass ratio of 8:(0.8-1.2):(0.5-0.8), stir and mix thoroughly, sterilize at 105-110℃ for 30-40 minutes, and cool to room temperature to obtain the pretreated carrier; S5. Mixing and drying: The compound fermentation broth obtained in step S3 and the pretreated carrier obtained in step S4 are mixed evenly at a mass ratio of 1:(5-8), and vacuum dried at a temperature of 45-55℃ and a vacuum degree of 0.06-0.08MPa until the moisture content is ≤10%. The mixture is then pulverized through a 40-60 mesh sieve to obtain the compound microbial feed additive.

[0012] Furthermore, in step S2, the seed culture medium consists of: glucose 15-20 g / L, peptone 8-12 g / L, yeast extract 4-6 g / L, sodium chloride 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.5 g / L, pH 7.0-7.5, and sterilization conditions of 121°C for 15-20 min.

[0013] Furthermore, the fermentation medium in step S3 consists of: 20-30 g / L corn flour, 15-20 g / L soybean meal, 10-15 g / L wheat bran, 3-5 g / L ammonium sulfate, 3 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate, and 0.05 g / L manganese sulfate, with a pH of 7.0-7.5, and sterilization conditions of 121℃ for 20-25 min.

[0014] An application of a compound microbial feed additive, wherein the compound microbial feed additive is added by feeding throughout the entire feeding period or by feeding in stages; when feeding in stages, the addition amount for broilers aged 1-7 days is 0.3% of the daily feed weight, and the addition amount for broilers aged 8-42 days is 0.1%-0.2% of the daily feed weight.

[0015] Furthermore, the compound microbial feed additive is used to promote broiler gut health, reduce ammonia emissions in the broiler farming environment, and improve broiler growth performance in one or more ways.

[0016] Furthermore, the broiler chicken is a white-feathered broiler chicken or a yellow-feathered broiler chicken.

[0017] Beneficial effects This invention discloses a compound microbial feed additive, its preparation method, and its application. Addressing the shortcomings of existing microbial feed additives—such as limited functionality, poor intestinal colonization stability, low controllability of preparation processes, and the inability to simultaneously regulate broiler intestinal health and reduce ammonia emissions in the breeding environment—this invention achieves a multi-effect combination of improved intestinal health, reduced ammonia emissions, and enhanced growth performance in broiler farming through synergistic design involving precise strain matching, compatible composite carriers, and optimized process parameters. Leveraging the ammonia nitrogen degradation and urease inhibition characteristics of three strains—*Priscilla megaterium*, *Bacillus laterosporus*, and *Bacillus licheniformis*—and their effective colonization capabilities in the broiler intestine, this invention ensures stable overall process, meets product viable count standards, and its application method is adapted to the growth patterns of broilers. Specific beneficial effects are as follows: 1. The strain has excellent inherent functions and is scientifically matched. Combined with the vector, it synergistically achieves multiple effects such as ammonia nitrogen degradation, urease inhibition and intestinal colonization. The three strains selected in this invention—*Priscilla gigantea* (CGMCC1.10466), *Bacillus laterosporus* (CGMCC1.2827), and *Bacillus licheniformis* (CGMCC28445)—all possess ammonia nitrogen degradation and urease inhibition capabilities. Furthermore, the three strains, when mixed and fermented in a volume ratio of (2-4):(1-3):5, form a complementary and synergistic microecological effect. Combined with the protective and proliferative effects of glucomannan and xylooligosaccharides in the carrier, this not only synergistically enhances the functions of ammonia nitrogen degradation and urease inhibition but also improves the colonization ability and stability of the strains in the broiler intestine. After colonization, the strains can simultaneously exert a dual effect: on the one hand, comprehensively regulating the intestinal microecological balance of broilers, inhibiting the reproduction of harmful bacteria and promoting the proliferation of beneficial bacteria, thus fundamentally improving broiler intestinal health; on the other hand, directly degrading ammonia nitrogen in the intestine and reducing the hydrolysis of urea into ammonia by inhibiting urease activity, thereby reducing the amount of ammonia generated in the intestine from the source. This solves the problem of existing technologies that only focus on intestinal antibacterial measures or provide isolated environmental improvements, resulting in a functional disconnect.

[0018] 2. Optimized preparation process ensures the activity and function of the strain, resulting in strong product stability. This invention employs a stepwise culture process of strain activation-scale-mixed fermentation, coupled with precise control of parameters such as temperature, rotation speed, and aeration rate, ensuring that the total viable count in the mixed fermentation broth is ≥2.0 × 10⁻⁶. 9 The CFU / g concentration ensured the efficient proliferation of the three strains, providing a viable bacterial quantity basis for their ammonia nitrogen degradation, urease inhibition, and intestinal colonization capabilities. Subsequent vacuum drying at 45-55℃ reduced the product's moisture content (≤10%) while minimizing viable bacterial loss, ensuring strain activity and functional stability, and improving product storage and transportation performance. Furthermore, the seed culture medium and fermentation medium formulations were specifically tailored to the growth requirements of the three strains, further guaranteeing fermentation efficiency and strain activity. The overall preparation process is highly controllable and reproducible, suitable for large-scale production.

[0019] 3. Innovative design of functional composite carriers to support the colonization and function of bacterial strains in the gut from multiple dimensions. This invention utilizes a functional composite carrier made from wheat bran, corn cob powder, rice bran, glucomannan, and xylooligosaccharides, balancing basic adsorption and microbial function promotion to achieve multiple advantages: ① The basic substrate has excellent adsorption properties, effectively adsorbing live bacteria in the composite fermentation broth and providing basic protection for the strains; ② Glucomannan and xylooligosaccharides form a "protection-proliferation" synergistic mechanism, improving the survival rate of strains during storage, transportation, and the digestive tract, while specifically promoting the proliferation of strains in the intestine, significantly enhancing the intestinal colonization efficiency and stability, and ensuring the full utilization of ammonia nitrogen degradation, urease inhibition, and intestinal microecological regulation functions; ③ All components in the carrier are natural raw materials, which can be used as natural nutritional supplements for broilers. Glucomannan and xylooligosaccharides can also supplement dietary fiber and optimize the intestinal environment, forming a dual complement of nutrition and function with microbial additives, further enhancing the nutritional value of the feed; ④ The carrier undergoes high-temperature sterilization pretreatment, eliminating the risk of contamination by other microorganisms, ensuring product purity, and all raw materials are readily available and inexpensive, balancing product performance and production economy.

[0020] 4. Based on colonization, this invention achieves multiple benefits in one, breaking the vicious cycle of broiler farming and significantly improving farming efficiency. The compound microbial feed additive of this invention takes the effective colonization of strains into the intestines as its core foundation, achieving a synergistic unity of three core functions: promoting broiler intestinal health, reducing ammonia emissions in the farming environment, and improving broiler growth performance. On the one hand, after colonization, the strains regulate the intestinal microecological balance, improve intestinal health, and secrete various digestive enzymes to improve the broiler's absorption efficiency of feed nutrients, thereby increasing daily weight gain, reducing the feed conversion ratio, and improving growth performance. On the other hand, the colonized strains directly decompose ammonia nitrogen in the intestines through their own ammonia nitrogen degradation capabilities, while simultaneously reducing ammonia production through urease inhibition. This dual action significantly reduces nitrogen emissions in broiler feces, thereby reducing ammonia accumulation in the chicken house, alleviating the irritation of ammonia on the broiler's respiratory tract, and preventing damage to the intestinal microecology due to environmental degradation. This fundamentally breaks the vicious cycle of "environmental degradation - intestinal damage - growth retardation" in existing farming practices, significantly improving the overall efficiency of broiler farming.

[0021] 5. Scientific application method, adapted to the growth pattern of broilers, and widely applicable. This invention is designed with a phased feeding method to address the physiological characteristics of broilers at different growth stages. The addition amount is 0.3% for broilers aged 1-7 days and 0.1%-0.2% for broilers aged 8-42 days. This takes into account both the colonization needs of strains during the critical period of intestinal development in chicks and the growth needs of adult chickens, ensuring that the functions of strains can be effectively exerted at different stages. It can also be used for full-process feeding according to breeding needs, making it flexible and practical. At the same time, this additive is suitable for both white-feathered and yellow-feathered broilers, covering the mainstream broiler breeds in China, and has a wide range of applications.

[0022] 6. Green and safe, with no residues, meeting the needs of sustainable development in the livestock industry. This invention's compound microbial feed additive uses three strains of Bacillus as its core, containing no chemical additives or drug residues. Its ammonia nitrogen degradation, urease inhibition, and intestinal regulation functions are all achieved through the microorganisms' own metabolism. After use, it has no adverse effects on broilers, the breeding environment, or livestock products. It can effectively replace traditional chemical additives, reducing secondary pollution of the breeding environment by chemical agents and improving the safety of livestock products. This aligns with the trend of large-scale, intensive, and green development in my country's broiler farming industry, and is of great significance to promoting the sustainable development of the broiler farming industry. Attached Figure Description

[0023] Figure 1 The image shows the culture results of *Priscilla megaterium* (CGMCC1.10466), *Bacillus laterosporus* (CGMCC1.2827), and *Bacillus licheniformis* (CGMCC28445) as verified by the plate confrontation method of this invention. Figure 2 The morphological structure of broiler jejunum in Embodiment 1 of the present invention (×100x magnification). Figure 3 Morphological structure of jejunum in broiler chickens (×100x magnification) as a blank control. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0025] Example 1 A compound microbial feed additive, made from Priestella megaterium ( Priestia megatherium ), Bacillus retroflexus ( Brevibacillus laterosporus ), Bacillus licheniformis ( Bacillus licheniformis The mixture was fermented at a volume ratio of 2:1:5, and then dried with a carrier at a mass ratio of 1:8.

[0026] The strain number of *Priscilla megaterium* is CGMCC1.10466, purchased from the China General Microbiological Culture Collection Center (CGMCC), with an original deposit date of March 18, 2010. The strain number of *Bacillus laterosporus* is CGMCC1.2827, purchased from the CGMCC, with an original deposit date of August 28, 2001. Both *Priscilla megaterium* and *Bacillus laterosporus* can be purchased through the collection center and do not require biological preservation. The *Bacillus licheniformis* has the accession number CGMCC28445, deposited at the CGMCC, with a deposit date of September 12, 2023. This *Bacillus licheniformis* was donated by another party.

[0027] The carrier is a composite carrier of wheat bran, corn cob powder, rice bran, glucomannan, and xylooligosaccharides. Wheat bran, corn cob powder, and rice bran are mixed in a mass ratio of 1:2:1 as the base material, and the base material is mixed with glucomannan and xylooligosaccharides in a mass ratio of 8:0.8:0.5.

[0028] A method for preparing a compound microbial feed additive includes the following steps: S1. Activation of bacterial strains: Priestella megaterium, Bacillus laterosporus, and Bacillus licheniformis were inoculated into sterilized LB liquid medium and cultured with shaking at 25-30℃ and 150-200 rpm for 18 hours to obtain activated seed solutions of each strain. S2. Seed culture expansion: The activated seed culture obtained in step S1 was transferred to the seed culture medium at an inoculation rate of 10% by volume. The culture was shaken and cultured for 16 hours at a temperature of 25-30℃ and a rotation speed of 150-200 rpm to obtain the secondary seed culture of each strain. S3. Mixed Fermentation: The secondary seed cultures of *Priscilla megaterium*, *Bacillus laterosporus*, and *Bacillus licheniformis* obtained in step S2 are mixed at a volume ratio of 2:1:5. This mixture is then inoculated into the fermentation medium at an inoculum volume of 5% of the total volume of the mixture. Fermentation is carried out at a temperature of 32-35℃, a rotation speed of 180-220 rpm, and an aeration rate of 1:0.5-1.0 vvm until the total viable cell count is ≥2.0 × 10⁻⁶. 9 CFU / mL was used to obtain a composite fermentation broth; S4. Carrier pretreatment: Mix wheat bran, corn cob powder, and rice bran evenly in a mass ratio of 1:2:1, and pulverize them through an 80-100 mesh sieve to obtain the base material; add glucomannan and xylooligosaccharide in the above base material in a mass ratio of 8:0.8:0.5, stir and mix thoroughly, sterilize at 105-110℃ for 30-40 minutes, and cool to room temperature to obtain the pretreated carrier; S5. Mixing and drying: The compound fermentation broth obtained in step S3 and the pretreated carrier obtained in step S4 are mixed evenly at a mass ratio of 1:8. The mixture is then vacuum dried at a temperature of 45-55℃ and a vacuum degree of 0.06-0.08MPa until the moisture content is ≤10%. The mixture is then pulverized through a 40-60 mesh sieve to obtain the compound microbial feed additive.

[0029] Test for antagonistic effects of strains: The compatibility of *Priscilla megaterium* (CGMCC1.10466), *Bacillus laterosporus* (CGMCC1.2827), and *Bacillus licheniformis* (CGMCC28445) was verified using the plate confrontation method. Activated bacterial suspensions of the three strains (1×10⁻⁶) were prepared. 8Inoculate two strains (CFU / mL) onto LB agar plates and incubate at 25-30℃ for 24-48 hours. Observe the growth of the strains and the formation of inhibition zones.

[0030] The results show that ( Figure 1 After the three strains were cultured in pairs, the colonies all grew normally, with no inhibition zones or inhibition bands forming in the contact areas. The colonies merged naturally without any growth inhibition. This indicates that there is no antagonistic effect among the three strains, they are well-compatible, and can be mixed for fermentation.

[0031] In step S2, the seed culture medium consists of: glucose 15-20 g / L, peptone 8-12 g / L, yeast extract 4-6 g / L, sodium chloride 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.5 g / L, pH 7.0-7.5, and sterilization conditions of 121℃ for 15-20 min.

[0032] The fermentation medium described in step S3 consists of: 20-30 g / L corn flour, 15-20 g / L soybean meal, 10-15 g / L wheat bran, 3-5 g / L ammonium sulfate, 3 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate, and 0.05 g / L manganese sulfate, with a pH of 7.0-7.5. The sterilization conditions are 121℃ for 20-25 min.

[0033] Example 2 A compound microbial feed additive, made from Priestella megaterium ( Priestia megatherium ), Bacillus retroflexus ( Brevibacillus laterosporus ), Bacillus licheniformis ( Bacillus licheniformis The mixture was fermented at a volume ratio of 4:3:5, and then dried with a carrier at a mass ratio of 1:5.

[0034] The strain number of *Priscilla megaterium* is CGMCC1.10466, purchased from the China General Microbiological Culture Collection Center (CGMCC), with an original deposit date of March 18, 2010. The strain number of *Bacillus laterosporus* is CGMCC1.2827, purchased from the CGMCC, with an original deposit date of August 28, 2001. Both *Priscilla megaterium* and *Bacillus laterosporus* can be purchased through the collection center and do not require biological preservation. The *Bacillus licheniformis* has the accession number CGMCC28445, deposited at the CGMCC, with a deposit date of September 12, 2023. This *Bacillus licheniformis* was donated by another party.

[0035] The carrier is a composite carrier of wheat bran, corn cob powder, rice bran, glucomannan, and xylooligosaccharides. Wheat bran, corn cob powder, and rice bran are mixed in a mass ratio of 1:2:1 as the base material, and the base material is mixed with glucomannan and xylooligosaccharides in a mass ratio of 8:1.2:0.8.

[0036] A method for preparing a compound microbial feed additive includes the following steps: S1. Activation of bacterial strains: Priestella megaterium, Bacillus laterosporus, and Bacillus licheniformis were inoculated into sterilized LB liquid medium and cultured with shaking at 25-30℃ and 150-200 rpm for 24 hours to obtain activated seed solutions of each strain. S2. Seed culture expansion: The activated seed culture obtained in step S1 was transferred to the seed culture medium at an inoculation rate of 5% by volume. The culture was shaken and cultured for 12 hours at a temperature of 25-30℃ and a rotation speed of 150-200 rpm to obtain the secondary seed culture of each strain. S3. Mixed Fermentation: The secondary seed cultures of *Priscilla megaterium*, *Bacillus laterosporus*, and *Bacillus licheniformis* obtained in step S2 are mixed at a volume ratio of 4:3:5. This mixture is then inoculated into the fermentation medium at an inoculum volume of 8% of the total volume of the mixture. Fermentation is carried out at a temperature of 32-35℃, a rotation speed of 180-220 rpm, and an aeration rate of 1:0.5-1.0 vvm until the total viable cell count is ≥2.0 × 10⁻⁶. 9 CFU / mL was used to obtain a composite fermentation broth; S4. Carrier pretreatment: Mix wheat bran, corn cob powder, and rice bran evenly in a mass ratio of 1:2:1, and pulverize them through an 80-100 mesh sieve to obtain the base material; add glucomannan and xylooligosaccharide in the above base material in a mass ratio of 8:1.2:0.8, stir and mix thoroughly, sterilize at 105-110℃ for 30-40 minutes, and cool to room temperature to obtain the pretreated carrier; S5. Mixing and drying: The compound fermentation broth obtained in step S3 and the pretreated carrier obtained in step S4 are mixed evenly at a mass ratio of 1:5. The mixture is then vacuum dried at a temperature of 45-55℃ and a vacuum degree of 0.06-0.08MPa until the moisture content is ≤10%. The mixture is then pulverized through a 40-60 mesh sieve to obtain the compound microbial feed additive.

[0037] In step S2, the seed culture medium consists of: glucose 15-20 g / L, peptone 8-12 g / L, yeast extract 4-6 g / L, sodium chloride 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.5 g / L, pH 7.0-7.5, and sterilization conditions of 121℃ for 15-20 min.

[0038] The fermentation medium described in step S3 consists of: 20-30 g / L corn flour, 15-20 g / L soybean meal, 10-15 g / L wheat bran, 3-5 g / L ammonium sulfate, 3 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate, and 0.05 g / L manganese sulfate, with a pH of 7.0-7.5. The sterilization conditions are 121℃ for 20-25 min.

[0039] Comparative Example 1 This comparative example is identical to Example 1 in all raw materials and steps except for the absence of *Priscilla megaterium*. That is: A compound microbial feed additive, composed of Bacillus laterosporus (Bacillus retroflexus) Brevibacillus laterite ), Bacillus licheniformis ( Bacillus licheniformis After fermentation by mixing in a volume ratio of 1:5, it is then mixed with a carrier in a mass ratio of 1:8 and dried to obtain the final product.

[0040] The strain of *Bacillus laterosporus*, with accession number CGMCC 1.2827, was purchased from the China General Microbiological Culture Collection Center (CGMCC), with an original accession date of August 28, 2001. *Bacillus laterosporus* can be purchased from the collection center without the need for biological preservation. The *Bacillus licheniformis*, with accession number CGMCC28445, is deposited at the CGMCC on September 12, 2023. This *Bacillus licheniformis* was donated by another party.

[0041] A method for preparing a compound microbial feed additive includes the following steps: S1. Activation of bacterial strains: Bacillus lateralis and Bacillus licheniformis were inoculated into sterilized LB liquid medium and cultured with shaking at 25-30℃ and 150-200 rpm for 18 hours to obtain activated seed solutions of each strain. S2. Seed culture expansion: The activated seed culture obtained in step S1 was transferred to the seed culture medium at an inoculation rate of 10% by volume. The culture was shaken and cultured for 16 hours at a temperature of 25-30℃ and a rotation speed of 150-200 rpm to obtain the secondary seed culture of each strain. S3. Mixed Fermentation: The secondary seed cultures of *Bacillus laterosporus* and *Bacillus licheniformis* obtained in step S2 are mixed at a volume ratio of 1:5. This mixture is then inoculated into the fermentation medium at an inoculum volume of 5% of the total volume. Fermentation is carried out at a temperature of 32-35℃, a rotation speed of 180-220 rpm, and an aeration rate of 1:0.5-1.0 vvm until the total viable cell count is ≥2.0 × 10⁻⁶. 9 CFU / mL was used to obtain a composite fermentation broth; S4. Carrier pretreatment: Mix wheat bran, corn cob powder, and rice bran evenly in a mass ratio of 1:2:1, and pulverize them through an 80-100 mesh sieve to obtain the base material; add glucomannan and xylooligosaccharide in the above base material in a mass ratio of 8:0.8:0.5, stir and mix thoroughly, sterilize at 105-110℃ for 30-40 minutes, and cool to room temperature to obtain the pretreated carrier; S5. Mixing and drying: The compound fermentation broth obtained in step S3 and the pretreated carrier obtained in step S4 are mixed evenly at a mass ratio of 1:8. The mixture is then vacuum dried at a temperature of 45-55℃ and a vacuum degree of 0.06-0.08MPa until the moisture content is ≤10%. The mixture is then pulverized through a 40-60 mesh sieve to obtain the compound microbial feed additive.

[0042] Comparative Example 2 This comparative example is identical to Example 1 in all raw materials and steps except for the absence of Bacillus retroflexus. That is: A compound microbial feed additive, made from Priestella megaterium ( Priestia megatherium ), Bacillus licheniformis ( Bacillus licheniformis After fermentation by mixing the two components at a volume ratio of 2:5, they are then mixed with a carrier at a mass ratio of 1:8 and dried to obtain the final product.

[0043] The strain number of *Priscilla megaterium* is CGMCC1.10466, purchased from the China General Microbiological Culture Collection Center (CGMCC), with an original deposit date of March 18, 2010. *Priscilla megaterium* can be purchased through the collection center without the need for biological preservation. The accession number of *Bacillus licheniformis* is CGMCC28445, deposited at the CGMCC, with a deposit date of September 12, 2023. This *Bacillus licheniformis* was donated by another party.

[0044] A method for preparing a compound microbial feed additive includes the following steps: S1. Activation of bacterial strains: Priestella megaterium and Bacillus licheniformis were inoculated into sterilized LB liquid medium and cultured with shaking at 25-30℃ and 150-200 rpm for 18 hours to obtain activated seed solutions of each bacterial strain. S2. Seed culture expansion: The activated seed culture obtained in step S1 was transferred to the seed culture medium at an inoculation rate of 10% by volume. The culture was shaken and cultured for 16 hours at a temperature of 25-30℃ and a rotation speed of 150-200 rpm to obtain the secondary seed culture of each strain. S3. Mixed Fermentation: The secondary seed cultures of *Priscilla megaterium* and *Bacillus licheniformis* obtained in step S2 are mixed at a volume ratio of 2:5. This mixture is then inoculated into the fermentation medium at an inoculum volume of 5% of the total volume. Fermentation is carried out at a temperature of 32-35℃, a rotation speed of 180-220 rpm, and an aeration rate of 1:0.5-1.0 vvm until the total viable cell count reaches ≥2.0 × 10⁻⁶. 9 CFU / mL was used to obtain a composite fermentation broth; S4. Carrier pretreatment: Mix wheat bran, corn cob powder, and rice bran evenly in a mass ratio of 1:2:1, and pulverize them through an 80-100 mesh sieve to obtain the base material; add glucomannan and xylooligosaccharide in the above base material in a mass ratio of 8:0.8:0.5, stir and mix thoroughly, sterilize at 105-110℃ for 30-40 minutes, and cool to room temperature to obtain the pretreated carrier; S5. Mixing and drying: The compound fermentation broth obtained in step S3 and the pretreated carrier obtained in step S4 are mixed evenly at a mass ratio of 1:8. The mixture is then vacuum dried at a temperature of 45-55℃ and a vacuum degree of 0.06-0.08MPa until the moisture content is ≤10%. The mixture is then pulverized through a 40-60 mesh sieve to obtain the compound microbial feed additive.

[0045] Comparative Example 3 This comparative example is identical to Example 1 in all raw materials and steps except for the absence of Bacillus licheniformis. A compound microbial feed additive, made from Priestella megaterium ( Priestia megatherium ), Bacillus retroflexus ( Brevibacillus laterosporus After fermentation by mixing in a volume ratio of 2:1, it is then mixed with a carrier in a mass ratio of 1:8 and dried to obtain the final product.

[0046] The strain number of *Priscilla megaterium* is CGMCC1.10466, purchased from the China General Microbiological Culture Collection Center (CGMCC), with an original deposit date of March 18, 2010; the strain number of *Bacillus laterosporus* is CGMCC1.2827, purchased from the CGMCC, with an original deposit date of August 28, 2001. Both *Priscilla megaterium* and *Bacillus laterosporus* can be purchased from the collection center and do not require biological preservation.

[0047] A method for preparing a compound microbial feed additive includes the following steps: S1. Activation of bacterial strains: Priestella megaterium and Bacillus laterosporus were inoculated into sterilized LB liquid medium and cultured with shaking at 25-30℃ and 150-200 rpm for 18 hours to obtain activated seed solutions of each strain. S2. Seed culture expansion: The activated seed culture obtained in step S1 was transferred to the seed culture medium at an inoculation rate of 10% by volume. The culture was shaken and cultured for 16 hours at a temperature of 25-30℃ and a rotation speed of 150-200 rpm to obtain the secondary seed culture of each strain. S3. Mixed Fermentation: The secondary seed cultures of *Priscilla megaterium* and *Bacillus laterosporus* obtained in step S2 are mixed at a volume ratio of 2:1. This mixture is then inoculated into the fermentation medium at an inoculum volume of 5% of the total volume of the mixture. Fermentation is carried out at a temperature of 32-35℃, a rotation speed of 180-220 rpm, and an aeration rate of 1:0.5-1.0 vvm until the total viable cell count is ≥2.0 × 10⁻⁶. 9 CFU / mL was used to obtain a composite fermentation broth; S4. Carrier pretreatment: Mix wheat bran, corn cob powder, and rice bran evenly in a mass ratio of 1:2:1, and pulverize them through an 80-100 mesh sieve to obtain the base material; add glucomannan and xylooligosaccharide in the above base material in a mass ratio of 8:0.8:0.5, stir and mix thoroughly, sterilize at 105-110℃ for 30-40 minutes, and cool to room temperature to obtain the pretreated carrier; S5. Mixing and drying: The compound fermentation broth obtained in step S3 and the pretreated carrier obtained in step S4 are mixed evenly at a mass ratio of 1:8. The mixture is then vacuum dried at a temperature of 45-55℃ and a vacuum degree of 0.06-0.08MPa until the moisture content is ≤10%. The mixture is then pulverized through a 40-60 mesh sieve to obtain the compound microbial feed additive.

[0048] Comparative Example 4 In this comparative example, except for the use of other species of *Priscilla megaterium*, the raw materials and procedures are the same as in Example 1. A compound microbial feed additive, made from Priestella megaterium ( Priestia megatherium ), Bacillus retroflexus ( Brevibacillus laterosporus ), Bacillus licheniformis ( Bacillus licheniformis The mixture was fermented at a volume ratio of 2:1:5, and then dried with a carrier at a mass ratio of 1:8.

[0049] The strain number of *Priscilla megaterium* is CGMCC1.16094, purchased from the China General Microbiological Culture Collection Center (CGMCC), with an original deposit date of March 11, 2017. The strain number of *Bacillus laterosporus* is CGMCC1.2827, purchased from the CGMCC, with an original deposit date of August 28, 2001. Both *Priscilla megaterium* and *Bacillus laterosporus* can be purchased through the collection center and do not require biological preservation. The *Bacillus licheniformis* has the accession number CGMCC28445, deposited at the CGMCC, with a deposit date of September 12, 2023. This *Bacillus licheniformis* was donated by another party.

[0050] Comparative Example 5 In this comparative example, except for the use of other types of Bacillus laterosporus, the raw materials and procedures are the same as in Example 1. A compound microbial feed additive, made from Priestella megaterium ( Priestia megatherium ), Bacillus retroflexus ( Brevibacillus laterosporus ), Bacillus licheniformis ( Bacillus licheniformis The mixture was fermented at a volume ratio of 2:1:5, and then dried with a carrier at a mass ratio of 1:8.

[0051] The strain number of *Priscilla megaterium* is CGMCC1.10466, purchased from the China General Microbiological Culture Collection Center (CGMCC), with an original deposit date of March 18, 2010. The strain number of *Bacillus laterosporus* is BNCC388948, purchased from the Henan Provincial Industrial Microbial Culture Collection Center of Beina Biotechnology. Both *Priscilla megaterium* and *Bacillus laterosporus* can be purchased through the collection center and do not require biological preservation. The *Bacillus licheniformis* has the accession number CGMCC28445, deposited at the CGMCC, with a deposit date of September 12, 2023. This *Bacillus licheniformis* was donated by another party.

[0052] Comparative Example 6 In this comparative example, except for the use of other types of Bacillus licheniformis, the raw materials and procedures are the same as in Example 1. A compound microbial feed additive, made from Priestella megaterium ( Priestia megatherium ), Bacillus retroflexus ( Brevibacillus laterosporus ), Bacillus licheniformis ( Bacillus licheniformis The mixture was fermented at a volume ratio of 2:1:5, and then dried with a carrier at a mass ratio of 1:8.

[0053] The strain number of *Priscilla megaterium* is CGMCC1.10466, purchased from the China General Microbiological Culture Collection Center (CGMCC), with an original deposit date of March 18, 2010. The strain number of *Bacillus laterosporus* is CGMCC1.2827, purchased from the CGMCC, with an original deposit date of August 28, 2001. The accession number of *Bacillus licheniformis* is CGMCC1.10257, purchased from the CGMCC, with an original deposit date of November 4, 2009. *Priscilla megaterium*, *Bacillus laterosporus*, and *Bacillus licheniformis* can all be purchased from the collection centers and do not require biological preservation.

[0054] Comparative Example 7 In this comparative example, except that glucomannan was not added to the carrier, all other raw materials and steps were the same as in Example 1. That is: A compound microbial feed additive, made from Priestella megaterium ( Priestia megatherium ), Bacillus retroflexus ( Brevibacillus laterosporus ), Bacillus licheniformis ( Bacillus licheniformis The mixture was fermented at a volume ratio of 2:1:5, and then dried with a carrier at a mass ratio of 1:8.

[0055] The strain number of *Priscilla megaterium* is CGMCC1.10466, purchased from the China General Microbiological Culture Collection Center (CGMCC), with an original deposit date of March 18, 2010. The strain number of *Bacillus laterosporus* is CGMCC1.2827, purchased from the CGMCC, with an original deposit date of August 28, 2001. Both *Priscilla megaterium* and *Bacillus laterosporus* can be purchased through the collection center and do not require biological preservation. The *Bacillus licheniformis* has the accession number CGMCC28445, deposited at the CGMCC, with a deposit date of September 12, 2023. This *Bacillus licheniformis* was donated by another party.

[0056] The carrier is a composite carrier of wheat bran, corn cob powder, rice bran, and xylooligosaccharides. Wheat bran, corn cob powder, and rice bran are mixed in a mass ratio of 1:2:1 as the base material, and the base material is mixed with xylooligosaccharides in a mass ratio of 8:1.3.

[0057] Comparative Example 8 This comparative example is identical to Example 1 in all raw materials and steps except that xylooligosaccharides are not added to the carrier. That is: A compound microbial feed additive, made from Priestella megaterium ( Priestia megatherium ), Bacillus retroflexus ( Brevibacillus laterosporus ), Bacillus licheniformis ( Bacillus licheniformis The mixture was fermented at a volume ratio of 2:1:5, and then dried with a carrier at a mass ratio of 1:8.

[0058] The strain number of *Priscilla megaterium* is CGMCC1.10466, purchased from the China General Microbiological Culture Collection Center (CGMCC), with an original deposit date of March 18, 2010. The strain number of *Bacillus laterosporus* is CGMCC1.2827, purchased from the CGMCC, with an original deposit date of August 28, 2001. Both *Priscilla megaterium* and *Bacillus laterosporus* can be purchased through the collection center and do not require biological preservation. The *Bacillus licheniformis* has the accession number CGMCC28445, deposited at the CGMCC, with a deposit date of September 12, 2023. This *Bacillus licheniformis* was donated by another party.

[0059] The carrier is a composite carrier of wheat bran, corn cob powder, rice bran, and glucomannan. Wheat bran, corn cob powder, and rice bran are mixed in a mass ratio of 1:2:1 as the base material, and the base material is mixed with glucomannan in a mass ratio of 8:1.3.

[0060] Experimental Test Ammonia nitrogen degradation capacity and urease inhibition effect of seed liquids of different strains Seed culture was prepared according to the method of Example 1 and the strain composition and proportions of the examples and comparative examples. S1. Activation of bacterial strains: The test strains (Priestella megaterium, Bacillus laterosporus, and Bacillus licheniformis) were inoculated into sterilized LB liquid medium and cultured with shaking at 25-30℃ and 150-200 rpm for 18 hours to obtain the activated seed liquid of each strain. S2. Seed culture expansion: The activated seed culture obtained in step S1 was transferred to seed culture medium at an inoculation rate of 10% (v / v). The culture was then shaken and cultured for 16 hours at 25-30℃ and 150-200 rpm to obtain secondary seed cultures for each strain. The seed cultures were mixed according to the proportions in Table 1, and their degradation rate of ammonia nitrogen and inhibition rate of urease activity were measured.

[0061] The composition of the seed liquid is shown in Table 1: Table 1. Composition of seed liquid in different experimental groups The seed culture to be tested was inoculated at a 10% inoculation rate into beef extract peptone medium containing 1% concentrated ammonia. A small container containing 10 mL of 1% boric acid was placed inside for ammonia absorption. After incubation at 25-30℃ and 180 r / min for 48 h, the culture was centrifuged at 8000 r / min for 10 min, and the supernatant was retained. The ammonia nitrogen content in the supernatant was determined by Nessler's reagent spectrophotometry. An equal volume of distilled water was used to replace the supernatant of the bacterial culture as a control group (CK). The ammonia nitrogen utilization rate was calculated according to the following formula.

[0062] .

[0063] The seed culture was inoculated into the urease inhibitor medium at a 6% inoculum size. After incubation at 25-30°C for 48 hours, it was centrifuged at 8000 rpm for 10 minutes, and the supernatant was used as the crude urease inhibitor extract. The crude extract was mixed with soybean urease at a 1:1 volume ratio, and the soybean urease activity was measured. An equal volume of distilled water was used to replace the crude extract as a blank control. Soil urease activity was measured using the phenol-sodium hypochlorite colorimetric method following the same procedure. The inhibition rates of different seed cultures of the inoculant on soybean urease and soil urease were calculated according to the formula. The composition of the urease inhibitor medium was as follows: corn flour 15.0 g / L, soybean meal 15.0 g / L, MgSO4·7H2O 3.0 g / L, Na2HPO4 2.0 g / L, KH2PO4 2.0 g / L.

[0064] .

[0065] Each experimental group was repeated three times, and the results were averaged. The experimental results are shown in Table 2.

[0066] Table 2 Test Results The results showed that Example 1, using a combination of *Priscilla megaterium* CGMCC1.10466, *Bacillus laterosporus* CGMCC1.2827, and *Bacillus licheniformis* CGMCC28445, achieved an ammonia nitrogen utilization rate of 87.5%, a soybean urease inhibition rate of 60.5%, and a soil urease inhibition rate as high as 89.6%, significantly superior to the single-strain controls and other comparative examples. The ammonia nitrogen utilization rates of single-strain controls 1-3 were 55.2%, 52.8%, and 58.6%, respectively; the soybean urease inhibition rates were 30.4%, 28.7%, and 33.2%, respectively; and the soil urease inhibition rates were 58.3%, 55.9%, and 61.4%, respectively, indicating that the ammonia nitrogen degradation capacity and urease inhibition effect of the single strains were significantly lower than those of the three-strain composite system.

[0067] Comparative Examples 1-3, lacking *Priscilla megaterium*, *Bacillus laterosporus*, and *Bacillus licheniformis* respectively, showed ammonia nitrogen utilization rates of 72.3%, 68.7%, and 63.4%, respectively; soybean urease inhibition rates of 45.2%, 42.6%, and 38.1%, respectively; and soil urease inhibition rates of 74.8%, 71.3%, and 66.2%, respectively. The results indicate that the absence of any one strain significantly reduced the function of the complex system. *Bacillus licheniformis* played a central role in ammonia nitrogen degradation and urease inhibition, while the participation of *Priscilla megaterium* and *Bacillus laterosporus* further enhanced the overall efficiency.

[0068] Comparative Examples 4-6 were replaced with different strain numbers. In Comparative Example 4, CGMCC1.16094 replaced CGMCC1.10466; in Comparative Example 5, BNCC388948 replaced CGMCC1.2827; and in Comparative Example 6, CGMCC1.10257 replaced CGMCC28445. The ammonia nitrogen utilization rates of the three strains were 78.9%, 76.4%, and 74.1%, respectively; the soybean urease inhibition rates were 52.3%, 50.7%, and 48.9%, respectively; and the soil urease inhibition rates were 81.5%, 79.8%, and 77.6%, respectively. Although the three-strain complex structure was maintained, all indicators after strain replacement were lower than in Example 1, indicating that the specific strain combination selected in this invention is irreplaceable, and that similar strains from different sources have significant differences in metabolic characteristics, enzyme activity expression, and interaction effects.

[0069] As shown in Table 2, this invention constructed a highly efficient synergistic system of three bacteria by screening specific numbered *Priscilla gigantea* CGMCC1.10466, *Bacillus laterosporus* CGMCC1.2827, and *Bacillus licheniformis* CGMCC28445. This system exhibited a significant synergistic effect in ammonia nitrogen degradation and urease inhibition, laying a foundation for the preparation of subsequent feed additives.

[0070] Aquaculture experiment: The experiment was conducted at a large-scale chicken farm in Yinan County, Linyi City, Shandong Province. One-day-old broiler chickens were used as the experimental subjects. The experiment consisted of 11 groups, with three replicates per group and 40 chickens per replicate.

[0071] One group served as the control group, fed with a basal diet; the remaining groups were fed with the supplemental diet and comparative additives.

[0072] Control group: fed a basal diet (without any microbial additives); Example 1 group: basal diet + compound microbial feed additive prepared in Example 1; Example 2 group: basal diet + compound microbial feed additive prepared in Example 2; Comparative Example 1: Basal diet + additive prepared in Comparative Example 1 (lacking Priestella megaterium). Comparative Example 2: Basal diet + additive prepared in Comparative Example 2 (Bacillus brevicaulis omitting Lateral spores). Comparative Example 3: Basal diet + additive (Bacillus licheniformis) prepared in Comparative Example 3. Comparative Example 4: basal diet + additive prepared in Comparative Example 4 (Priscilla megaterium replaced with CGMCC1.16094). Comparative Example 5: Basal diet + additive prepared in Comparative Example 5 (Bacillus laterosporus was replaced with BNCC388948). Comparative Example 6: basal diet + additive prepared in Comparative Example 6 (Bacillus licheniformis replaced with CGMCC1.10257). Comparative Example 7: basal diet + additive prepared in Comparative Example 7 (carrier without glucomannan). Comparative Example 8: Basal diet + additive prepared in Comparative Example 8 (no xylooligosaccharides in the carrier).

[0073] The antibiotic-free basal diet was formulated according to the "Chicken Feeding Standard" (NY / T 33). The formalization period was 42 days. During the trial, the chickens were fed and vaccinated normally, and had free access to feed and water. The additives were added in stages according to this invention: 0.3% of the diet weight for chickens aged 1-7 days, and 0.2% of the diet weight for chickens aged 8-42 days (evenly mixed into the diet).

[0074] The nutritional components and nutritional levels of the diet are shown in Table 3.

[0075] Table 3 Composition and nutrient levels of broiler diets Note: The premix provides 13000 IU of vitamin A, 1000 IU of vitamin D3, 30 IU of vitamin E, 3.00 mg of vitamin K3, 1000 mg of vitamin B2, 5.00 mg of vitamin B6, and 1000 mg of vitamin B1 per kg of diet. 12 0.03 mg, calcium pantothenate 30 mg, niacin 70 mg, folic acid 1 mg, biotin 0.5 mg, Fe 100 mg, Cu 10 mg, Zn 80 mg, Mn 100 mg, I 0.3 mg, Se 0.1 mg, choline chloride 2000 mg.

[0076] Detection indicators and methods: Growth performance On day 42 of the experiment, the broilers were fasted for 8 hours, and their feed intake and body weight were recorded for each replicate group. The average daily gain (ADG), average daily feed intake (ADFI), and feed / gain (F / G) ratio of the 42-day-old broilers in each replicate group were calculated.

[0077] ADG (g) = Weight gain per group during the trial period ( / number of trial days × number of chickens per group); ADFI(g) = Feed intake per group during the trial period ( / number of trial days × number of chickens per group); F / G = Feed consumption / Weight gain.

[0078] ammonia concentration At 21 and 42 days of age, the ammonia concentration (unit: mg / m³) was measured using a portable ammonia detector at a height of 30 cm above the ground in each experimental group of chicken houses.

[0079] Gut health indicators On day 42 of the experiment, five chickens were randomly selected from each replicate and slaughtered. The middle section of the jejunum was taken, paraffin sections were prepared, and the villus height (VH) and crypt depth (CD) were measured. The villus height / crypt depth ratio (VH / CD) was calculated.

[0080] Histological examination: Jejunal tissue fixed in 4% paraformaldehyde solution was sent to Wuhan Saiwei Biotechnology Co., Ltd. for routine paraffin embedding and sectioning. After dewaxing and hydration, the sections were stained with hematoxylin-eosin (HE) and alcian blue-periodica acid schiff (AB-PAS). The stained sections were scanned and photographed, and observed using Case Viewer (V2.43). Data were analyzed using SPSS 26.0 and are expressed as mean ± standard error. Differences between groups were analyzed using one-way ANOVA and Duncan's multiple comparisons, with P < 0.05 considered statistically significant. Different letters in the same column indicate statistically significant differences.

[0081] Table 4 Effects of different treatments on broiler growth performance (1-42 days old) Note: Different lowercase letters in the superscript of data in the same column indicate significant differences (P<0.05), and the same applies below.

[0082] As shown in Table 4, compared with the control group, the ADG of broilers in each additive group was increased, while the F / G ratio was decreased. Among them, the Example 1 and Example 2 groups showed the best results, with ADG reaching 58.7g and 58.4g respectively, which were 12.2% and 11.7% higher than the control group (52.3g); the F / G ratios were 1.68 and 1.69 respectively, which were significantly lower than the control group (1.88).

[0083] The ADG values ​​of Comparative Examples 1-3 (which lacked *Priscilla megaterium*, *Bacillus laterosporus*, and *Bacillus licheniformis*, respectively) were 54.6 g, 54.1 g, and 53.5 g, respectively, and their F / G ratios were 1.81, 1.82, and 1.84, respectively. Their growth performance was lower than that of the Example Group, indicating that all three strains were indispensable, with the absence of *Bacillus licheniformis* (Comparative Example 3) having the greatest impact on growth performance. The ADG values ​​of Comparative Examples 4-6 (with strain replacement) were 56.2 g, 55.8 g, and 55.5 g, respectively, and their F / G ratios were 1.76, 1.77, and 1.78, respectively. Although their growth performance was better than that of Comparative Examples 1-3, it was still lower than that of the Example Group, demonstrating that the specific strain numbering selected in this invention is irreplaceable.

[0084] The ADG values ​​of comparative groups 7 and 8 (vectors lacking glucomannan or xylooligosaccharides) were 57.1 g and 56.9 g, respectively, with an F / G ratio of 1.73. Their growth performance was between that of the example group and comparative groups 1-3, indicating that glucomannan and xylooligosaccharides have a synergistic enhancing effect on the function of the strain.

[0085] Table 5. Effects of different treatments on ammonia concentration in chicken houses (mg / m³) 3 ) As shown in Table 5, all additive groups reduced the ammonia concentration in the chicken house, with the most significant effects observed in groups 1 and 2. At 21 days of age, the ammonia concentrations in groups 1 and 2 were 8.9 mg / m³ and 9.1 mg / m³, respectively, representing reductions of 41.4% and 40.1% compared to the control group (15.2 mg / m³). At 42 days of age, the concentrations were 10.5 mg / m³ and 10.7 mg / m³, respectively, representing reductions of 43.2% and 42.2% compared to the control group (18.5 mg / m³) (P<0.05).

[0086] The ammonia concentrations in Comparative Examples 1-3 at 21 days of age were 12.5, 12.8, and 13.3 mg / m³, respectively, and at 42 days of age were 15.4, 15.7, and 16.3 mg / m³, respectively, which were significantly higher than those in the Example Group (P<0.05). This indicates that the absence of any strain weakens the ammonia emission reduction effect, with the absence of Bacillus licheniformis having the greatest impact.

[0087] The ammonia concentrations in comparative examples 4-6 were slightly higher than those in the example groups, ranging from 11.2 to 11.9 mg / m³ at 21 days of age and from 13.8 to 14.6 mg / m³ at 42 days of age, indicating that the strain source affects functional expression.

[0088] The ammonia concentrations in comparative examples 7 and 8 were 10.5 and 10.7 mg / m³ at 21 days of age, and 12.9 and 13.1 mg / m³ at 42 days of age, respectively, which were also higher than those in the example group. This indicates that glucomannan and xylooligosaccharides help the strain colonize and thus enhance ammonia emission reduction.

[0089] Table 6. Effects of different treatments on gut health in broilers (42 days old) Table 6 shows that Groups 1 and 2 of Example 1 exhibited the best performance in intestinal health indicators. In Group 1, the villus height reached 1112.5 μm, the crypt depth was only 165.8 μm, and the VH / CD ratio was as high as 6.71; in Group 2, the villus height was 1105.8 μm, the crypt depth was 167.2 μm, and the VH / CD ratio was 6.61. Both groups were significantly better than the control group (P<0.05). Compared with the control group, Group 1 showed a 34.8% increase in villus height, a 26.2% decrease in crypt depth, and an 82.3% increase in the VH / CD ratio, indicating that this compound microbial feed additive can significantly improve the intestinal morphology and structure of broilers and enhance nutrient absorption. Figure 2-3 The morphological diagram of the jejunum also shows that the villi in the blank control are short and sparse, with some villi having irregular shapes / slight breaks and a disordered arrangement; the mucosa layer is thinner, the villi are flattened, and the overall structural integrity is poor. In contrast, the villi in Example 1 are long, slender, and most regularly arranged, with uniform spacing and no obvious damage. The mucosa layer is thick, the structure is intact, and the villi are upright and spread out.

[0090] The gut health indicators of Comparative Examples 1-3 (lacking a single strain) were significantly worse than those of the Example groups. Comparative Example 1 (lacking *Priscilla megaterium*) had a VH of 898.6 μm, a CD of 209.5 μm, and a VH / CD ratio of 4.29; Comparative Example 2 (lacking *Bacillus laterosporus*) had a VH of 885.3 μm, a CD of 213.6 μm, and a VH / CD ratio of 4.15; Comparative Example 3 (lacking *Bacillus licheniformis*) had the lowest VH (858.2 μm) and the highest CD (218.9 μm), with a VH / CD ratio of only 3.92, making it the worst performing group among the three-strain deletion groups. This further confirms the necessity of synergistic effects of the three strains for gut health, and that *Bacillus licheniformis* plays a crucial role in maintaining intestinal barrier function.

[0091] The gut health indicators of Comparative Examples 4-6 (strain replacement groups) were between those of the Example Group and Comparative Examples 1-3. Comparative Example 4 (replaced with *Priscilla megaterium*) had a VH of 968.5 μm and a VH / CD ratio of 4.95; Comparative Example 5 (replaced with *Bacillus laterosporus*) had a VH of 955.2 μm and a VH / CD ratio of 4.81; and Comparative Example 6 (replaced with *Bacillus licheniformis*) had a VH of 942.8 μm and a VH / CD ratio of 4.68. Although the replaced strains still exhibited some probiotic effects, they were significantly lower than the specific strain combinations screened in this invention, indicating that specific strain selection is crucial for gut health regulation.

[0092] The gut health indicators of comparative groups 7 and 8 (carrier component-deficient groups) were better than those of comparative groups 1-6, but not as good as those of the example group. The VH of comparative group 7 (without glucomannan) was 1048.6 μm, with a VH / CD ratio of 5.75; the VH of comparative group 8 (without xylooligosaccharides) was 1042.3 μm, with a VH / CD ratio of 5.66. Glucomannan, as a prebiotic, can promote the colonization of probiotics, while xylooligosaccharides can selectively stimulate the proliferation of beneficial bacteria in the gut. The synergistic effect of the two can significantly improve gut health.

[0093] Considering three indicators—growth performance, ammonia emission reduction, and gut health—the compound microbial feed additives prepared in Examples 1 and 2 both demonstrated excellent effects, with Example 1 showing a slight improvement over Example 2. The synergistic system of the three bacteria (Priscilla megaterium, Bacillus laterosporus, and Bacillus licheniformis) is irreplaceable. The selection of specific strain numbers and the addition of glucomannan and xylooligosaccharide carriers both significantly promote its functional performance. This additive achieves the dual goals of healthy broiler farming and improved farming environment through multiple mechanisms, including ammonia nitrogen degradation and improved gut microbiota.

[0094] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A compound microbial feed additive, characterized in that, By Priestella megaterium ( Priestia megaterium ), Bacillus retroflexus ( Brevibacillus laterosporus ), Bacillus licheniformis ( Bacillus licheniformis The mixture was fermented at a volume ratio of (2-4):(1-3):5, and then mixed with a carrier at a mass ratio of 1:(5-8) and dried to obtain the final product.

2. The compound microbial feed additive according to claim 1, characterized in that, The strain number of *Priestella megaterium* is CGMCC1.10466, purchased from the China General Microbiological Culture Collection Center; the strain number of *Bacillus laterosporus* is CGMCC1.2827, purchased from the China General Microbiological Culture Collection Center; and the preservation number of *Bacillus licheniformis* is CGMCC28445, deposited at the China General Microbiological Culture Collection Center on September 12, 2023.

3. The compound microbial feed additive according to claim 1, characterized in that, The carrier is a composite carrier of wheat bran, corn cob powder, rice bran, glucomannan, and xylooligosaccharide. Wheat bran, corn cob powder, and rice bran are mixed in a mass ratio of 1:2:1 as the base material. The base material is mixed with glucomannan and xylooligosaccharide in a mass ratio of 8:(0.8-1.2):(0.5-0.8).

4. A method for preparing the compound microbial feed additive according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Activation of bacterial strains: Inoculate *Priestella megaterium*, *Bacillus laterosporus*, and *Bacillus licheniformis* into sterilized LB liquid medium and culture with shaking at 25-30℃ and 150-200 rpm for 18-24 hours to obtain activated seed solutions of each strain. S2. Seed culture expansion: The activated seed culture obtained in step S1 is transferred to the seed culture medium at an inoculation rate of 5%-10% by volume. The culture is shaken and cultured for 12-16 hours at a temperature of 25-30℃ and a rotation speed of 150-200 rpm to obtain the secondary seed culture of each strain. S3. Mixed Fermentation: The secondary seed cultures of *Priscilla megaterium*, *Bacillus laterosporus*, and *Bacillus licheniformis* obtained in step S2 are mixed at a volume ratio of (2-4):(1-3):

5. This mixture is then inoculated into the fermentation medium at an inoculum volume of 5%-8% of the total volume. Fermentation is carried out at a temperature of 32-35℃, a rotation speed of 180-220 rpm, and an aeration rate of 1:0.5-1.0 vvm until the total viable cell count is ≥2.0 × 10⁻⁶. 9 CFU / mL was used to obtain a composite fermentation broth; S4. Carrier pretreatment: Mix wheat bran, corn cob powder, and rice bran evenly in a mass ratio of 1:2:1, and pulverize them through an 80-100 mesh sieve to obtain the base material; add glucomannan and xylooligosaccharide in the above base material in a mass ratio of 8:(0.8-1.2):(0.5-0.8), stir and mix thoroughly, sterilize at 105-110℃ for 30-40 minutes, and cool to room temperature to obtain the pretreated carrier; S5. Mixing and drying: The compound fermentation broth obtained in step S3 and the pretreated carrier obtained in step S4 are mixed evenly at a mass ratio of 1:(5-8), and vacuum dried at a temperature of 45-55℃ and a vacuum degree of 0.06-0.08MPa until the moisture content is ≤10%. The mixture is then pulverized through a 40-60 mesh sieve to obtain the compound microbial feed additive.

5. The preparation method of the compound microbial feed additive according to claim 4, characterized in that, In step S2, the seed culture medium consists of: glucose 15-20 g / L, peptone 8-12 g / L, yeast extract 4-6 g / L, sodium chloride 5 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.5 g / L, pH 7.0-7.5, and sterilization conditions of 121℃ for 15-20 min.

6. The method for preparing the compound microbial feed additive according to claim 4, characterized in that, The fermentation medium described in step S3 consists of: 20-30 g / L corn flour, 15-20 g / L soybean meal, 10-15 g / L wheat bran, 3-5 g / L ammonium sulfate, 3 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate, and 0.05 g / L manganese sulfate, with a pH of 7.0-7.

5. The sterilization conditions are 121℃ for 20-25 min.

7. The application of the compound microbial feed additive according to any one of claims 1-3, characterized in that, The compound microbial feed additive is added either throughout the entire feeding process or in stages. When feeding in stages, the addition amount for broilers aged 1-7 days is 0.3% of the daily feed weight, and the addition amount for broilers aged 8-42 days is 0.1%-0.2% of the daily feed weight.

8. The application of the compound microbial feed additive according to claim 7, characterized in that, The compound microbial feed additive is used to promote broiler gut health, reduce ammonia emissions in the broiler breeding environment, and improve broiler growth performance in one or more ways.

9. The application of the compound microbial feed additive according to claim 7, characterized in that, The broiler chickens are either white-feathered or yellow-feathered.

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