Complex microbial inoculant and application thereof in breeding early weaned lambs

Abstract

The invention discloses a complex microbial inoculant and application of the complex microbial inoculant in breeding early weaned lambs. The composite microbial inoculant is prepared from pediococcus pentosaceus, bacillus amyloliquefaciens, enterobacter ludwigii, lactobacillus gasseri and delftia delphinii. Experiments prove that after the compound microbial agent is uniformly sprayed on the surface of starter feed and then fed to early weaned lambs, the survival rate of the lambs can be increased, the feed intake and daily gain can be increased, growth and development can be promoted, the diarrhea rate can be reduced, intestinal mucosa can be protected, inflammatory response induced by weaning stress can be reduced, and a micro-ecological environment in intestinal tracts of the lambs after weaning can be quickly established. The complex microbial inoculant prepared by the invention can guarantee the breed source of the replacement lambs, and is of great significance to popularization of an early weaning technology in a large-scale sheep farm. The method has an important application value.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a composite bacterial agent and application thereof in breeding early weaned lambs. Background Art

[0002] As the country with the largest scale of sheep farming in the world, my country has an annual sheep inventory of about 300 million in the past decade. In 2022, China's sheep inventory reached 326.2726 million, the output reached 336.24 million, and the mutton output was 5.2453 million tons. As of 2020, the proportion of sheep farms with more than 100 sheep in my country increased from 0.6% in 2003 to 67.87% of the total number of sheep farms in the country, and the proportion of large-scale sheep farming has been greatly improved. Optimizing the industrial structure and vigorously promoting grain-saving herbivorous animal husbandry are the strategic directions for the development of my country's animal husbandry. The scale and standardization of mutton sheep are accelerating, and the implementation of early weaning is an important measure to achieve high-frequency breeding and large-scale breeding. The quality of lamb breeding is a key link in determining the production efficiency of modern mutton sheep. Early weaning has become a common practice for lamb breeding in many countries. At present, many years of exploration and research have been carried out on the early weaning technology of lambs. The results show that feeding strategies can effectively improve the utilization efficiency of ewes, shorten the lactation period of lambs, and give full play to production potential. However, the stress response caused by early weaning can easily cause diseases such as lamb diarrhea, leading to decreased lamb production performance and survival rate. The annual lamb mortality rate due to weaning stress syndrome and other reasons is over 25%, and the shortage of reserve sheep sources has seriously restricted the sustainable development of the industry.

[0003] The intestinal tissue of lactating lambs is not fully developed, and the intestinal microecological system is in the early stages of establishment and has not reached a mature ecological balance period; the number and function of matrix cells and immune cells are still developing. If weaning is given during lactation, the lambs will lack the supply of maternal antibodies, and the body's immune system and antioxidant capacity will be insufficient, which can easily cause damage to the intestinal mucosal barrier; at the same time, the nature of food changes greatly, and the imperfect intestinal microecological system is very susceptible to invasion by external pathogens, thereby exacerbating the threat to the development and survival of lambs. Summary of the invention

[0004] The invention aims to reduce the mortality rate and diarrhea rate of early weaned lambs and promote growth and development.

[0005] The invention firstly protects a composite bacterial agent, which may include Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter ludwigii, Lactobacillus graminis and Delftia foetida.

[0006] The composite bacterial agent can specifically be composed of Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter ludwigii, Lactobacillus gestellt and Delftia foetida.

[0007] The composite bacterial agent can be used for breeding early weaned lambs.

[0008] In any of the above-mentioned composite bacterial agents, the Pediococcus pentosaceus may be Pediococcus pentosaceus M3; Pediococcus pentosaceus M3, whose deposit number in the General Microbiological Center of China National Microbiological Culture Collection Administration is CGMCC No.30882.

[0009] In any of the above-mentioned composite bacterial agents, the Bacillus amyloliquefaciens may be Bacillus amyloliquefaciens CK4; Bacillus amyloliquefaciens CK4, whose deposit number in the General Microbiological Center of China National Microbiological Culture Collection Committee is CGMCC No.31178.

[0010] In any of the above-mentioned composite bacterial agents, the Ludwigii Enterobacter may be Ludwigii Enterobacter (Enterobacter ludwigii) CK6; Ludwigii Enterobacter (Enterobacter ludwigii) CK6, whose preservation number in the General Microbiological Center of China National Microbiological Culture Collection Administration is CGMCC No.31179.

[0011] In any of the above-mentioned composite bacterial agents, the viable counts of Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter Ludwigii, Lactobacillus gestellinus and Delftia foetida are all 2×10 11 CFU / mL and above.

[0012] In any of the above-mentioned composite bacterial agents, the ratio of the number of live bacteria of Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter Ludwigii, Lactobacillus gestellinus and Delftia foetida can be 4.40×10 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 14 CFU / mL: (5.00-8.80)×10 12 CFU / mL: (5.00-5.90)×10 13 CFU / mL: (1.00-2.52)×10 12CFU / mL: (5.00-5.10)×10 13 CFU / mL or 4.40×

[0013] 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).

[0014] In any of the above-mentioned composite bacterial agents, the ratio of the number of live bacteria of Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter Ludwigii, Lactobacillus gestellinus and Delftia foetida can be 4.40×10 14 CFU / mL:

[0015] 8.80×10 12 CFU / mL: 5.90×10 13 CFU / mL: 2.52×10 12 CFU / mL: 5.10×10 13 CFU / mL.

[0016] When preparing any of the above-mentioned composite bacterial agents, Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter Ludwigii, Lactobacillus gestellii and Delftia foetida can all be added in the form of bacterial liquid.

[0017] The preparation method of the bacterial liquid can be: inoculating Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter Ludwigii, Lactobacillus gestellii or Delftia foetida into a culture medium, and fermenting to obtain the bacterial liquid. The culture medium can be a common broth culture medium.

[0018] The preparation method of any of the above-mentioned composite bacterial agents can be obtained by mixing the bacterial solution of Pediococcus pentosaceus, the bacterial solution of Bacillus amyloliquefaciens, the bacterial solution of Enterobacter ludwigii, the bacterial solution of Lactobacillus gestellii and the bacterial solution of Delftia foetida, and then adding an appropriate amount of water to obtain the composite bacterial agent.

[0019] The present invention also protects the application of any of the above-mentioned composite bacterial agents, which may be at least one of A1)-A14):

[0020] A1) Improve the survival rate of weaned lambs;

[0021] A2) preparing a product for improving the survival rate of weaned lambs;

[0022] A3) Reduce diarrhea rate and / or mortality of weaned lambs;

[0023] A4) preparing a product for reducing diarrhea rates and / or mortality in weaned lambs;

[0024] A5) Increase feed intake and / or daily feed intake of weaned lambs;

[0025] A6) Preparation of products for increasing feed intake and / or daily feed intake of weaned lambs;

[0026] A7) Increase the body weight and / or daily weight gain of weaned lambs;

[0027] A8) preparing a product for increasing body weight and / or daily weight gain of weaned lambs;

[0028] A9) Promote the growth and development of weaned lambs;

[0029] A10) preparing products for promoting growth and development of weaned lambs;

[0030] A11) reducing the inflammatory response of weaned lambs and / or alleviating intestinal inflammation of weaned lambs;

[0031] A12) preparing a product for reducing inflammatory response in weaned lambs and / or alleviating intestinal inflammation in weaned lambs;

[0032] A13) raising weaned lambs;

[0033] A14) Preparation of products for rearing weaned lambs.

[0034] In the above application, the weaned lamb can be an early weaned lamb. The early weaned lamb can preferably be a lamb of 28 to 35 days of age (such as 28 to 30 days of age, 31 to 33 days of age, 34 to 35 days of age, 28 days of age, 29 days of age, 30 days of age, 31 days of age, 32 days of age, 33 days of age, 34 days of age or 35 days of age).

[0035] In any of the above applications, the method for using any of the above composite bacterial agents can be: spray any of the above composite bacterial agents evenly on the surface of the starter feed, and then feed it to early weaned lambs. The feeding frequency of the composite probiotic preparation is preferably once a day. The ratio of any of the above composite bacterial agents to the starter feed can be 3-5mL:1kg (such as 3-4mL:1kg, 4-5mL:1kg, 3mL:1kg, 4mL:1kg or 5mL:1kg). Each lamb is fed about 500-1000g of starter feed per day.

[0036] In the above application, the lamb can be a sheep lamb or a goat lamb. The sheep can be a meat sheep. The meat sheep can be a Hu sheep. The goat can be a Boer goat.

[0037] The inventors of the present application have found through a large number of experiments that early weaning can induce iron accumulation in the intestinal mucosal epithelial cells and lamina propria macrophages of lambs, and ferroptosis occurs, which confirms that it is an important cause of intestinal mucosal damage in early weaned lambs. Early weaning can obviously cause the death and shedding of lamb jejunal mucosal epithelial cells, and early weaning can cause the number of lactobacilli in the jejunum of 35-42-day-old lambs to decrease significantly, and the products related to lipid metabolism are also significantly reduced. At this time, the physical barrier of the intestinal mucosa of early weaned lambs is severely damaged, and the ability to absorb nutrients is reduced, which is manifested as diarrhea and growth retardation, and even death occurs. Microecological preparations have the effect of adjusting intestinal flora imbalance and improving intestinal microecological environment. The composite bacterial agent prepared in this application is reasonably matched with the starter feed and fed to early weaned lambs, which can improve the survival rate of lambs (reduce mortality), increase feed intake and daily weight gain, promote growth and development, improve diarrhea symptoms (reduce diarrhea rate), protect intestinal mucosa, reduce the inflammatory response induced by weaning stress, and help lambs quickly establish a microecological environment in the intestine after weaning. The composite bacterial agent prepared in this application can guarantee the provenance of reserve lambs, which is of great significance to the promotion of early weaning technology in large-scale sheep farms. The present invention has important application value.

[0038] Collection Instructions

[0039] Bacteria name: Pediococcus pentosaceus

[0040] Latin name: Pediococcus pentosaceus

[0041] Strain ID: M3

[0042] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0043] Abbreviation of depository institution: CGMCC

[0044] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0045] Storage date: June 7, 2024

[0046] CGMCC Registration Number: CGMCC No.30882

[0047] Bacteria name: Bacillus amyloliquefaciens

[0048] Latin name: Bacillus amyloliquefaciens

[0049] Strain ID: CK4

[0050] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0051] Abbreviation of depository institution: CGMCC

[0052] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0053] Storage date: July 5, 2024

[0054] CGMCC registration number: CGMCC No.31178

[0055] Bacterial species name: Enterobacter ludwigii

[0056] Latin name: Enterobacter ludwigii

[0057] Strain ID: CK6

[0058] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0059] Abbreviation of depository institution: CGMCC

[0060] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0061] Storage date: July 5, 2024

[0062] CGMCC Registration Number: CGMCC No.31179 BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is the growth status of the bacterial M3 colony.

[0064] Figure 2 This is the growth status of the bacterial CK4 colony.

[0065] Figure 3 This is the growth status of the bacterial CK6 colony.

[0066] Figure 4 The figure shows the change in diarrhea rate of early weaned lambs after feeding the bacterial agent in Example 3.

[0067] Figure 5 The figure shows the change in daily feed intake of early weaned lambs after feeding the bacterial agent in Example 3.

[0068] Figure 6 The figure shows the changes in body weight of early weaned lambs after feeding the bacterial agent in Example 3. DETAILED DESCRIPTION

[0069] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0070] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0071] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0072] The starter feed in the following embodiments is a mixture of commercially available corn, whey powder, soybean meal, alfalfa and additive premix. In this application, corn, soybean meal and alfalfa were purchased from Linqing Runlin Animal Husbandry Co., Ltd.; whey powder was purchased from Beijing Precision Animal Nutrition Research Center Co., Ltd.; the additive premix contains iron, manganese, copper, zinc, selenium, iodine, cobalt, VA, VD and VE, all provided by Beijing Precision Animal Nutrition Research Center Co., Ltd.

[0073] The culture medium involved in the following examples is as follows:

[0074] The solutes and their concentrations of ordinary nutrient agar culture medium are 15 g / L agar, 10 g / L peptone, 3 g / L beef extract powder and 5 g / L sodium chloride, the solvent is distilled water, and the pH value is 7.2±0.2.

[0075] The solutes and their concentrations of the common broth culture medium are 20 g / L peptone, 5 g / L beef extract powder and 5 g / L sodium chloride, the solvent is distilled water, and the pH value is 7.2±0.2.

[0076] The solutes and their concentrations of the YPDA medium are 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose and 0.03 g / L adenine sulfate, the solvent is distilled water, and the pH value is 6.0-6.5.

[0077] Example 1. Obtaining Pediococcus pentosaceus M3, Bacillus amyloliquefaciens CK4 and Enterobacter ludwigii CK6

[0078] 1. Isolation, identification and preservation of Pediococcus pentosaceus M3 CGMCC No.30882

[0079] 1. Separation

[0080] 1. Add 1g of home fermented dough (from farmers in Anqiu City, Weifang City, Shandong Province) and an appropriate amount of glass beads to 9mL of sterile saline, shake for 10min, let stand for 30s, and make a dough stock solution (the dilution at this time is recorded as 10 -1 ). Take 1mL of the supernatant of the dough stock solution and add it into a test tube containing 9mL of sterile saline and mix thoroughly (the dilution is 10 at this time). -2 ), then draw 1mL from this test tube and add it to another test tube containing 9mL of sterile saline and mix well, and so on to make a dilution solution (dilution degree is 10 -4 ).

[0081] 2. Use a pipette to take 100 μL of dilutions of different dilutions and spread them evenly on ordinary nutrient agar medium. Culture at 28°C for 5-7 days and observe the growth status of the colonies.

[0082] 3. After completing step 2, inoculate each colony into common broth culture medium, culture, and obtain the corresponding bacterial solution.

[0083] 4. Take 10 μL of the above bacterial solution, add 900 μL of sterile saline, inoculate 100 μL of the bacterial solution into ordinary nutrient agar medium, and culture at 37°C for 24 hours.

[0084] 5. After completing step 4, use a sterile inoculation loop to pick up a single colony and inoculate it into ordinary broth culture medium, culture it, and obtain the corresponding bacterial solution.

[0085] The screened bacteria was named bacteria M3.

[0086] The growth status of the bacterial M3 colony is shown in Figure 1 .

[0087] (II) Molecular identification

[0088] The genomic DNA of bacteria M3 was extracted and used as a template. Primer 27F: 5'

[0089] PCR amplification was performed using a primer pair consisting of primer 1492R: 5'-TACGACTTAACCCCAATCGC-3' to obtain 16S rDNA.

[0090] The nucleotide sequence of 16S rDNA of bacterium M3 is shown in SEQ ID NO:1.

[0091] The sequence shown in SEQ ID NO: 1 was subjected to BLAST in the NCBI Standard Nucleotide database to finally determine the genus and species of the bacterium M3.

[0092] The results showed that the bacteria M3 had the highest homology with Pediococcus pentosaceus. Therefore, the bacteria M3 was identified as Pediococcus pentosaceus.

[0093] (III) Preservation

[0094] The bacteria M3 isolated in step 1 has been deposited in the General Microbiological Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​June 7, 2024, with a deposit number of CGMCC No. 30882. The full name of the bacteria M3 is Pediococcus pentosaceus M3 CGMCC No. 30882, abbreviated as Pediococcus pentosaceus M3.

[0095] 2. Isolation, identification and storage of Bacillus amyloliquefaciens CK4 CGMCC No.31178

[0096] 1. Separation

[0097] 1. Add 1 g of home fermented dough (from farmers in Ju County, Rizhao City, Shandong Province) and an appropriate amount of glass beads to 9 mL of sterile saline, shake for 10 min, let stand for 30 s, and make a dough stock solution (the dilution at this time is recorded as 10 -1 ). Take 1mL of the supernatant of the dough stock solution and add it into a test tube containing 9mL of sterile saline and mix thoroughly (the dilution is 10 at this time). -2 ), then draw 1mL from this test tube and add it to another test tube containing 9mL of sterile saline and mix well, and so on to make a dilution solution (dilution degree is 10 -4 ).

[0098] 2. Use a pipette to take 100 μL of dilutions of different dilutions and spread them evenly on ordinary nutrient agar medium. Culture at 28°C for 5-7 days and observe the growth status of the colonies.

[0099] 3. After completing step 2, inoculate each colony into common broth culture medium, culture, and obtain the corresponding bacterial solution.

[0100] 4. Take 10 μL of the above bacterial solution, add 900 μL of sterile saline, inoculate 100 μL of the bacterial solution into ordinary nutrient agar medium, and culture at 37°C for 24 hours.

[0101] 5. After completing step 4, use a sterile inoculation loop to pick up a single colony and inoculate it into ordinary broth culture medium, culture it, and obtain the corresponding bacterial solution.

[0102] One of the screened bacteria was named bacteria CK4.

[0103] The growth status of bacterial CK4 colonies is shown in Figure 2 .

[0104] (II) Molecular identification

[0105] The genomic DNA of bacterial CK4 was extracted and used as a template. The primers 27F: 5'

[0106] PCR amplification was performed using a primer pair consisting of primer 1492R: 5'-TACGACTTAACCCCAATCGC-3' to obtain 16S rDNA.

[0107] The nucleotide sequence of 16S rDNA of bacteria CK4 is shown in SEQ ID NO:2.

[0108] The sequence shown in SEQ ID NO:2 was subjected to BLAST in the NCBI Standard Nucleotide database to finally determine the genus and species of the bacterium CK4.

[0109] The results showed that the bacterial strain CK4 had the highest homology with Bacillus amyloliquefaciens. Therefore, the bacterial strain CK4 was identified as Bacillus amyloliquefaciens.

[0110] (III) Preservation

[0111] The bacteria CK4 isolated in step 1 has been deposited in the General Microbiological Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on ​​July 5, 2024, with a deposit number of CGMCC No. 31178. The full name of the bacteria CK4 is Bacillus amyloliquefaciens CK4 CGMCC No. 31178, abbreviated as Bacillus amyloliquefaciens CK4.

[0112] 3. Isolation, identification and storage of Enterobacter ludwigii CK6 CGMCC No.31179

[0113] 1. Separation

[0114] 1. Add 1 g of herbivorous insect feces (from Fangzi District, Weifang City, Shandong Province) to 9 mL of sterile saline, shake for 10 min, let stand for 30 s, and prepare a feces suspension (the dilution at this time is recorded as 10-1 ). Centrifuge at 2000 rpm for 5 minutes. Pipette 1 mL of the supernatant of the fecal suspension into a test tube containing 9 mL of sterile saline and mix thoroughly (the dilution is 10 at this time). -2 ), then draw 1mL from this test tube and add it to another test tube containing 9mL of sterile saline and mix well, and so on to make a dilution solution (dilution degree is 10 -4 ).

[0115] 2. Use a pipette to take 100 μL of dilutions of different dilutions and spread them evenly on ordinary nutrient agar medium. Culture at 28°C for 5-7 days and observe the growth status of the colonies.

[0116] 3. After completing step 2, inoculate each colony into common broth culture medium, culture, and obtain the corresponding bacterial solution.

[0117] 4. Take 10 μL of the above bacterial solution, add 900 μL of sterile saline, inoculate 100 μL of the bacterial solution into ordinary nutrient agar medium, and culture at 37°C for 24 hours.

[0118] 5. After completing step 4, use a sterile inoculation loop to pick up a single colony and inoculate it into ordinary broth culture medium, culture it, and obtain the corresponding bacterial solution.

[0119] One of the screened bacteria was named bacteria CK6.

[0120] The growth status of bacterial CK6 colonies is shown in Figure 3 .

[0121] (II) Molecular identification

[0122] The genomic DNA of bacterial CK6 was extracted and used as a template. Primer 27F: 5'

[0123] PCR amplification was performed using a primer pair consisting of primer 1492R: 5'-TACGACTTAACCCCAATCGC-3' to obtain 16S rDNA.

[0124] The nucleotide sequence of 16S rDNA of bacteria CK6 is shown in SEQ ID NO:3.

[0125] The sequence shown in SEQ ID NO:3 was subjected to BLAST in the NCBI Standard Nucleotide database to finally determine the genus and species of the bacterium CK6.

[0126] The results showed that bacterial CK6 had the highest homology with Enterobacter ludwigii. Therefore, bacterial CK6 was identified as Enterobacter ludwigii.

[0127] (III) Preservation

[0128] The bacteria CK6 isolated in step 1 has been deposited in the General Microbiological Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on ​​July 5, 2024, with a deposit number of CGMCC No. 31179. The full name of the bacteria CK6 is Enterobacter ludwigii CK6 CGMCC No. 31179, abbreviated as Enterobacter ludwigii CK6.

[0129] Example 2: Preparation of bacterial agent

[0130] 1. Preparation of composite bacterial agents (also known as composite probiotic agents)

[0131] 1. Preparation of Pediococcus pentosaceus M3 inoculant

[0132] (1) inoculating a single colony of Pediococcus pentosaceus M3 into 50 mL of common broth medium, culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain a primary seed solution of Pediococcus pentosaceus M3;

[0133] (2) inoculating the primary seed solution of Pediococcus pentosaceus M3 into 50 mL of common broth medium (for the purpose of secondary activation), culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain the secondary seed solution of Pediococcus pentosaceus M3;

[0134] (3) The secondary seed liquid of Pediococcus pentosaceus M3 was inoculated into 100 mL of common broth medium, and fermented and cultured at 33-37° C. and 50-100 rpm for 18-24 h to obtain Pediococcus pentosaceus M3 fermentation liquid. During the fermentation and culture, the ventilation rate was 0.3-1 L / min.

[0135] In the fermentation broth of Pediococcus pentosaceus M3, the viable bacterial count of Pediococcus pentosaceus M3 is ≥ 2×10 11 CFU / mL.

[0136] The fermentation broth of Pediococcus pentosaceus M3 is the Pediococcus pentosaceus M3 bacterial agent.

[0137] 2. Preparation of Bacillus amyloliquefaciens CK4 inoculant

[0138] (1) inoculating a single colony of Bacillus amyloliquefaciens CK4 into 50 mL of ordinary broth medium, culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain a primary seed solution of Bacillus amyloliquefaciens CK4;

[0139] (2) inoculating the primary seed liquid of Bacillus amyloliquefaciens CK4 into 50 mL of ordinary broth medium (for the purpose of secondary activation), culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain the secondary seed liquid of Bacillus amyloliquefaciens CK4;

[0140] (3) The secondary seed liquid of Bacillus amyloliquefaciens CK4 was inoculated into 100 mL of common broth medium, and fermented at 33-37° C. and 50-100 rpm for 18-24 h to obtain Bacillus amyloliquefaciens CK4 fermentation liquid. During the fermentation, the ventilation rate was 0.3-1 L / min.

[0141] The viable cell count of Bacillus amyloliquefaciens CK4 in the fermentation broth is ≥2×10 11 CFU / mL.

[0142] The fermentation broth of Bacillus amyloliquefaciens CK4 is the Bacillus amyloliquefaciens CK4 bacterial agent.

[0143] 3. Preparation of Enterobacter Ludwigii CK6 inoculum

[0144] (1) A single colony of Enterobacter Ludwigii CK6 was inoculated into 50 mL of ordinary broth medium, and cultured at 33-37° C. and 50-100 rpm for 18-24 h to obtain a primary seed solution of Enterobacter Ludwigii CK6;

[0145] (2) inoculating the primary seed solution of Enterobacter Ludwigii CK6 into 50 mL of ordinary broth medium (for the purpose of secondary activation), culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain the secondary seed solution of Enterobacter Ludwigii CK6;

[0146] (3) The secondary seed liquid of Enterobacter Ludwigii CK6 was inoculated into 100 mL of ordinary broth medium, and fermented at 33-37° C. and 50-100 rpm for 18-24 h to obtain Enterobacter Ludwigii CK6 fermentation liquid. During the fermentation, the ventilation rate was 0.3-1 L / min.

[0147] In the fermentation broth of Enterobacter Ludwigii CK6, the viable bacterial count of Enterobacter Ludwigii CK6 is ≥2×

[0148] 10 11 CFU / mL.

[0149] The fermentation broth of Enterobacter Ludwigii CK6 is the Enterobacter Ludwigii CK6 bacterial agent.

[0150] 4. Preparation of Lactobacillus gefitinib

[0151] Lactobacillus formatus is specifically a product of Beina Chuanglian Biotechnology Co., Ltd., and the product catalog number is BNCC339385.

[0152] (1) inoculating a single colony of Lactobacillus gestellens into 50 mL of common broth medium, culturing at 33-37° C. and 50-100 rpm for 18-24 hours to obtain a primary seed solution of Lactobacillus gestellens;

[0153] (2) inoculating the primary seed liquid of Lactobacillus gesneri into 50 mL of common broth medium (for the purpose of secondary activation), culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain the secondary seed liquid of Lactobacillus gesneri;

[0154] (3) The secondary seed liquid of Lactobacillus gesneri is inoculated into 100 mL of common broth medium, and fermented and cultured at 33-37° C. and 50-100 rpm for 18-24 hours to obtain Lactobacillus gesneri fermentation liquid. During the fermentation and culture, the ventilation rate is 0.3-1 L / min.

[0155] The viable cell count of Lactobacillus gesneri in the fermented broth is ≥2×10 11 CFU / mL.

[0156] The Lactobacillus gesneri fermentation liquid is the Lactobacillus gesneri agent.

[0157] 5. Preparation of pond-grown Delftia bacteria agent

[0158] The pond-grown Delftia bacteria is specifically a product of Unico Life Sciences Co., Ltd., and the product catalog number is YLK-jz3731.

[0159] (1) inoculating a single colony of the pond-grown Delftia into 50 mL of common broth medium, culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain a primary seed solution of the pond-grown Delftia;

[0160] (2) inoculating the primary seed liquid of the pond-grown Delftia into 50 mL of common broth medium (for the purpose of secondary activation), culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain the secondary seed liquid of the pond-grown Delftia;

[0161] (3) The secondary seed liquid of the pond-grown Delftia bacteria was inoculated into 100 mL of common broth medium, and fermented at 33-37° C. and 50-100 rpm for 18-24 h to obtain a fermentation liquid of the pond-grown Delftia bacteria. During the fermentation, the ventilation rate was 0.3-1 L / min.

[0162] The viable bacterial count of pool-grown Delftia in the fermentation broth of pool-grown Delftia is ≥ 2×10 11 CFU / mL.

[0163] The pond-grown Delftia fermentation liquid is the pond-grown Delftia bacterial agent.

[0164] 6. Preparation of composite bacterial agent

[0165] The bacterial agent of Pediococcus pentosaceus M3, the bacterial agent of Bacillus amyloliquefaciens CK4, the bacterial agent of Enterobacter Ludwigii CK6, the bacterial agent of Lactobacillus gesneri and the bacterial agent of Delftia foetida are mixed to obtain a composite bacterial agent. In the composite bacterial agent, the volume ratio of the bacterial agent of Pediococcus pentosaceus M3, the bacterial agent of Bacillus amyloliquefaciens CK4, the bacterial agent of Enterobacter Ludwigii CK6, the bacterial agent of Lactobacillus gesneri and the bacterial agent of Delftia foetida is 1:1:1:1:1.

[0166] 2. Preparation of comparative probiotic 1

[0167] The comparative probiotic 1 is Bacillus licheniformis, which is specifically a product of Beina Chuanglian Biotechnology Co., Ltd., with a product catalog number of BNCC132665.

[0168] (1) Inoculating a single colony of Bacillus licheniformis into 50 mL of ordinary broth medium, culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain a primary seed solution of Bacillus licheniformis;

[0169] (2) inoculating the primary seed liquid of Bacillus licheniformis into 50 mL of common broth medium (for the purpose of secondary activation), culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain the secondary seed liquid of Bacillus licheniformis;

[0170] (3) The secondary seed liquid of Bacillus licheniformis is inoculated into 100 mL of common broth medium, and fermented at 33-37° C. and 50-100 rpm for 18-24 h to obtain Bacillus licheniformis fermentation liquid. During the fermentation, the ventilation rate is 0.3-1 L / min.

[0171] The concentration of viable Bacillus licheniformis in the fermentation broth is ≥2×10 11 CFU / mL.

[0172] The fermented liquid of Bacillus licheniformis is the Bacillus licheniformis agent, that is, the comparative probiotic 1 agent.

[0173] 3. Preparation of Probiotic 2

[0174] The comparative probiotic 2 is Pichia pastoris, which is specifically a product of Beina Chuanglian Biotechnology Co., Ltd., with a product catalog number of BNCC380011.

[0175] (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 a primary seed solution of Pichia pastoris;

[0176] (2) inoculating the primary seed solution of Pichia pastoris into 50 mL of YPDA medium (for secondary activation), culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain the secondary seed solution of Pichia pastoris;

[0177] (3) The secondary seed liquid of Pichia pastoris was inoculated into 100 mL of YPDA medium, and fermented at 33-37° C. and 50-100 rpm for 18-24 h to obtain Pichia pastoris fermentation liquid. During the fermentation, the ventilation rate was 0.3-1 L / min.

[0178] The concentration of viable Pichia yeast in the fermentation broth is ≥2×10 11 CFU / mL.

[0179] The Pichia yeast fermentation broth is the Pichia yeast agent, that is, the comparative probiotic 2 agent.

[0180] 4. Preparation of 3 comparative probiotic agents

[0181] The probiotic 3 agent is a composite agent, and the specific preparation method is as follows:

[0182] 1. Preparation of Lactobacillus amylovora

[0183] The starch-eating lactobacillus is specifically a product of Beina Chuanglian Biotechnology Co., Ltd., and the product catalog number is BNCC132483.

[0184] (1) inoculating a single colony of Lactobacillus amylovora into 50 mL of common broth medium, culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain a primary seed solution of Lactobacillus amylovora;

[0185] (2) inoculating the primary seed liquid of Lactobacillus amylovora into 50 mL of common broth medium (for the purpose of secondary activation), culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain the secondary seed liquid of Lactobacillus amylovora;

[0186] (3) The secondary seed liquid of Lactobacillus amylovora was inoculated into 50 mL of common broth medium, and fermented at 33-37° C. and 50-100 rpm for 18-24 h to obtain Lactobacillus amylovora fermentation liquid. During the fermentation, the ventilation rate was 0.3-1 L / min.

[0187] The viable cell count of Lactobacillus amylovora in the fermentation broth is ≥2×1011 CFU / mL.

[0188] The fermentation liquid of Lactobacillus amylovora is the Lactobacillus amylovora agent.

[0189] 2. Preparation of Lactobacillus reuteri agent

[0190] Lactobacillus reuteri is specifically a product of Beina Chuanglian Biotechnology Co., Ltd., and the product catalog number is BNCC337178.

[0191] (1) inoculating a single colony of Lactobacillus reuteri into 50 mL of common broth medium, culturing at 33-37° C. and 50-100 rpm for 18-24 hours to obtain a primary seed solution of Lactobacillus reuteri;

[0192] (2) inoculating the primary seed liquid of Lactobacillus reuteri into 50 mL of common broth medium (for the purpose of secondary activation), culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain the secondary seed liquid of Lactobacillus reuteri;

[0193] (3) The secondary seed liquid of Lactobacillus reuteri is inoculated into 100 mL of common broth medium, and fermented and cultured at 33-37° C. and 50-100 rpm for 18-24 hours to obtain Lactobacillus reuteri fermentation liquid. During the fermentation and culture, the ventilation rate is 0.3-1 L / min.

[0194] The viable cell count of Lactobacillus reuteri in the fermented broth is ≥ 2 × 10 11 CFU / mL.

[0195] The Lactobacillus reuteri fermentation broth is the Lactobacillus reuteri agent.

[0196] 3. Preparation of Lactobacillus johnsonii agent

[0197] Lactobacillus johnsonii is specifically a product of Beina Chuanglian Biotechnology Co., Ltd., and the product catalog number is BNCC186110.

[0198] (1) inoculating a single colony of Lactobacillus johnsonii into 50 mL of common broth medium, culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain a primary seed solution of Lactobacillus johnsonii;

[0199] (2) inoculating the primary seed liquid of Lactobacillus johnsonii into 50 mL of common broth medium (for the purpose of secondary activation), culturing at 33-37° C. and 50-100 rpm for 18-24 h to obtain the secondary seed liquid of Lactobacillus johnsonii;

[0200] (3) The secondary seed liquid of Lactobacillus johnsonii is inoculated into 100 mL of common broth medium, and fermented and cultured at 33-37° C. and 50-100 rpm for 18-24 hours to obtain Lactobacillus johnsonii fermentation liquid. During the fermentation and culture, the ventilation rate is 0.3-1 L / min.

[0201] The viable cell count of Lactobacillus johnsonii in the fermented broth is ≥ 2 × 10 11 CFU / mL.

[0202] Lactobacillus johnsonii fermentation broth is the Lactobacillus johnsonii agent.

[0203] 4. Comparative preparation of three probiotic agents

[0204] The Lactobacillus amylovora, the Lactobacillus reuteri and the Lactobacillus johnsonii were mixed to obtain the comparative probiotic 3 agent; in the comparative probiotic 3 agent, the volume ratio of the Lactobacillus amylovora, the Lactobacillus reuteri and the Lactobacillus johnsonii agent was 1:1:1.

[0205] Application of the composite probiotic prepared in Example 3 and Example 2 in the cultivation of early weaned sheep lambs

[0206] In the composite probiotic agent of the present invention, the viable bacterial count concentration of Pediococcus pentosaceus M3 is specifically 4.40×10 14 CFU / mL, the viable bacterial count of Bacillus amyloliquefaciens CK4 is 8.80×10 12 CFU / mL, the viable bacterial count of Ludwig's Enterobacter CK6 is 5.90×10 13 CFU / mL, the viable bacterial count concentration of Lactobacillus Formatus is

[0207] 2.52×10 12 CFU / mL, the viable bacterial count of pool-grown Delftia is 5.10×10 13 CFU / mL.

[0208] In the comparative probiotic 1 of the present invention, the viable bacterial count concentration of Bacillus licheniformis is specifically 6.60×10 14 CFU / mL.

[0209] In the comparative probiotic 2 agent of the present invention, the viable bacterial count concentration of Pichia pastoris is specifically 2.10×10 12 CFU / mL.

[0210] In the comparative probiotic 3 agent of the present invention, the viable bacterial count concentration of Lactobacillus amylovora is specifically 3.40×10 12 CFU / mL, the specific concentration of viable bacteria of Lactobacillus reuteri is 4.60×10 14CFU / mL, the specific concentration of viable bacteria of Lactobacillus johnsonii is 8.30×10 14 CFU / mL.

[0211] Experimental location: A Huyang farm in Shijiazhuang, Hebei. The experiment was conducted in a semi-open sheep house with good lighting and ventilation conditions.

[0212] Test subjects: Hu sheep, sheep. All sheep were tested for foot-and-mouth disease, Brucella, anthrax and peste des petits ruminants to confirm that all sheep were healthy. All lambs were kept in the same pen with their mothers after birth and were fed with starter feed at 7 days of age; all test lambs were ear-tagged, and the immunization procedure was implemented according to the regular procedures of the sheep farm.

[0213] Experimental process: Huyang lambs with a weaning age of 28 days were randomly selected and then randomly divided into five groups, namely early weaning group, compound probiotic group, comparison probiotic group 1, comparison probiotic group 2 and comparison probiotic group 3, with 12 lambs in each group (half male and half female). That is, at the age of 28 days, each group of lambs was separated from the ewes (the lambs stayed in place and the ewes were moved away) and weaned once, and then the following experiments were carried out:

[0214] Early weaning group: starter feed was fed at 8:00 and 17:00 every day.

[0215] Compound probiotic group: fed with starter feed containing compound probiotics at 8:00 a.m. and feed with starter feed at 5:00 p.m. every day.

[0216] The preparation method of the starter containing the composite probiotic is as follows: add an appropriate amount of drinking water to the composite probiotic, mix well and spray it on the surface of the starter to obtain the starter containing the composite probiotic; in the starter containing the composite probiotic, the ratio of the composite probiotic to the starter is 3-5 mL: 1 kg (specifically 5 mL: 1 kg in this embodiment).

[0217] Comparative probiotic group 1: The starter feed containing comparative probiotic 1 was fed at 8:00 a.m. and the starter feed was fed at 17:00 p.m. every day.

[0218] Add an appropriate amount of drinking water to the comparative probiotic 1 agent, mix well and spray it on the surface of the starter to obtain a starter containing the comparative probiotic 1 agent; in the starter containing the comparative probiotic 1 agent, the ratio of the comparative probiotic 1 agent to the starter is 1-2 mL: 1 kg (specifically 1 mL: 1 kg in this embodiment).

[0219] Comparative probiotic group 2: The starter feed containing comparative probiotic 2 was fed at 8:00 a.m. and the starter feed was fed at 17:00 p.m. every day.

[0220] Add an appropriate amount of drinking water to the comparative probiotic 2 agent, mix well and spray it on the surface of the starter to obtain a starter containing the comparative probiotic 2 agent; in the starter containing the comparative probiotic 2 agent, the ratio of the comparative probiotic 2 agent to the starter is 1-2 mL: 1 kg (specifically 1 mL: 1 kg in this embodiment).

[0221] Comparative probiotic group 3: The starter feed containing comparative probiotic group 3 was fed at 8:00 a.m. and the starter feed was fed at 17:00 p.m. every day.

[0222] Add an appropriate amount of drinking water to the comparative probiotic 3 agent, mix well and spray it on the surface of the starter to obtain a starter containing the comparative probiotic 3 agent; in the starter containing the comparative probiotic 3 agent, the ratio of the comparative probiotic 3 agent to the starter is 3-4 mL: 1 kg (specifically 3 mL: 1 kg in this embodiment).

[0223] The test lasted for 28 days. During the test, all lambs were allowed to drink water freely and were fed starter feed twice a day at 8:00 and 17:00 respectively. The amount of feed was used until the lambs stopped actively eating. Both the composite probiotics and the control probiotics were sprayed when the starter feed was fed at 8:00. Each lamb was fed about 500-1000g of starter feed per day.

[0224] During the experiment, the lambs' feeding and diarrhea conditions were observed at 9:00 and 18:00 every day, and the diarrhea rate was further calculated according to the ear number of the lambs with diarrhea recorded by ear tags. The amount of starter feed added and the remaining amount were recorded every day, and the changes in daily feed intake were calculated and analyzed. In addition, the body weight of lambs was measured before morning feeding at 21, 28, 35, 42, 49 and 56 days of age. Anticoagulated blood was collected from the jugular vein before morning feeding at 35, 42 and 49 days of age for routine blood tests.

[0225] The experimental results are as follows:

[0226] Some statistical results of the daily diarrhea rate of lambs in each group are shown in Figure 4 (Day 0 after weaning means lambs are 28 days old, day 1 after weaning means lambs are 29 days old, and so on; early weaning refers to the early weaning group, compound probiotics refers to the compound probiotics group, comparison probiotic 1 refers to comparison probiotics group 1, comparison probiotics 2 refers to comparison probiotics group 2, and comparison probiotics 3 refers to comparison probiotics group 3). The results showed that compared with the early weaning group, the diarrhea rates of lambs in the compound probiotics group, comparison probiotics group 1, comparison probiotics group 2, and comparison probiotics group 3 were significantly reduced; and the diarrhea rate of lambs in the compound probiotics group was the lowest, which was significantly lower than that of comparison probiotics group 1, comparison probiotics group 2, and comparison probiotics group 3. It can be seen that the effect of compound probiotics in preventing and treating lamb diarrhea is significantly better than that of comparison probiotics 1, comparison probiotics 2, and comparison probiotics 3.

[0227] Some statistical results of the average daily feed intake of lambs in each group are shown in Figure 5 (Day 0 after weaning means lambs are 28 days old, day 1 after weaning means lambs are 29 days old, and so on; early weaning is the early weaning group, compound probiotics is the compound probiotics group, comparison probiotic 1 is the comparison probiotics group 1, comparison probiotics 2 is the comparison probiotics group 2, and comparison probiotics 3 is the comparison probiotics group 3). The results showed that compared with the early weaning group, the daily feed intake of lambs in the compound probiotics group, comparison probiotics group 1, comparison probiotics group 2, and comparison probiotics group 3 increased significantly; and the daily feed intake of lambs in the compound probiotics group was significantly higher than that of the comparison probiotics group 1, comparison probiotics group 2, and comparison probiotics group 3. It can be seen that the effect of compound probiotics on increasing the daily feed intake of lambs is significantly better than that of comparison probiotics 1, comparison probiotics 2, and comparison probiotics 3.

[0228] The statistical results of the weight changes of lambs in each group are shown in Figure 6 (* indicates p < 0.05, i.e., there is a significant difference; -7 days after weaning means lambs are 21 days old, 0 days after weaning means lambs are 28 days old, and so on; early weaning is the early weaning group, compound probiotics is the compound probiotics group, comparative probiotic 1 is the comparative probiotics group 1, comparative probiotics 2 is the comparative probiotics group 2, and comparative probiotics 3 is the comparative probiotics group 3). The results showed that 14 days after weaning and 21 days after weaning, the weight of lambs in the compound probiotics group, comparative probiotics group 1, comparative probiotics group 2, and comparative probiotics group 3 increased significantly compared with the early weaning group. It can be seen that compound probiotics, comparative probiotics 1, comparative probiotics 2, and comparative probiotics 3 can alleviate the weight loss caused by weaning stress.

[0229] The blood routine test results (average value) of lambs in each group are shown in Table 1 (7 days after weaning, that is, lambs are 35 days old, 14 days after weaning, that is, lambs are 42 days old, and 21 days after weaning, that is, lambs are 49 days old; early weaning is the early weaning group, compound probiotics is the compound probiotics group, comparison probiotics 1 is the comparison probiotics group 1, comparison probiotics 2 is the comparison probiotics group 2, and comparison probiotics 3 is the comparison probiotics group 3). 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 probiotics group, comparison probiotics group 1, comparison probiotics group 2 and comparison probiotics group 3 were all reduced to a certain extent. It can be seen that the composite probiotics, comparative probiotics 1, comparative probiotics 2 and comparative probiotics 3 can all improve the body's inflammatory response; and the composite probiotics have a more obvious regulatory effect on granulocytes 7 days after weaning, and its effect on improving the body's inflammatory response is significantly better than that of comparative probiotics 1, comparative probiotics 2 and comparative probiotics 3. Since granulocytes are the main effector cells in the intestines of early weaned lambs, composite probiotics are more effective in alleviating intestinal inflammation in early weaned lambs.

[0230] Table 1

[0231]

[0232]

[0233] Note: Data in the same row with different letters a, b on their shoulders indicate significant differences between different groups (p<0.05), while data with the same letters indicate no significant differences (p>0.05).

[0234] Application of the composite probiotic prepared in Example 4 and Example 2 in the cultivation of early weaned goat lambs

[0235] In the composite probiotic agent of the present invention, the viable bacterial count concentration of Pediococcus pentosaceus M3 is specifically 4.40×10 14 CFU / mL, the viable bacterial count of Bacillus amyloliquefaciens CK4 is 8.80×10 12 CFU / mL, the viable bacterial count of Ludwig's Enterobacter CK6 is 5.90×10 13 CFU / mL, the viable bacterial count concentration of Lactobacillus Formatus is

[0236] 2.52×10 12 CFU / mL, the viable bacterial count of pool-grown Delftia is 5.10×10 13 CFU / mL.

[0237] The test site is a Boer goat farm in Shijiazhuang, Hebei. The test site is a semi-open goat house with good lighting and ventilation conditions.

[0238] Test subjects: Boer goats, goats. Before the test, blood was collected from all goats, serum was separated, and foot-and-mouth disease, Brucella, anthrax and peste des petits ruminants were tested to confirm that all goats were healthy. All lambs were kept in the same pen with their mothers after birth and were fed with starter feed at 7 days of age. All test lambs were ear-tagged, and the immunization procedure was implemented according to the regular procedures of the goat farm.

[0239] Experimental process: Boer goat lambs with a weaning age of 28 days were randomly selected and then randomly divided into two groups: a weaning group and a weaning + compound probiotics group, with 15 lambs in each group (half male and half female). That is, at the age of 28 days, each group of lambs was separated from the ewes (the lambs stayed in place and the ewes were moved away), and weaned once, and then the following experiments were carried out:

[0240] Weaning group: Feed starter feed at 8:00 and 17:00 every day.

[0241] Weaning + compound probiotics group: feed the starter feed containing compound probiotics at 8:00 a.m. and feed the starter feed at 5:00 p.m. every day.

[0242] The preparation method of the starter containing the composite probiotic is as follows: add an appropriate amount of drinking water to the composite probiotic, mix well and spray it on the surface of the starter to obtain the starter containing the composite probiotic; in the starter containing the composite probiotic, the ratio of the composite probiotic to the starter is 3-5 mL: 1 kg (specifically 3 mL: 1 kg in this embodiment).

[0243] The experiment lasted for 21 days. During the experiment, all lambs had free access to water and were fed starter feed twice a day at 8:00 and 17:00 respectively. The amount of feed was used until the lambs stopped actively eating. Each lamb was fed about 500-1000g of starter feed per day.

[0244] During the experiment, the lambs' feeding and diarrhea were observed at 9:00 and 18:00 every day, and the ear numbers of the dead lambs were recorded according to the ear tags. The mortality rate was further calculated. The amount of starter feed added and the remaining amount were recorded every day, and the daily feed intake was calculated and analyzed. The average daily feed intake and average daily feed intake were further calculated. In addition, the body weight of the lambs was measured before morning feeding at 28 and 49 days of age.

[0245] The experimental results are as follows:

[0246] The statistical results of the initial weight, final weight, daily weight gain and mortality of the two groups of lambs are shown in Table 2. The results showed that compared with the weaning group, the weight of the lambs in the weaning + compound probiotics group increased significantly, with an average daily weight gain of 151.19g, and the difference was significant (* indicates p < 0.05). Three lambs died in the weaning group, with a mortality rate of 20%; while there was no death in the weaning + compound probiotics group. It can be seen that feeding the composite probiotics to lambs can significantly increase the weight of the lambs and reduce the mortality rate of the lambs (that is, increase the survival rate of the lambs).

[0247] Table 2

[0248] Group Initial body weight (kg) Ending weight (kg) Daily weight gain (g) mortality rate(%) Weaning + compound probiotics group 14.57 17.75 151.19 0.0 Weaning group 13.95 15.05 52.38 20.0 p-value 0.72 0.11 <![CDATA[0.03 * ]]>

[0249] The results of the average daily feed intake of the two groups of lambs are shown in Table 3. The results show that during the entire experimental period, the average daily feed intake of the lambs in the weaning + compound probiotics group was as high as 650.02g, which was significantly different from the weaning group (*** indicates p < 0.001); in the first week, second week and third week after weaning, the average daily feed intake of the lambs in the weaning + compound probiotics group was significantly different from the weaning group (* indicates p < 0.05), significantly different (* indicates p < 0.05) and extremely significant (** indicates p < 0.01). It can be seen that feeding lambs with compound probiotics can significantly increase the daily feed intake of lambs.

[0250] Table 3

[0251]

[0252] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A composite bacterial agent comprising Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter ludwigii, Lactobacillus gestellt and Delftia foetida.

2. The composite bacterial agent according to claim 1, characterized in that: The composite bacterial agent consists of Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter Ludwigii, Lactobacillus gestellii and Delftia foetida.

3. The composite bacterial agent according to claim 1 or 2, characterized in that: The Pediococcus pentosaceus is Pediococcus pentosaceus M3; The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens CK4; The Ludwig Enterobacter is Ludwig Enterobacter (Enterobacter ludwigii) CK6; Pediococcus pentosaceus M3, whose deposit number in the General Microbiological Center of China Culture Collection Administration is CGMCC No.30882; Bacillus amyloliquefaciens CK4, whose deposit number is CGMCC No.31178 in the General Microbiological Center of China Culture Collection Administration; Enterobacter ludwigii CK6, whose deposit number in the General Microbiological Center of China Culture Collection Administration is CGMCC No.31179.

4. The composite bacterial agent according to any one of claims 1 to 3, characterized in that: In the composite bacterial agent, the viable counts of Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter Ludwigii, Lactobacillus gestellii and Delftia foetida were all 2×10 11 CFU / mL and above.

5. The composite bacterial agent according to any one of claims 1 to 3, characterized in that: In the composite bacterial agent, the ratio of the number of live bacteria of Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter Ludwigii, Lactobacillus gestellii and Delftia foetida is 4.40×10 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.

6. The composite bacterial agent according to claim 5, characterized in that: In the composite bacterial agent, the ratio of the number of live bacteria of Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter Ludwigii, Lactobacillus gestellii and Delftia foetida is 4.40×10 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.

7. The composite bacterial agent according to any one of claims 1 to 6, characterized in that: When preparing the composite bacterial agent, Pediococcus pentosaceus, Bacillus amyloliquefaciens, Enterobacter Ludwigii, Lactobacillus gestellii and Delftia foetida are all added in the form of bacterial liquid.

8. The use of the composite bacterial agent according to any one of claims 1 to 7, which is at least one of A1) to A14): A1) Improve the survival rate of weaned lambs; A2) preparing a product for improving the survival rate of weaned lambs; A3) Reduce diarrhea rate and / or mortality of weaned lambs; A4) preparing a product for reducing diarrhea rates and / or mortality in weaned lambs; A5) Increase feed intake and / or daily feed intake of weaned lambs; A6) Preparation of products for increasing feed intake and / or daily feed intake of weaned lambs; A7) Increase the body weight and / or daily weight gain of weaned lambs; A8) preparing a product for increasing body weight and / or daily weight gain of weaned lambs; A9) Promote the growth and development of weaned lambs; A10) preparing products for promoting growth and development of weaned lambs; A11) reducing the inflammatory response of weaned lambs and / or alleviating intestinal inflammation of weaned lambs; A12) preparing a product for reducing inflammatory response in weaned lambs and / or alleviating intestinal inflammation in weaned lambs; A13) raising weaned lambs; A14) Preparation of products for rearing weaned lambs.

9. The use according to claim 8, characterized in that: The weaned lambs are early weaned lambs; The early weaned lambs are preferably 28 to 35 days old lambs.

10. The use according to claim 8 or 9, characterized in that: The lamb is a sheep lamb or a goat lamb.

Images