Complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention discloses a complex microbial inoculant as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. The complex microbial inoculant disclosed by the invention is prepared by co-fermentation of lactobacillus plantarum CGMCC (China General Microbiological Culture Collection Center) NO.12849 and enterococcus faecium CGMCC The complex microbial inoculant has the characteristics of high antibacterial activity, safety, no toxicity, high stability and the like, and can be used for killing clinically common gram-negative pathogenic bacteria. By utilizing the high-density fermentation culture medium, the fermentation process and the preparation process provided by the invention, the complex microbial inoculant can be produced on a large scale and is low in cost, and the complex microbial inoculant is expected to replace antibiotics to become a safe, green and efficient ideal antibacterial agent and has a very wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a composite bacterial agent and a preparation method and application thereof. Background Art

[0002] In the animal breeding process, animal intestinal inflammation and diarrhea caused by Gram-negative pathogens such as Escherichia coli and Salmonella are very serious problems. Adding antibiotics to feed can play a role in disease prevention and disease promotion and animal growth. However, long-term use of antibiotics can lead to increased drug resistance of animal intestinal pathogens, antibiotic residues in livestock and poultry products, and environmental pollution, posing a threat to animal and even human health. In the current context of antibiotic prohibition, research on the efficient and scientific application of antibiotic substitutes in animal breeding production is of great significance and has broad prospects.

[0003] At present, probiotics and prebiotics are very promising alternatives to antibiotics. Many studies have shown that lactic acid bacteria have significant effects in resisting pathogen infection. Lactic acid bacteria antagonize the growth, reproduction, and colonization of pathogens by producing antibacterial metabolites, enhancing the barrier function of the intestinal epithelium, competing with pathogens for adhesion sites, regulating the structure of the intestinal flora, and regulating intestinal immunity, thereby resisting the infection of pathogens to the body or reducing inflammatory responses and alleviating the symptoms of related intestinal diseases. In addition, some studies have found that under the premise of equal doses, the combined use of probiotics of different species is better than the use of a single probiotic. This may be because mixed probiotic preparations can combine the probiotic effects of different strains and produce a synergistic effect. Therefore, the development of composite lactic acid bacteria microecological preparations has important practical application value. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a composite bacterial agent, which is prepared by co-fermentation of Lactobacillus plantarum CGMCC NO.12849 and Enterococcus faecium CGMCC NO.9134.

[0005] Furthermore, the composite bacterial agent is prepared by inoculating Lactobacillus plantarum CGMCC NO.12849 and Enterococcus faecium CGMCC NO.9134 into a fermentation medium at a ratio of 1:1.

[0006] Furthermore, the viable bacterial count of the composite bacterial agent is 1×10 9 CFU / mL~4×10 10 CFU / mL.

[0007] The present invention also provides a fermentation method of the composite bacterial agent, and the fermentation steps are as follows:

[0008] (1) Primary seed liquid culture: single colonies of Lactobacillus plantarum CGMCC No. 12849 and Enterococcus faecium CGMCC No. 9134 were taken from plates and inoculated into MRS liquid medium, and then placed in anaerobically at 37° C. for 24 hours to obtain primary culture fermentation liquid;

[0009] (2) Secondary seed solution culture: the primary seed solution of Lactobacillus plantarum CGMCC No. 12849 and Enterococcus faecium CGMCC No. 9134 cultured in step 1 is inoculated into MRS liquid culture medium at an inoculation amount of 3% and cultured at 37° C. overnight to obtain a secondary seed solution;

[0010] (3) Liquid fermentation: After the fermentation medium is sterilized, cooling water is passed through to keep the temperature at 32° C. The secondary seed liquid is inoculated at a 10% inoculum into a 5L fermentation tank containing 3.5L of liquid fermentation medium. The pH is adjusted to 5.5 by pH-ammonia coupling. The fermentation is cultured for 16-24 hours.

[0011] Furthermore, the components and contents of the liquid fermentation medium are as follows:

[0012] Yeast powder 4g / L, beef powder 6g / L, peptone 10g / L, molasses 20g / L, dipotassium hydrogen phosphate 2g / L, triammine citrate 2g / L, manganese sulfate 0.05g / L, magnesium sulfate 0.2g / L, sodium acetate 5g / L, Tween 80 1mL / L.

[0013] The present invention also provides a spray-dried powder of the composite bacterial agent, and the preparation method of the spray-dried powder is as follows:

[0014] The fermentation liquid obtained by co-fermentation of Lactobacillus plantarum CGMCC No. 12849 and Enterococcus faecium CGMCC No. 9134 is centrifuged to obtain a supernatant, and the supernatant is mixed with a carrier and spray-dried to obtain the product.

[0015] Furthermore, the carrier is made of 10% corn starch, 10% talcum powder and 0.1% white carbon black by mass.

[0016] The present invention also provides a microecological preparation of the composite bacterial agent, wherein the microecological preparation comprises the composite bacterial agent.

[0017] The present invention also provides the use of the composite bacterial agent in preparing feed or medicine for inhibiting Gram-negative pathogenic bacteria, wherein the Gram-negative pathogenic bacteria are Escherichia coli and / or Salmonella.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The composite bacterial agent provided by the present invention has the characteristics of fast acid production, broad antibacterial spectrum, non-cytotoxicity, non-hemolytic, high stability, etc., and can kill microorganisms including common clinical drug-resistant strains, bacteria and fungi. After testing, the co-fermentation of Lactobacillus plantarum CGMCC NO.12849 and Enterococcus faecium CGMCC NO.9134 in the composite bacterial agent of the present invention can synergize and achieve excellent bactericidal effects. After the composite bacterial agent of the present invention is spray-dried to form a powder, it has the advantages of heat resistance, pH resistance and gastrointestinal environment resistance. Utilizing the high-density fermentation medium, fermentation process and preparation process provided by the present invention, the composite bacterial agent is easy to produce on a large scale and is low in cost, and is expected to replace antibiotics to become a safe, green and efficient ideal antibacterial agent, and has a very broad application prospect in the fields of preparing antibacterial drugs, antibacterial preparations and antibacterial products. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Results of the minimum inhibitory concentration (maximum dilution multiple) of the composite bacterial agent against different pathogenic bacteria. DETAILED DESCRIPTION

[0021] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased from the market.

[0022] Example 1 Screening and combination of composite bacterial agents

[0023] 1. Culture medium preparation:

[0024] MRS liquid culture medium, the composition and content are as follows: yeast powder 5g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, diammonium citrate 2g / L, sodium acetate 3g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, Tween 80 1mL / L.

[0025] 2. Primary activation:

[0026] Single colonies of Lactobacillus plantarum CGMCC NO.12849, Enterococcus faecium CGMCC NO.9134, Lactobacillus reuteri CGMCC NO.19493 and Lactobacillus acidophilus CGMCC No.5093 were inoculated into MRS liquid culture medium, and cultured anaerobically at 37°C for 24 hours to obtain primary culture fermentation liquid.

[0027] Among them, Lactobacillus plantarum CGMCC NO.12849 is a strain owned by this laboratory, which is named Lactobacillus plantarum DBNZW02 (Lactobacillus plantarum DBNZW02), and was deposited on August 15, 2016 in the General Microbiological Center of China Culture Collection Administration, referred to as CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC No. 12849, see patent CN108570421B for details.

[0028] Enterococcus faecium CGMCC NO.9134 is a strain of our laboratory, named Enterococcus faecium UE01, which was deposited on May 8, 2014 at the General Microbiology Center of China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.9134. For details, see patent CN104388327B.

[0029] Lactobacillus reuteri CGMCC NO.19493 is a strain of our laboratory, named Lactobacillus reuteri DBN-JP, which was deposited at the General Microbiology Center of China National Committee for the Preservation of Microorganisms on March 19, 2020, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 19493. For details, see patent CN112011481B.

[0030] Lactobacillus acidophilus CGMCC No. 5093 is a strain owned by our laboratory. The applicant has deposited it in the General Microbiological Center of China National Committee for Microbiological Culture Collection (hereinafter referred to as CGMCC) on July 26, 2011. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 5093. For details, see patent CN102559539A.

[0031] 3. Antibacterial scheme and effect evaluation:

[0032] The first-level fermentation broth of Lactobacillus plantarum CGMCC NO.12849, Lactobacillus acidophilus CGMCC NO.5093, Enterococcus faecium CGMCC NO.9134 and Lactobacillus reuteri CGMCC NO.19493 was inoculated into MRS liquid culture medium with an inoculation amount of 1% and placed in static anaerobic culture at 37°C for 24 hours to obtain the second-level fermentation broth. After the fermentation was completed, the number of viable bacteria in the fermentation broth was (1-3)×10 9 CFU / mL, and the antibacterial evaluation was performed by the Oxford cup method.

[0033] Antibacterial evaluation method:

[0034] The double-layer agar Oxford cup method was used to determine the inhibitory diameter of the samples against Escherichia coli K88 and Salmonella SL1344.

[0035] In the clean bench, pour about 7-10mL of sterilized 0.8% plain agar into each sterilized petri dish to make the bottom layer uniform. After the plain agar cools and solidifies, place the sterilized Oxford cup on the agar. Dilute the indicator bacteria cultured overnight by 200 times and add it to the culture medium at a constant temperature of 50±5℃ at a ratio of 1%. Shake it gently to avoid foaming. Pour the culture medium with bacterial solution into the water agar culture dish with the Oxford cup placed. Pour 20mL into each culture dish. The thickness of the bacterial layer in each culture dish should be uniform.

[0036] After the above culture medium cools and solidifies, use tweezers to take out the Oxford cup, mark it on the culture dish, and draw 150μL of the sample solution to be tested into the corresponding well. Diffusion at room temperature for about 1 to 2 hours. Transfer the culture dish with the sample to a 37℃ incubator for incubation, and observe the results after 12-16 hours of incubation. Use a vernier caliper to measure the diameter of the antibacterial zone in the plate to determine the antibacterial effect of the sample.

[0037] The antibacterial scheme and ratio are as follows:

[0038] (1) Secondary fermentation broth of Lactobacillus plantarum CGMCC NO.12849;

[0039] (2) Lactobacillus acidophilus CGMCC NO.5093 secondary fermentation broth;

[0040] (3) Secondary fermentation broth of Enterococcus faecium CGMCC No.9134;

[0041] (4) Lactobacillus reuteri CGMCC NO.19493 secondary fermentation broth;

[0042] (5) inoculating the primary fermentation broth with Lactobacillus plantarum CGMCC NO.12849 and Lactobacillus acidophilus CGMCC NO.5093 (1:1);

[0043] (6) Inoculate the primary fermentation broth with Lactobacillus plantarum CGMCC NO.12849 and Enterococcus faecium CGMCC NO.9134 (1:1);

[0044] (7) inoculating the primary fermentation broth with Lactobacillus plantarum CGMCC NO.12849 and Lactobacillus reuteri CGMCC NO.19493 (1:1);

[0045] (8) Lactobacillus acidophilus CGMCC No.5093 and Enterococcus faecium CGMCC No.9134 (1:1) were inoculated into the primary fermentation broth;

[0046] (9) inoculating the primary fermentation broth with Lactobacillus acidophilus CGMCC No.5093 and Lactobacillus reuteri CGMCC No.19493 (1:1);

[0047] (10) Enterococcus faecium CGMCC NO.9134 and Lactobacillus reuteri CGMCC NO.19493 (1:1) were inoculated into the primary fermentation broth;

[0048] (11) Inoculate the primary fermentation broth with Lactobacillus plantarum CGMCC NO.12849, Lactobacillus acidophilus CGMCC NO.5093, and Enterococcus faecium CGMCC NO.9134 (1:1:1);

[0049] (12) Inoculate the primary fermentation broth with Lactobacillus plantarum CGMCC NO.12849, Lactobacillus acidophilus CGMCC NO.5093, and Lactobacillus reuteri CGMCC NO.19493 (1:1:1);

[0050] (13) Lactobacillus acidophilus CGMCC No.5093, Enterococcus faecium CGMCC No.9134, and Lactobacillus reuteri CGMCC No.19493 (1:1:1) were inoculated into the primary fermentation broth;

[0051] (14) Inoculate the primary fermentation broth with Lactobacillus plantarum CGMCC NO.12849, Lactobacillus acidophilus CGMCC NO.5093, Enterococcus faecium CGMCC NO.9134, and Lactobacillus reuteri CGMCC NO.19493 (1:1:1:1);

[0052] The antibacterial results of the above treatments are shown in Table 1. The results show that Scheme 13, i.e., inoculating the primary fermentation broth with Lactobacillus acidophilus CGMCC NO.5093, Enterococcus faecium CGMCC NO.9134 and Lactobacillus reuteri CGMCC NO.19493 (1:1:1), has the best antibacterial effect. However, considering the production cost, Scheme 6, i.e., inoculating the primary fermentation broth with Lactobacillus plantarum CGMCC NO.12849 and Enterococcus faecium CGMCC NO.9134 (1:1), is adopted.

[0053] Table 1 Comparison of antibacterial activity of different lactic acid bacteria alone and in combination

[0054]

[0055]

[0056] Example 2 Preparation of composite bacterial agent

[0057] 1. Culture medium preparation:

[0058] (1) The seed culture medium is MRS medium, and its composition and content are as follows: yeast powder 5 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium citrate 2 g / L, sodium acetate 3 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1 mL / L.

[0059] (2) Screening of fermentation medium for tank fermentation, the composition and content are as follows:

[0060] Solution 1: sucrose 29g / L, peptone 25g / L, yeast extract powder 31g / L, sodium acetate 6g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, manganese sulfate tetrahydrate 0.1g / L, magnesium sulfate 1g / L, Tween 80 1g / L.

[0061] Solution 2: Yeast powder 4g / L, beef powder 6g / L, peptone 10g / L, molasses 20g / L, dipotassium hydrogen phosphate 2g / L, triammine citrate 2g / L, manganese sulfate 0.05g / L, magnesium sulfate 0.2g / L, sodium acetate 5g / L, Tween 80 1mL / L.

[0062] 2. High-density fermentation method

[0063] (1) Primary seed liquid culture

[0064] Single colonies of Lactobacillus plantarum CGMCC NO.12849 and Enterococcus faecium CGMCC NO.9134 were taken from the plates, inoculated into MRS liquid culture medium, and cultured anaerobically at 37°C for 24 hours to obtain primary culture fermentation liquid.

[0065] (2) Secondary seed liquid culture

[0066] The first-level seed solution (1:1) of Lactobacillus plantarum CGMCC NO.12849 and Enterococcus faecium CGMCC NO.9134 cultured in step 1 was inoculated into MRS liquid culture medium with an inoculation amount of 3% and cultured at 37° C. overnight to obtain a second-level seed solution.

[0067] (3) Liquid fermentation

[0068] After the fermentation medium is sterilized, cooling water is passed to keep it warm to 32°C, and the secondary seed liquid is inoculated at a 10% inoculation rate into a 5L fermentation tank containing 3.5L of liquid fermentation medium. The pH is coupled with ammonia water, and the pH is adjusted at 5.5. The fermentation culture is carried out for 16-24 hours, and the fermentation is completed.

[0069] The antibacterial activity of the fermentation liquid of the composite bacteria system cultured in different tank culture media is compared in Table 2. The results show that the antibacterial activity of Scheme 1 is higher. After determination, the number of viable bacteria in the fermentation liquid after fermentation using the fermentation medium of Schemes 1 and 2 was 3.7×10 10 CFU / mL and 5.63×10 9 CFU / mL.

[0070] Table 2 Comparison of antibacterial activity of composite bacterial fermentation liquid cultured in different tank culture media

[0071] Escherichia coli K88 Salmonella SL1344 1 20.03mm 19.49mm 2 13.27mm 10.98mm

[0072] 3. Spray-dried powder preparation

[0073] The carrier formula composition and ratio are as follows (substance concentration in 1L fermentation broth):

[0074] (1) Corn starch 20%, white carbon black 0.1%;

[0075] (2) talc 20%;

[0076] (3) Corn starch 10%, talcum powder 10%, white carbon black 0.1%.

[0077] The fermentation broth obtained in step 2 was centrifuged (8000rpm, 10min), different carriers were added to the supernatant, the inlet air temperature was 160°C, the outlet air temperature was 80°C, and the powder was spray-dried. 1.000g of the spray-dried powder was weighed, 9mL of sterile water was added, and the mixture was shaken at 220rpm and 37°C for 0.5h to obtain a 10-fold dilution, which was then diluted 10 times to obtain a 100-fold dilution to be tested. The antibacterial test was then performed according to the antibacterial evaluation method in Example 1.

[0078] The antibacterial results of different carrier formulas are shown in Table 3. The results show that the antibacterial effect of formula 3 is the best. Under this formula, the minimum inhibitory concentration of various pathogens was determined. The minimum inhibitory concentrations of Escherichia coli and Salmonella were 62.5μg / mL, which is 16000 times diluted ( Figure 1 ).

[0079] Table 3 Comparison of antibacterial activity of different spray-dried carriers

[0080] Escherichia coli K88 Salmonella SL1344 1 20.00mm 19.98mm 2 19.27mm 20.03mm 3 22.03mm 20.83mm

[0081] Example 3 Study on the stability of composite bacteria

[0082] The composite bacterial agent of this embodiment was prepared according to the method of Example 2, and the fermentation medium in the upper tank included: sucrose 29 g / L, peptone 25 g / L, yeast extract powder 31 g / L, sodium acetate 6 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, manganese sulfate tetrahydrate 0.1 g / L, magnesium sulfate 1 g / L, and Tween 80 1 g / L.

[0083] 1. High temperature resistance

[0084] Weigh 5 portions of composite bacterial agents, one of which is a control, and the other three are placed in a water bath at 80, 90, and 100°C for 0.5 h; the last portion is placed in an autoclave and treated at 121°C for 0.5 h. After the sample is cooled, the antibacterial changes are detected. The results are shown in Table 4. There is no significant change in the antibacterial activity of the lactic acid bacteria composite bacteria under different temperature treatments, indicating that the lactic acid bacteria composite bacteria are resistant to high temperatures.

[0085] Table 4 Comparison of antibacterial activity at different temperatures

[0086] CK 80℃ 90℃ 100℃ 121℃ Escherichia coli K88 19.99mm 19.47mm 19.03mm 18.88mm 18.03mm Salmonella SL1344 19.44mm 19.45mm 19.12mm 19.50mm 18.97mm

[0087] 2. pH resistance

[0088] The composite bacterial agent was adjusted to pH 2.0, 4.0, 6.0, 8.0, and 10.0 with 1 mol / L HCl and 1 mol / L NaOH, respectively, and the pH was adjusted to neutral after standing at room temperature for 2 hours. The antibacterial activity was determined with the untreated composite bacterial agent as the control. The results are shown in Table 5, and it can be seen that the pH change has no effect on the antibacterial activity of the composite bacterial agent.

[0089] Table 5 Comparison of antibacterial activity at different pH treatments

[0090]

[0091]

[0092] 3. Simulated gastrointestinal tolerance test

[0093] Simulated gastric fluid: Pepsin was dissolved in PBS (pH 3.0) to a final concentration of 1 g / L and sterilized by filtration using a 0.22 μm filter membrane.

[0094] Simulated intestinal fluid: Trypsin was dissolved in PBS (pH 8.0) to a final concentration of 1 g / L, and sterilized by filtration using a 0.22 μm filter membrane.

[0095] The composite bacterial agent was digested with trypsin and pepsin respectively, and the antibacterial activity was determined in a water bath at 37°C for 4 hours. The results are shown in Table 6, which show that the composite bacterial agent can tolerate the gastrointestinal tract.

[0096] Table 6 Simulated gastrointestinal tolerance results

[0097] CK Pepsin Trypsin Escherichia coli K88 19.79mm 19.68mm 19.39mm Salmonella SL1344 20.06mm 19.98mm 19.61mm

[0098] 4.37℃ accelerated test

[0099] The composite bacterial agent was placed in an aluminum foil bag, sealed and packaged, and placed in an incubator at 25°C and 37°C for 1 month, and the antibacterial activity was tested every 5 days. The results are shown in Table 7, and it can be seen that the antibacterial activity of the composite bacterial agent at room temperature and 37°C for 1 month did not change significantly, indicating that the composite bacterial agent of the present invention is relatively stable.

[0100] Table 7 Accelerated test results

[0101]

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite bacterial agent, characterized in that: The composite bacterial agent is prepared by co-fermentation of Lactobacillus plantarum CGMCC NO.12849 and Enterococcus faecium CGMCC NO.9134.

2. The composite bacterial agent according to claim 1, characterized in that The composite bacterial agent is prepared by inoculating Lactobacillus plantarum CGMCC NO.12849 and Enterococcus faecium CGMCC NO.9134 into a fermentation medium in a ratio of 1:

1.

3. The composite bacterial agent according to claim 1, characterized in that The viable bacterial count of the composite bacterial agent is 1×10 9 CFU / mL~4×10 10 CFU / mL.

4. The fermentation method of the composite bacterial agent according to any one of claims 1 to 3, characterized in that: The fermentation steps are as follows: (1) Primary seed liquid culture: single colonies of Lactobacillus plantarum CGMCC No. 12849 and Enterococcus faecium CGMCC No. 9134 were taken from plates and inoculated into MRS liquid medium, and then placed in anaerobically at 37° C. for 24 hours to obtain primary culture fermentation liquid; (2) Secondary seed solution culture: the primary seed solution of Lactobacillus plantarum CGMCC No. 12849 and Enterococcus faecium CGMCC No. 9134 cultured in step 1 is inoculated into MRS liquid culture medium at an inoculation amount of 3% and cultured at 37° C. overnight to obtain a secondary seed solution; (3) Liquid fermentation: After the fermentation medium is sterilized, cooling water is passed through to keep the temperature at 32° C. The secondary seed liquid is inoculated at a 10% inoculum into a 5L fermentation tank containing 3.5L of liquid fermentation medium. The pH is adjusted to 5.5 by pH-ammonia coupling. The fermentation is cultured for 16-24 hours.

5. The fermentation method according to claim 4, characterized in that The components and contents of the liquid fermentation medium are as follows: Yeast powder 4g / L, beef powder 6g / L, peptone 10g / L, molasses 20g / L, dipotassium hydrogen phosphate 2g / L, triammine citrate 2g / L, manganese sulfate 0.05g / L, magnesium sulfate 0.2g / L, sodium acetate 5g / L, Tween 80 1mL / L.

6. A spray-dried powder containing the composite bacterial agent according to any one of claims 1 to 3, characterized in that: The fermentation liquid obtained by co-fermentation of Lactobacillus plantarum CGMCC No. 12849 and Enterococcus faecium CGMCC No. 9134 is centrifuged to obtain a supernatant, and the supernatant is mixed with a carrier and spray-dried to obtain the product.

7. The spray-dried powder according to claim 6, characterized in that The carrier is made of 10% corn starch, 10% talcum powder and 0.1% white carbon black by mass.

8. A microecological preparation containing the composite bacterial agent according to any one of claims 1 to 3.

9. Use of the composite bacterial agent according to any one of claims 1 to 3 in the preparation of feed or medicine for inhibiting Gram-negative pathogenic bacteria.

10. The use according to claim 9, characterized in that The Gram-negative pathogenic bacteria are Escherichia coli and / or Salmonella.

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