Compound microbial inoculant and application thereof in breeding early weaned lambs

Abstract

The application discloses a kind of compound microbial inoculant and its application in the cultivation of early weaned lamb;The compound microbial inoculant is composed of Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter ludwigii, Lactobacillus gasseri and Delftia tsuruhatensis.Experiments show that the compound microbial inoculant is evenly sprayed on the surface of starter feed, and then the early weaned lamb is fed, which can improve the survival rate of the lamb, increase the feed intake and daily weight gain, promote growth and development, reduce the diarrhea rate, protect the intestinal mucosa and reduce the inflammatory response induced by weaning stress, and help the lamb quickly establish the intestinal microecological environment after weaning.The compound microbial inoculant prepared by the application can guarantee the reserve lamb source, and has important significance for the popularization of early weaning technology in large-scale sheep farms.The application has important application value.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a compound microbial agent and its application in the breeding of early-weaned lambs. Background Technology

[0002] The rapid development of large-scale and standardized meat sheep farming has made early weaning a crucial measure for achieving high-frequency reproduction and large-scale breeding. Lamb rearing quality is a key factor determining the efficiency of modern meat sheep production, and early weaning has become a common practice in many countries. Years of exploration and research have been conducted on early weaning techniques for lambs, showing that feeding strategies can effectively improve ewe utilization efficiency, shorten the lamb's lactation period, and maximize its production potential. However, the stress response caused by early weaning easily leads to diseases such as diarrhea in lambs, resulting in decreased lamb production performance and survival rates. Annual lamb mortality due to weaning stress syndrome and other causes exceeds 25%, and the insufficient supply of replacement lambs severely restricts the sustainable development of the industry.

[0003] The intestinal tissue of suckling lambs is not fully developed, and the intestinal micro-ecosystem is in its early stages of establishment and has not yet reached a mature ecological balance. Furthermore, the number and function of stromal cells and immune cells are still developing. If weaning occurs during the suckling period, the lambs will lack access to maternal antibodies, and their immune system and antioxidant capacity will be insufficient, making them susceptible to damage to the intestinal mucosal barrier. Simultaneously, the drastic change in diet makes the underdeveloped intestinal micro-ecosystem highly vulnerable to external pathogens, thus exacerbating the threat to the lambs' development and survival. Summary of the Invention

[0004] The purpose of this invention is to reduce the mortality and diarrhea rates of early-weaned lambs and promote their growth and development.

[0005] This invention first protects a compound microbial agent, which may include Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter ludwig's enterobacter, Lactobacillus graminearum, and Delftobacterium tumefaciens.

[0006] The compound microbial agent may specifically consist of Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter ludwig's brevis, Lactobacillus geranioli, and Delftobacterium tumefaciens.

[0007] The compound microbial agent can be used to breed early-weaned lambs.

[0008] In any of the above-mentioned compound bacterial agents, the *Pediococcus pentosaceus* may be *Pediococcus pentosaceus* (…). Pediococcus pentosaceus M3; Pediococcus pentosaceus ( Pediococcus pentosaceus M3, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 30882.

[0009] In any of the above-mentioned compound bacterial agents, the Bacillus amyloliquefaciens may be Bacillus amyloliquefaciens (B. amyloliquefaciens). Bacillus amyloliquefaciens CK4; Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens CK4, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 31178.

[0010] In any of the above-mentioned compound bacterial agents, the Ludwig Culex can be Ludwig Culex (… Enterobacter ludwigii CK6; Ludwig's Enterobacter ( Enterobacter ludwigii CK6, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 31179.

[0011] In any of the above-mentioned compound microbial agents, the viable counts of Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter ludwig's bacterium, Lactobacillus geranioli, and Delfordia chebula are all 2 × 10⁻⁶. 11 CFU / mL or higher.

[0012] In any of the aforementioned compound microbial agents, the viable count ratio of Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter ludwig's bacterium, Lactobacillus graminearum, and Delftobacterium tumefaciens can be 4.40 × 10⁻⁶. 14 CFU / mL: (5.00-10.00) × 10 12 CFU / mL: (5.00-10.00) × 10 13 CFU / mL: (1.00-5.00) × 10 12 CFU / mL: (5.00-10.00) × 10 13 CFU / mL (e.g., 4.40 × 10⁻⁶) 14 CFU / mL: (5.00-8.80) × 10 12 CFU / mL: (5.00-5.90) ​​× 10 13 CFU / mL: (1.00-2.52)×10 12 CFU / mL: (5.00-5.10) × 10 13 CFU / mL or 4.40×10 14 CFU / mL: (8.80-10.00) × 10 12 CFU / mL: (5.90-10.00) × 10 13 CFU / mL: (2.52-5.00) × 10 12 CFU / mL: (5.10-10.00) × 10 13 (CFU / mL).

[0013] In any of the aforementioned compound microbial agents, the specific ratio of viable bacteria of *Pediococcus pentosaceus*, *Bacillus amyloliquefaciens*, *Enterobacter ludwig's bacterium*, *Lactobacillus geranioli*, and *Delfordia chebula* can be 4.40 × 10⁻⁶. 14 CFU / mL: 8.80×10 12 CFU / mL: 5.90×10 13 CFU / mL: 2.52×10 12 CFU / mL: 5.10×10 13 CFU / mL.

[0014] When preparing any of the above-mentioned compound bacterial agents, Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter ludwig's bacillus, Lactobacillus graminearum, and Delftobacterium tumefaciens can all be added in the form of bacterial suspension.

[0015] The bacterial culture can be prepared by inoculating *Pediococcus pentosaceus*, *Bacillus amyloliquefaciens*, *Enterobacter ludwig vannamei*, *Lactobacillus graminearum*, or *Delfordia chebula* into a culture medium, followed by fermentation to obtain the bacterial culture. Specifically, the culture medium can be a regular broth medium.

[0016] The preparation method of any of the above-mentioned compound microbial agents may be to mix the bacterial suspensions of Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter ludwig's bacterium, Lactobacillus graminearum, and Delfordia chebula, and then add an appropriate amount of water.

[0017] This invention also protects the application of any of the above-described compound microbial agents, which may be at least one of A1)-A14): A1) Improve the survival rate of weaned lambs; A2) Prepare products to improve the survival rate of weaned lambs; A3) Reduce the rate of diarrhea and / or mortality in weaned lambs; A4) Prepare products for reducing the rate of diarrhea and / or mortality in weaned lambs; A5) Increase feed intake and / or daily feed intake of weaned lambs; A6) Prepare products for increasing feed intake and / or daily feed intake in weaned lambs; A7) Increase the weight and / or daily weight gain of weaned lambs; A8) Prepare products for increasing the weight and / or daily weight gain of weaned lambs; A9) Promotes the growth and development of weaned lambs; A10) Prepare products to promote the growth and development of weaned lambs; A11) Reduces the inflammatory response in weaned lambs and / or alleviates intestinal inflammation in weaned lambs; A12) Prepare products for reducing inflammatory response in weaned lambs and / or alleviating intestinal inflammation in weaned lambs; A13) Breeding weaned lambs; (A14) Prepare products for breeding weaned lambs.

[0018] In the above applications, the weaned lamb can be an early-weaned lamb. The early-weaned lamb is preferably a lamb aged 28 to 35 days (e.g., 28 to 30 days, 31 to 33 days, 34 to 35 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, or 35 days).

[0019] In any of the above-described applications, the method of using any of the above-described compound probiotic agents is as follows: The compound probiotic agent is evenly sprayed onto the surface of the starter feed, and then fed to early-weaned lambs. The preferred feeding frequency for the compound probiotic preparation is once per day. The ratio of the compound probiotic agent to the starter feed can be 3-5 mL:1 kg (e.g., 3-4 mL:1 kg, 4-5 mL:1 kg, 3 mL:1 kg, 4 mL:1 kg, or 5 mL:1 kg). Each lamb is fed approximately 500-1000 g of starter feed per day.

[0020] In the above applications, the lamb can be a sheep lamb or a goat lamb. Specifically, the sheep can be a meat sheep. The meat sheep can be a Hu sheep. The goat can be a Boer goat.

[0021] The inventors of this application discovered through extensive experiments that early weaning can induce iron accumulation in the intestinal mucosal epithelial cells and macrophages of the lamina propria of lambs, leading to ferroptosis, confirming it as a significant cause of intestinal mucosal damage in early-weaned lambs. Early weaning can significantly lead to the death and shedding of jejunal mucosal epithelial cells in lambs. Furthermore, early weaning can cause a significant decrease in the number of lactobacilli in the jejunum of lambs aged 35-42 days, and a significant reduction in lipid metabolism-related products. At this time, the physical barrier of the intestinal mucosa of early-weaned lambs is severely damaged, resulting in reduced nutrient absorption capacity, manifested as diarrhea, stunted growth, and even death. Microecological preparations have the effect of adjusting intestinal flora imbalance and improving the intestinal microecological environment. Feeding early-weaned lambs with the compound microbial agent prepared in this application, when properly combined with starter feed, can improve lamb survival rate (reduce mortality), increase feed intake and daily weight gain, promote growth and development, improve diarrhea symptoms (reduce diarrhea rate), protect the intestinal mucosa, reduce inflammatory responses induced by weaning stress, and help lambs quickly establish a post-weaning intestinal microecological environment. The compound microbial agent prepared in this application can ensure the breeding stock of replacement lambs and is of great significance for the promotion of early weaning technology in large-scale sheep farms. This invention has significant application value.

[0022] Deposit Description Strain name: Pediococcus pentosaceus Latin name: Pediococcus pentosaceus Strain number: M3 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: June 7, 2024 CGMCC Registration Number: CGMCC No. 30882 Bacterial strain name: Bacillus amyloliquefaciens Latin name: Bacillus amyloliquefaciens Strain number: CK4 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: July 5, 2024 CGMCC Registration Number: CGMCC No. 31178 Bacterial strain name: Ludwig's Enterobacter Latin name: Enterobacter ludwigii Strain number: CK6 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: July 5, 2024 CGMCC Registration Number: CGMCC No. 31179 Attached Figure Description Figure 1 This represents the growth status of bacterial colonies of M3.

[0023] Figure 2 This shows the growth status of bacterial colonies of CK4.

[0024] Figure 3 This shows the growth status of bacterial colonies of CK6.

[0025] Figure 4 The change in diarrhea rate of early-weaned lambs after feeding with the bacterial agent in Example 3.

[0026] Figure 5 The change in daily feed intake of early-weaned lambs after feeding with the microbial agent in Example 3 is shown.

[0027] Figure 6The change in body weight of early-weaned lambs after feeding with the microbial agent in Example 3. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0031] The starter feed in the following examples is a mixture of commercially available corn, whey powder, soybean meal, alfalfa, and additive premix. In this application, the corn, soybean meal, and alfalfa were purchased from Linqing Runlin Animal Husbandry Co., Ltd.; the whey powder was purchased from Beijing Precision Animal Nutrition Research Center Co., Ltd.; and the additive premix, containing iron, manganese, copper, zinc, selenium, iodine, cobalt, vitamin A, vitamin D, and vitamin E, was provided by Beijing Precision Animal Nutrition Research Center Co., Ltd.

[0032] The culture media involved in the following examples are as follows: The solutes and their concentrations in a standard nutrient agar medium are: agar 15 g / L, peptone 10 g / L, beef extract 3 g / L, and sodium chloride 5 g / L. The solvent is distilled water, and the pH value is 7.2 ± 0.2.

[0033] The solutes and their concentrations in the ordinary broth culture medium were 20 g / L peptone, 5 g / L beef extract powder, and 5 g / L sodium chloride, with distilled water as the solvent and a pH of 7.2 ± 0.2.

[0034] The solutes and their concentrations in the YPDA medium were 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 0.03 g / L adenine sulfate, with distilled water as the solvent and a pH of 6.0-6.5.

[0035] Example 1: Obtaining Pediococcus pentosaceus M3, Bacillus amyloliquefaciens CK4, and Enterobacter ludwig's CK6 I. Pediococcus pentosaceus ( Pediococcus pentosaceus Isolation, identification and preservation of M3 CGMCC No. 30882 (a) Separation 1. Add 1g of homemade fermented dough (from a farmer in Anqiu City, Weifang City, Shandong Province) and an appropriate amount of glass beads to 9mL of sterile physiological saline, shake for 10min, let stand for 30s, and make the dough stock solution (the dilution at this time is recorded as 10). -1 Add 1 mL of the supernatant from the dough stock solution to a test tube containing 9 mL of sterile physiological saline and mix thoroughly (the dilution is now 10). -2 Then, take 1 mL from this test tube and add it to another test tube containing 9 mL of sterile saline solution, mix well, and so on to prepare a diluted solution (dilution factor of 10). -4 ).

[0036] 2. Using a pipette, take 100µL of the diluted solution at different concentrations and spread it evenly on ordinary nutrient agar medium. Incubate at 28℃ for 5-7 days and observe the growth status of the colonies.

[0037] 3. After completing step 2, inoculate each colony into ordinary broth medium and culture them to obtain the corresponding bacterial solution.

[0038] 4. Take 10µL of the above bacterial solution and add 900µL of sterile physiological saline. Inoculate 100µL of the bacterial solution onto ordinary nutrient agar medium and incubate at 37℃ for 24 h.

[0039] 5. After completing step 4, use a sterile inoculation loop to pick up a single colony and inoculate it into ordinary broth medium, then culture it to obtain the corresponding bacterial solution.

[0040] One of the selected bacteria was named Bacterium M3.

[0041] The growth status of bacterial colonies of M3 is shown in [the image]. Figure 1 .

[0042] (ii) Molecular identification Genomic DNA was extracted from bacteria M3 and used as a template. PCR amplification was performed using primer pair 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and primer 1492R: 5'-TACGACTTAACCCCAATCGC-3' to obtain 16S rDNA.

[0043] The nucleotide sequence of the 16S rDNA of bacterial M3 is shown in SEQ ID NO:1.

[0044] The sequence shown in SEQ ID NO:1 was BLASTed in the NCBI Standard Nucleotide database to finally determine the genus and species of bacteria M3.

[0045] The results showed that bacteria M3 and Pediococcus pentosaceus ( Pediococcus pentosaceusIt showed the highest homology with *Pediococcus pentosaceus*. Therefore, bacterial M3 was identified as *Pediococcus pentosaceus*. Pediococcus pentosaceus ).

[0046] (III) Preservation The bacterium M3 isolated in step one was deposited on June 7, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 30882. The full name of bacterium M3 is *Pediococcus pentosaceus*. Pediococcus pentosaceus M3 CGMCCNo.30882, abbreviated as Pediococcus pentosaceus M3.

[0047] II. Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens Separation, identification and preservation of CK4 CGMCC No. 31178 (a) Separation 1. Add 1g of homemade fermented dough (from a farmer in Ju County, Rizhao City, Shandong Province) and an appropriate amount of glass beads to 9mL of sterile physiological saline, shake for 10min, let stand for 30s, and prepare the dough stock solution (the dilution at this time is recorded as 10). -1 Add 1 mL of the supernatant from the dough stock solution to a test tube containing 9 mL of sterile physiological saline and mix thoroughly (the dilution is now 10). -2 Then, take 1 mL from this test tube and add it to another test tube containing 9 mL of sterile saline solution, mix well, and so on to prepare a diluted solution (dilution factor of 10). -4 ).

[0048] 2. Using a pipette, take 100µL of the diluted solution at different concentrations and spread it evenly on ordinary nutrient agar medium. Incubate at 28℃ for 5-7 days and observe the growth status of the colonies.

[0049] 3. After completing step 2, inoculate each colony into ordinary broth medium and culture them to obtain the corresponding bacterial solution.

[0050] 4. Take 10µL of the above bacterial solution and add 900µL of sterile physiological saline. Inoculate 100µL of the bacterial solution onto ordinary nutrient agar medium and incubate at 37℃ for 24 h.

[0051] 5. After completing step 4, use a sterile inoculation loop to pick up a single colony and inoculate it into ordinary broth medium, then culture it to obtain the corresponding bacterial solution.

[0052] One of the selected bacteria was named Bacterium CK4.

[0053] The growth status of bacterial colonies of CK4 is shown in [the image]. Figure 2 .

[0054] (ii) Molecular identification Genomic DNA was extracted from bacteria CK4 and used as a template. PCR amplification was performed using primer pair 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and primer 1492R: 5'-TACGACTTAACCCCAATCGC-3' to obtain 16S rDNA.

[0055] The nucleotide sequence of the 16S rDNA of bacteria CK4 is shown in SEQ ID NO:2.

[0056] The sequence shown in SEQ ID NO:2 was BLASTed in the NCBI Standard Nucleotide database to finally determine the genus and species of bacteria CK4.

[0057] The results showed that bacteria CK4 and Bacillus amyloliquefaciens (Bacillus) Bacillus amyloliquefaciens The highest homology was found with *Bacillus amyloliquefaciens*. Therefore, bacterium CK4 was identified as *Bacillus amyloliquefaciens*. Bacillus amyloliquefaciens ).

[0058] (III) Preservation The bacterium CK4 isolated in step one was deposited on July 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 31178. The full name of bacterium CK4 is *Bacillus amyloliquefaciens* (…). Bacillus amyloliquefaciens CK4CGMCC No.31178, abbreviated as Bacillus amyloliquefaciens CK4.

[0059] III. Ludwig's Colombi ( Enterobacter ludwigii Separation, identification and preservation of CK6 CGMCC No. 31179 (a) Separation 1. Add 1g of herbivorous insect excrement (from Fangzi District, Weifang City, Shandong Province) to 9mL of sterile physiological saline, shake for 10min, let stand for 30s, and prepare a fecal suspension (the dilution at this time is recorded as 10). -1 Centrifuge at 2000 rpm for 5 minutes. Add 1 mL of the supernatant from the fecal suspension to a test tube containing 9 mL of sterile saline and mix thoroughly (the dilution is now 10). -2 Then, take 1 mL from this test tube and add it to another test tube containing 9 mL of sterile saline solution, mix well, and so on to prepare a diluted solution (dilution factor of 10). -4 ).

[0060] 2. Using a pipette, take 100µL of the diluted solution at different concentrations and spread it evenly on ordinary nutrient agar medium. Incubate at 28℃ for 5-7 days and observe the growth status of the colonies.

[0061] 3. After completing step 2, inoculate each colony into ordinary broth medium and culture them to obtain the corresponding bacterial solution.

[0062] 4. Take 10µL of the above bacterial solution and add 900µL of sterile physiological saline. Inoculate 100µL of the bacterial solution onto ordinary nutrient agar medium and incubate at 37℃ for 24 h.

[0063] 5. After completing step 4, use a sterile inoculation loop to pick up a single colony and inoculate it into ordinary broth medium, then culture it to obtain the corresponding bacterial solution.

[0064] One of the selected bacteria was named Bacterium CK6.

[0065] The growth status of bacterial colonies of CK6 is shown in [the image / document]. Figure 3 .

[0066] (ii) Molecular identification Genomic DNA was extracted from bacteria CK6 and used as a template. PCR amplification was performed using primer pair 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and primer 1492R: 5'-TACGACTTAACCCCAATCGC-3' to obtain 16S rDNA.

[0067] The nucleotide sequence of the 16S rDNA of bacteria CK6 is shown in SEQ ID NO:3.

[0068] The sequence shown in SEQ ID NO:3 was BLASTed in the NCBI Standard Nucleotide database to finally determine the genus and species of bacteria CK6.

[0069] The results showed that bacteria CK6 and Ludwig's Enterobacter ( Enterobacter ludwigii It showed the highest homology with *Enterobacter ludwigii*. Therefore, bacteria CK6 was identified as *Enterobacter ludwigii*. Enterobacter ludwigii ).

[0070] (III) Preservation The bacterium CK6 isolated in step one was deposited on July 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 31179. The full name of bacterium CK6 is Ludwig's Enterobacter (…). Enterobacter ludwigiiCK6 CGMCC No. 31179, abbreviated as Ludwig's Enterobacter CK6.

[0071] Example 2: Preparation of the microbial agent I. Preparation of Compound Microbial Agents (also known as Compound Probiotic Agents) 1. Preparation of Pediococcus pentosaceus M3 inoculum (1) Inoculate a single colony of Pediococcus pentosacchari M3 into 50 mL of ordinary broth medium and incubate at 33-37℃ and 50-100 rpm for 18-24 h to obtain primary seed culture of Pediococcus pentosacchari M3; (2) Inoculate the primary seed culture of Pediococcus pentosacchari M3 into 50 mL of ordinary broth medium (for secondary activation), and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the secondary seed culture of Pediococcus pentosacchari M3; (3) Inoculate the secondary seed culture of Pediococcus pentosaceus M3 into 100 mL of ordinary broth medium and ferment at 33–37 °C and 50–100 rpm for 18–24 h to obtain the fermentation broth of Pediococcus pentosaceus M3. During fermentation, the aeration rate is 0.3–1 L / min.

[0072] The viable cell concentration of *Pediococcus pentosaceus* M3 in the fermentation broth is ≥2×10⁻⁶. 11 CFU / mL.

[0073] The fermentation broth of Pediococcus pentosacchari M3 is the Pediococcus pentosacchari M3 inoculum.

[0074] 2. Preparation of Bacillus amyloliquefaciens CK4 inoculum (1) Inoculate a single colony of Bacillus amyloliquefaciens CK4 into 50 mL of ordinary broth medium and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the primary seed culture of Bacillus amyloliquefaciens CK4; (2) Inoculate the primary seed culture of Bacillus amyloliquefaciens CK4 into 50 mL of ordinary broth medium (for secondary activation), and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the secondary seed culture of Bacillus amyloliquefaciens CK4; (3) Inoculate the secondary seed culture of Bacillus amyloliquefaciens CK4 into 100 mL of ordinary broth medium and ferment at 33–37 °C and 50–100 rpm for 18–24 h to obtain the fermentation broth of Bacillus amyloliquefaciens CK4. During fermentation, the aeration rate is 0.3–1 L / min.

[0075] The viable count concentration of Bacillus amyloliquefaciens CK4 in the fermentation broth was ≥2×10⁻⁶. 11 CFU / mL.

[0076] The fermentation broth of Bacillus amyloliquefaciens CK4 is the Bacillus amyloliquefaciens CK4 inoculum.

[0077] 3. Preparation of Ludwig's Enterobacter CK6 bacterial agent (1) Inoculate a single colony of Ludwig's Enterobacter CK6 into 50 mL of ordinary broth medium and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the primary seed culture of Ludwig's Enterobacter CK6; (2) Inoculate the primary seed culture of Ludwig's Enterobacter CK6 into 50 mL of ordinary broth medium (for secondary activation), and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the secondary seed culture of Ludwig's Enterobacter CK6. (3) Inoculate the secondary seed culture of Ludwig's Enterobacter CK6 into 100 mL of ordinary broth medium and ferment at 33-37℃ and 50-100 rpm for 18-24 h to obtain Ludwig's Enterobacter CK6 fermentation broth. During fermentation, the aeration rate is 0.3-1 L / min.

[0078] The viable count concentration of Ludwig's Enterobacter CK6 in the fermentation broth is ≥2×10⁻⁶. 11 CFU / mL.

[0079] The fermentation broth of Enterobacter ludwig's CK6 is the inoculum of Enterobacter ludwig's CK6.

[0080] 4. Preparation of Lactobacillus gravidarum inoculum Lactobacillus genomica is a product of Beina Chuanglian Biotechnology Co., Ltd., with product catalog number BNCC339385.

[0081] (1) Inoculate a single colony of Lactobacillus gracilistylus into 50 mL of ordinary broth medium and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the primary seed culture of Lactobacillus gracilistylus; (2) Inoculate the primary seed culture of Lactobacillus geraniol into 50 mL of ordinary broth medium (for secondary activation), and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the secondary seed culture of Lactobacillus geraniol; (3) Inoculate the secondary seed culture of Lactobacillus gravidarum into 100 mL of ordinary broth medium and ferment at 33-37℃ and 50-100 rpm for 18-24 h to obtain Lactobacillus gravidarum fermentation broth. During fermentation, the aeration rate is 0.3-1 L / min.

[0082] The viable count concentration of Lactobacillus geraniol in the fermentation broth is ≥2×10⁻⁶. 11 CFU / mL.

[0083] Lactobacillus fermentation broth is Lactobacillus inoculum.

[0084] 5. Preparation of *Delfordia ulmoides* inoculant The specific product of Chisheng Delftella is Unico Life Sciences Co., Ltd., with product catalog number YLK-jz3731.

[0085] (1) Inoculate a single colony of Delfordia sericulture into 50 mL of ordinary broth medium and incubate at 33-37℃ and 50-100 rpm for 18-24 h to obtain the primary seed culture of Delfordia sericulture. (2) Inoculate the primary seed culture of Delfordia chebula into 50 mL of ordinary broth medium (for secondary activation), and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the secondary seed culture of Delfordia chebula. (3) Inoculate the secondary seed culture of *Delfordia chebula* into 100 mL of ordinary broth medium and ferment at 33–37 °C and 50–100 rpm for 18–24 h to obtain *Delfordia chebula* fermentation broth. During fermentation, the aeration rate is 0.3–1 L / min.

[0086] The viable cell concentration of *Dalfordia chebula* in the fermentation broth was ≥2×10⁻⁶. 11 CFU / mL.

[0087] The fermentation broth of *Delfordia micrantha* is the inoculum of *Delfordia micrantha*.

[0088] 6. Preparation of compound microbial agents A compound bacterial agent was prepared by mixing *Pediococcus pentosaceus* M3 inoculum, *Bacillus amyloliquefaciens* CK4 inoculum, *Enterobacter ludwig vannamei* CK6 inoculum, *Lactobacillus geranioli* inoculum, and *Delfordia chebula* inoculum. The volume ratio of these compounds in the compound bacterial agent was 1:1:1:1:1.

[0089] II. Comparison of the preparation of probiotic agent 1 In contrast, probiotic 1 is Bacillus licheniformis, specifically a product of Beina Chuanglian Biotechnology Co., Ltd., with product catalog number BNCC132665.

[0090] (1) Inoculate a single colony of Bacillus licheniformis into 50 mL of ordinary broth medium and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the primary seed culture of Bacillus licheniformis; (2) Inoculate the primary seed culture of Bacillus licheniformis into 50 mL of ordinary broth medium (for secondary activation), and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the secondary seed culture of Bacillus licheniformis; (3) Inoculate the secondary seed culture of Bacillus licheniformis into 100 mL of ordinary broth medium and ferment at 33-37℃ and 50-100 rpm for 18-24 h to obtain Bacillus licheniformis fermentation broth. During fermentation, the aeration rate is 0.3-1 L / min.

[0091] The viable count concentration of Bacillus licheniformis in the fermentation broth is ≥2×10⁻⁶. 11 CFU / mL.

[0092] Bacillus licheniformis fermentation broth is Bacillus licheniformis inoculum, which is the same as the comparative probiotic inoculum 1.

[0093] III. Comparison of the preparation of probiotic 2 agents In contrast, Probiotic 2 is Pichia pastoris, specifically a product of Beina Chuanglian Biotechnology Co., Ltd., with product catalog number BNCC380011.

[0094] (1) Inoculate a single colony of Pichia pastoris into 50 mL of YPDA medium and culture at 33-37 °C and 50-100 rpm for 18-24 h to obtain the primary seed culture of Pichia pastoris; (2) Inoculate the primary seed culture of Pichia pastoris into 50 mL of YPDA medium (for secondary activation), and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the secondary seed culture of Pichia pastoris. (3) Inoculate the secondary seed culture of Pichia pastoris into 100 mL of YPDA medium and ferment at 33–37 °C and 50–100 rpm for 18–24 h to obtain the Pichia pastoris fermentation broth. During fermentation, the aeration rate is 0.3–1 L / min.

[0095] The viable cell count concentration of Pichia pastoris in the fermentation broth is ≥2×10⁻⁶. 11 CFU / mL.

[0096] Pichia pastoris fermentation broth is Pichia pastoris inoculum, which is also known as comparative probiotic inoculum.

[0097] IV. Comparison of the preparation of probiotic preparations 3 In contrast, probiotic 3 is a compound probiotic agent, and its specific preparation method is as follows: 1. Preparation of Lactobacillus amyloliquefaciens inoculum Lactobacillus amyloliquefaciens is a product of Beina Chuanglian Biotechnology Co., Ltd., with product catalog number BNCC132483.

[0098] (1) Inoculate a single colony of Lactobacillus amyloliquefaciens into 50 mL of ordinary broth medium and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the primary seed culture of Lactobacillus amyloliquefaciens; (2) Inoculate the primary seed culture of Lactobacillus amyloliquefaciens into 50 mL of ordinary broth medium (for secondary activation), and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the secondary seed culture of Lactobacillus amyloliquefaciens; (3) Inoculate the secondary seed culture of Lactobacillus amyloliquefaciens into 50 mL of ordinary broth medium and ferment at 33–37 °C and 50–100 rpm for 18–24 h to obtain Lactobacillus amyloliquefaciens fermentation broth. During fermentation, the aeration rate is 0.3–1 L / min.

[0099] The viable count concentration of Lactobacillus amyloliquefaciens in the fermentation broth is ≥2×10⁻⁶. 11 CFU / mL.

[0100] The fermentation broth of Lactobacillus amyloliquefaciens is the Lactobacillus amyloliquefaciens inoculum.

[0101] 2. Preparation of Lactobacillus reuteri inoculum Lactobacillus reuteri is a product of Beina Chuanglian Biotechnology Co., Ltd., with product catalog number BNCC337178.

[0102] (1) Inoculate a single colony of Lactobacillus reuteri into 50 mL of ordinary broth medium and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the primary seed culture of Lactobacillus reuteri; (2) Inoculate the primary seed culture of Lactobacillus reuteri into 50 mL of ordinary broth medium (for secondary activation), and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the secondary seed culture of Lactobacillus reuteri; (3) Inoculate the secondary seed culture of Lactobacillus reuteri into 100 mL of ordinary broth medium and ferment at 33–37 °C and 50–100 rpm for 18–24 h to obtain Lactobacillus reuteri fermentation broth. During fermentation, the aeration rate is 0.3–1 L / min.

[0103] The viable count concentration of Lactobacillus reuteri in the fermentation broth is ≥2×10⁻⁶. 11 CFU / mL.

[0104] Lactobacillus reuteri fermentation broth is Lactobacillus reuteri inoculum.

[0105] 3. Preparation of Lactobacillus johnsonii inoculum Lactobacillus johnsonii is a product of Beina Chuanglian Biotechnology Co., Ltd., with product catalog number BNCC186110.

[0106] (1) Inoculate a single colony of Lactobacillus johnsonii into 50 mL of ordinary broth medium and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the primary seed culture of Lactobacillus johnsonii; (2) Inoculate the primary seed culture of Lactobacillus johnsonii into 50 mL of ordinary broth medium (for secondary activation), and culture at 33-37℃ and 50-100 rpm for 18-24 h to obtain the secondary seed culture of Lactobacillus johnsonii. (3) Inoculate the secondary seed culture of Lactobacillus johnsonii into 100 mL of ordinary broth medium and ferment at 33–37 °C and 50–100 rpm for 18–24 h to obtain Lactobacillus johnsonii fermentation broth. During fermentation, the aeration rate is 0.3–1 L / min.

[0107] The viable count concentration of Lactobacillus johnsonii in the fermentation broth is ≥2×10⁻⁶. 11 CFU / mL.

[0108] Lactobacillus johnsonii fermentation broth is Lactobacillus johnsonii inoculum.

[0109] 4. Comparison of the preparation of probiotic preparations 3 The Lactobacillus amyloliquefaciens, Lactobacillus reuteri, and Lactobacillus johnsonii were mixed to obtain the comparative probiotic 3-strain preparation; the volume ratio of Lactobacillus amyloliquefaciens, Lactobacillus reuteri, and Lactobacillus johnsonii in the comparative probiotic 3-strain preparation was 1:1:1.

[0110] Application of the compound probiotic preparations prepared in Example 3 and Example 2 in the early weaning of lambs In the compound probiotic agent of this invention, the viable count concentration of Pediococcus pentosaceus M3 is specifically 4.40 × 10⁻⁶. 14 The specific viable count concentration of Bacillus amyloliquefaciens CK4 was 8.80 × 10⁻⁶ CFU / mL. 12 The specific viable count concentration of Ludwig's Enterobacter CK6 was 5.90 × 10⁻⁶ CFU / mL. 13 The specific viable count concentration of *Lactobacillus graminearum* was 2.52 × 10⁻⁶ CFU / mL. 12 The specific viable cell count concentration of *Delfordia gravidarum* was 5.10 × 10⁻⁶ CFU / mL. 13 CFU / mL.

[0111] In the comparative probiotic preparation of this invention, the viable count concentration of Bacillus licheniformis is specifically 6.60 × 10⁻⁶. 14 CFU / mL.

[0112] In the comparative probiotic preparation of this invention, the viable count concentration of Pichia pastoris is specifically 2.10 × 10⁻⁶. 12CFU / mL.

[0113] In the comparative probiotic preparation of this invention, the specific live bacteria concentration of *Lactobacillus amyloliquefaciens* is 3.40 × 10⁻³. 12 The specific viable count concentration of Lactobacillus reuteri was 4.60 × 10⁻⁶ CFU / mL. 14 The specific viable count concentration of *Lactobacillus johnsonii* was 8.30 × 10⁻⁶ CFU / mL. 14 CFU / mL.

[0114] Experimental location: A sheep farm in Shijiazhuang, Hebei Province. The experimental site was a semi-open sheepfold with good lighting and ventilation.

[0115] Experimental subjects: Hu sheep, a species of sheep. All sheep underwent testing for foot-and-mouth disease, Brucella, anthrax, and peste des petits ruminants (PPR) to confirm their health. All lambs were nursed by their mothers in the same pen after birth and were given supplemental feed starting at 7 days of age. All experimental lambs were ear-tagged, and their immunization program was implemented according to the standard procedures of the sheep farm.

[0116] Experimental Procedure: Hu sheep lambs at 28 days of weaning were randomly selected and then randomly divided into five groups: an early weaning group, a compound probiotic group, a control probiotic group 1, a control probiotic group 2, and a control probiotic group 3, with 12 lambs in each group (half male and half female). Each group of lambs was separated from their ewes at 28 days of age (lambs remained in place, ewes were removed) for a single weaning, followed by the following experiment: Early weaning group: fed starter food at 8:00 and 17:00 daily.

[0117] Compound probiotic group: Feed starter food containing compound probiotic agent at 8:00 am and feed starter food at 5:00 pm daily.

[0118] The preparation method of feed containing compound probiotic agent is as follows: add an appropriate amount of drinking water to the compound probiotic agent, mix well and spray it on the surface of the feed to obtain feed containing compound probiotic agent; in the feed containing compound probiotic agent, the ratio of compound probiotic agent to feed is 3-5 mL: 1 kg (specifically 5 mL: 1 kg in this example).

[0119] Group 1 (comparison probiotics): Feed the starter food containing the comparison probiotic 1 agent at 8:00 am daily and feed the starter food at 5:00 pm daily.

[0120] Add an appropriate amount of drinking water to the comparative probiotic 1 agent, mix well, and spray it onto the surface of the starter food to obtain starter food containing comparative probiotic 1 agent; in the starter food containing comparative probiotic 1 agent, the ratio of comparative probiotic 1 agent to starter food is 1-2 mL: 1 kg (specifically 1 mL: 1 kg in this example).

[0121] Group 2 (comparison probiotics): Feed the starter food containing the comparison probiotic 2 agent at 8:00 am daily and feed the starter food at 5:00 pm daily.

[0122] Add an appropriate amount of drinking water to the comparative probiotic 2 agent, mix well, and spray it onto the surface of the starter food to obtain starter food containing comparative probiotic 2 agent; in the starter food containing comparative probiotic 2 agent, the ratio of comparative probiotic 2 agent to starter food is 1-2 mL: 1 kg (specifically 1 mL: 1 kg in this example).

[0123] Group 3 (comparative probiotics): Feed the starter food containing the comparative probiotic 3 at 8:00 AM and feed the starter food at 5:00 PM daily.

[0124] Add an appropriate amount of drinking water to the comparative probiotic 3 agent, mix well, and spray it onto the surface of the starter food to obtain starter food containing the comparative probiotic 3 agent; in the starter food containing the comparative probiotic 3 agent, the ratio of the comparative probiotic 3 agent to the starter food is 3-4 mL: 1 kg (specifically 3 mL: 1 kg in this example).

[0125] The trial lasted 28 days. During the trial, all lambs had free access to water throughout the entire period and were fed starter feed twice daily, at 8:00 AM and 5:00 PM, until the lambs stopped actively eating. Both the compound probiotic and the control probiotic were sprayed on at the 8:00 AM starter feed feeding. Each lamb was fed approximately 500-1000g of starter feed per day.

[0126] During the experiment, lambs' feed intake and diarrhea status were observed at 9:00 AM and 6:00 PM daily. The ear tags of lambs exhibiting diarrhea were used to further calculate the diarrhea rate. The amount of initial feed added and the amount remaining were recorded daily, and changes in daily feed intake were calculated and analyzed. Additionally, lambs were weighed before their morning feed at 21, 28, 35, 42, 49, and 56 days of age. Anticoagulated blood samples were collected from the jugular vein before the morning feed at 35, 42, and 49 days of age for complete blood count testing.

[0127] The experimental results are as follows: Partial statistical results of the daily diarrhea rate of lambs in each group are shown below. Figure 4(Lambs were 28 days old at weaning, lambs were 29 days old at weaning, and so on; early weaning was the early weaning group, compound probiotics were the compound probiotic group, control probiotic 1 was the control probiotic 1 group, control probiotic 2 was the control probiotic 2 group, and control probiotic 3 was the control probiotic 3 group). The results showed that compared with the early weaning group, the diarrhea rate of lambs in the compound probiotic group, control probiotic 1 group, control probiotic 2 group, and control probiotic 3 group was significantly lower; and the diarrhea rate of lambs in the compound probiotic group was the lowest, significantly lower than that of control probiotic 1 group, control probiotic 2 group, and control probiotic 3 group. Therefore, the compound probiotic agent is significantly more effective than control probiotic 1, control probiotic 2, and control probiotic 3 in preventing and treating lamb diarrhea.

[0128] Partial statistical results of the average daily feed intake of lambs in each group are shown below. Figure 5 (Weaning days 0 corresponds to lambs at 28 days old, weaning days 1 corresponds to lambs at 29 days old, and so on; early weaning is the early weaning group, compound probiotics is the compound probiotic group, control probiotic 1 is the control probiotic group, control probiotic 2 is the control probiotic group, and control probiotic 3 is the control probiotic group). The results showed that compared with the early weaning group, the daily feed intake of lambs in the compound probiotic group, control probiotic group 1, control probiotic group 2, and control probiotic group 3 was significantly increased; and the daily feed intake of lambs in the compound probiotic group was significantly higher than that of control probiotic groups 1, 2, and 3. Therefore, the compound probiotic agent is significantly more effective than control probiotic 1, 2, and 3 in increasing the daily feed intake of lambs.

[0129] The statistical results of weight changes in lambs in each group are shown below. Figure 6 (* indicates p < 0.05, i.e., significant difference; days post-weaning minus 7 equals 21 days old for lambs, days post-weaning 0 equals 28 days old for lambs, and so on; early weaning is the early weaning group, compound probiotics is the compound probiotic group, control probiotic 1 is the control probiotic 1 group, control probiotic 2 is the control probiotic 2 group, and control probiotic 3 is the control probiotic 3 group). The results showed that at 14 days and 21 days post-weaning, compared with the early weaning group, the weight of lambs in the compound probiotic group, control probiotic 1 group, control probiotic 2 group, and control probiotic 3 group all significantly increased. Therefore, compound probiotics, control probiotic 1, control probiotic 2, and control probiotic 3 can all alleviate weight loss caused by weaning stress.

[0130] The blood routine test results (mean values) of lambs in each group are shown in Table 1 (lambs 7 days after weaning, i.e., 35 days old; lambs 14 days after weaning, i.e., 42 days old; lambs 21 days after weaning, i.e., 49 days old; early weaning group; compound probiotic group; control probiotic 1 group; control probiotic 2 group; control probiotic 3 group). The results showed that compared with the early weaning group, the number of white blood cells, lymphocytes, and granulocytes in the blood of lambs in the compound probiotic group, control probiotic 1 group, control probiotic 2 group, and control probiotic 3 group were all reduced to some extent. Therefore, it is evident that the compound probiotic, comparative probiotic 1, comparative probiotic 2, and comparative probiotic 3 all improved the body's inflammatory response. Furthermore, the compound probiotic showed a more pronounced regulatory effect on granulocytes 7 days post-weaning, and its effect in improving the body's inflammatory response was significantly superior to that of comparative probiotic 1, comparative probiotic 2, and comparative probiotic 3. Since granulocytes are the main effector cells in the intestines of early-weaned lambs, the compound probiotic is more effective in alleviating intestinal inflammation in early-weaned lambs.

[0131]

[0132] Application of the compound probiotic preparations prepared in Example 4 and Example 2 in the early weaning of goat lambs In the compound probiotic agent of this invention, the viable count concentration of Pediococcus pentosaceus M3 is specifically 4.40 × 10⁻⁶. 14 The specific viable count concentration of Bacillus amyloliquefaciens CK4 was 8.80 × 10⁻⁶ CFU / mL. 12 The specific viable count concentration of Ludwig's Enterobacter CK6 was 5.90 × 10⁻⁶ CFU / mL. 13 The specific viable count concentration of *Lactobacillus graminearum* was 2.52 × 10⁻⁶ CFU / mL. 12 The specific viable cell count concentration of *Delfordia gravidarum* was 5.10 × 10⁻⁶ CFU / mL. 13 CFU / mL.

[0133] Experimental location: A Boer goat farm in Shijiazhuang, Hebei Province. The experimental site was a semi-open sheepfold with good lighting and ventilation.

[0134] Experimental subjects: Boer goats, a species of capelin. Blood samples were collected from all goats before the start of the experiment, and serum was separated for testing against foot-and-mouth disease, Brucella, anthrax, and peste des petits ruminants (PPR) to confirm the health of all goats. All lambs were nursed by their mothers in the same pen after birth and were given supplemental feed starting at 7 days of age. All experimental lambs were ear-tagged, and their immunization program was implemented according to the farm's standard procedures.

[0135] Experimental Procedure: Boer goat lambs at 28 days of weaning were randomly selected and then randomly divided into two groups: a weaning group and a weaning + probiotic group, with 15 lambs in each group (half male and half female). That is, at 28 days of age, each group of lambs was separated from their mothers (lambs remained in place, mothers were removed) for a single weaning, followed by the following experiment: Weaning group: Feed starter food at 8:00 and 17:00 daily.

[0136] Weaning + compound probiotic group: Feed starter food containing compound probiotic agent at 8:00 am and feed starter food at 5:00 pm daily.

[0137] The preparation method of feed containing compound probiotic agent is as follows: add an appropriate amount of drinking water to the compound probiotic agent, mix well and spray it on the surface of the feed to obtain feed containing compound probiotic agent; in the feed containing compound probiotic agent, the ratio of compound probiotic agent to feed is 3-5 mL: 1 kg (specifically 3 mL: 1 kg in this example).

[0138] The trial lasted 21 days. During the trial, all lambs had free access to water throughout the entire period and were fed starter feed twice a day at 8:00 and 17:00, until the lambs stopped eating on their own. Each lamb was fed approximately 500-1000g of starter feed per day.

[0139] During the experiment, lambs' feed intake and diarrhea status were observed at 9:00 AM and 6:00 PM daily. Ear tags of deceased lambs were recorded to further calculate mortality rates. The amount of initial feed added and the amount remaining were recorded daily, and daily feed intake was calculated and analyzed. Weekly average daily feed intake and average daily feed consumption were also calculated. Additionally, lambs' body weight was measured before morning feeding at 28 and 49 days of age.

[0140] The experimental results are as follows: The initial weight, final weight, daily weight gain, and mortality rate of the two groups of lambs are shown in Table 2. The results showed that compared with the weaning group, the lambs in the weaning + probiotic group had a significantly increased weight, with an average daily weight gain of 151.19 g, a significant difference (* indicates p < 0.05). Three lambs died in the weaning group, resulting in a mortality rate of 20%; while no lambs died in the weaning + probiotic group. Therefore, feeding lambs with probiotics can significantly increase their weight and reduce their mortality rate (i.e., improve their survival rate).

[0141]

[0142] The average daily feed intake of the two groups of lambs is shown in Table 3. The results showed that throughout the entire experimental period, the average daily feed intake of lambs in the weaning + probiotic group was as high as 650.02 g, which was significantly different from the weaning group (*** indicates p < 0.001). In the first, second, and third weeks after weaning, the average daily feed intake of lambs in the weaning + probiotic group was significantly different from the weaning group (* indicates p < 0.05), significantly different (* indicates p < 0.05), and highly significant (** indicates p < 0.01), respectively. Therefore, feeding lambs with probiotics can significantly increase their daily feed intake.

[0143]

[0144] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A compound microbial agent, comprising *Pediococcus pentosaceus*, *Bacillus amyloliquefaciens*, *Enterobacter ludwig vannamei*, *Lactobacillus geranioli*, and *Delfordia chebula*; wherein the viable count of *Pediococcus pentosaceus*, *Bacillus amyloliquefaciens*, *Enterobacter ludwig vannamei*, *Lactobacillus geranioli*, and *Delfordia chebula* in the compound microbial agent is 2 × 10⁻⁶. 11 CFU / mL or higher; The viable count ratio of *Pediococcus pentosaceus*, *Bacillus amyloliquefaciens*, *Enterobacter ludwig's bacterium*, *Lactobacillus geranioli*, and *Delfordia chebula* in the compound microbial agent is 4.40 × 10⁻⁶. 14 CFU / mL: (5.00-10.00) × 10 12 CFU / mL: (5.00-10.00) × 10 13 CFU / mL: (1.00-5.00) × 10 12 CFU / mL: (5.00-10.00) × 10 13 CFU / mL; The Pediococcus pentosaceus is Pediococcus pentosaceus ( Pediococcus pentosaceus M3; Pediococcus pentosaceus ( Pediococcus pentosaceus M3 has the accession number CGMCC No. 30882 at the China General Microbiological Culture Collection Center. The amyloliquefaciens is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Bacillus amyloliquefaciens CK4; Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens CK4 has the accession number CGMCC No. 31178 from the China General Microbiological Culture Collection Center. The Ludwig's Enterobacter is Ludwig's Enterobacter ( Enterobacter ludwigii CK6; Ludwig's Enterobacter ( Enterobacter ludwigii CK6 has the accession number CGMCC No.31179 at the China General Microbiological Culture Collection Center.

2. The compound microbial agent according to claim 1, characterized in that: The viable count ratio of *Pediococcus pentosaceus*, *Bacillus amyloliquefaciens*, *Enterobacter ludwig's bacterium*, *Lactobacillus geranioli*, and *Delfordia chebula* in the compound microbial agent is 4.40 × 10⁻⁶. 14 CFU / mL: 8.80×10 12 CFU / mL: 5.90×10 13 CFU / mL: 2.52×10 12 CFU / mL: 5.10×10 13 CFU / mL.

3. The compound microbial agent according to claim 1 or 2, characterized in that: In preparing the compound bacterial agent, Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter ludwig's bacillus, Lactobacillus graminearum, and Delftobacterium tumefaciens are all added in the form of bacterial liquid.

4. The application of the compound microbial agent according to claim 1 or 2 is at least one of A1)-A7): A1) Prepare products to improve the survival rate of weaned lambs; A2) Prepare products for reducing the rate of diarrhea and / or mortality in weaned lambs; A3) Prepare products to increase the daily feed intake of weaned lambs; A4) Prepare products for increasing the weight and / or daily weight gain of weaned lambs; A5) Prepare products to promote the growth and development of weaned lambs; A6) Prepare products for reducing inflammatory response in weaned lambs and / or alleviating intestinal inflammation in weaned lambs; A7) Prepare products for breeding weaned lambs.

5. The application according to claim 4, characterized in that: The weaned lambs are early-weaned lambs; The early-weaned lambs are lambs aged 28 to 35 days.

6. The application according to claim 4 or 5, characterized in that: The lamb is either a sheep lamb or a goat lamb.

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