Microecological preparation as well as preparation method and application thereof

Abstract

The invention discloses a microecological preparation as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. The microecological preparation disclosed by the invention is prepared from bacillus subtilis CGMCC (China General Microbiological Culture Collection Center) NO.25499 and a related carrier. The microecological preparation disclosed by the invention has the characteristics of efficient antibacterial activity, no cytotoxicity, no hemolysis, high stability and the like, and can be used for killing microorganisms including clinically common drug-resistant strains, bacteria and the like. By utilizing the high-density fermentation culture medium, the fermentation process and the preparation process provided by the invention, the microecological preparation is convenient for large-scale production and low in cost, and is expected to replace antibiotics to become a safe, green and efficient ideal antibacterial product.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a microecological preparation and a preparation method and application thereof. Background Art

[0002] Adding antibiotic feed additives to animal feed can play a role in disease prevention and disease prevention and promote animal growth. However, long-term use of antibiotics can lead to problems such as increased resistance of animal intestinal pathogens, antibiotic residues in livestock and poultry products, and environmental pollution, posing a threat to animal and even human health. Under the current situation, from the perspectives of animal breeding safety and consumer health, it is of great significance to deeply study the efficient and scientific application of antibiotic substitutes in animal breeding production. Bacillus subtilis, also known as Bacillus subtilis, is a type of aerobic, endogenous, resistant spore rod-shaped Gram-positive bacteria that is non-pathogenic and can secrete enzymes such as amylase, protease, lipase and cellulase. In addition, studies have shown that fermentation of Bacillus subtilis can produce antimicrobial peptide substances such as polypeptides and lipopeptides. However, most antimicrobial peptides are not resistant to high temperatures, which greatly increases the cost of preparations (lyophilization); at the same time, the application of antimicrobial peptides is often restricted by poor stability (intolerant to the gastrointestinal tract). Therefore, how to develop efficient and stable antimicrobial preparations has very important practical application value. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a probiotic preparation, which is prepared by adding a carrier to Bacillus fermentation liquid and spray-drying it to form a powder, wherein the carrier is made of one of gum arabic, white carbon black, manganese sulfate and diatomaceous earth, light calcium carbonate, yeast cell wall, and enhanced powder.

[0004] Furthermore, the carrier is made of diatomaceous earth, gum arabic, white carbon black and manganese sulfate.

[0005] Furthermore, 1-5% diatomaceous earth, 1-10% gum arabic, 0.1-0.5% white carbon black and 0.1-1% manganese sulfate are added to each liter of bacillus fermentation liquid.

[0006] Furthermore, 2.5% diatomaceous earth, 1% gum arabic, 0.1% white carbon black and 0.5% manganese sulfate were added to each liter of the bacillus fermentation liquid.

[0007] Further, the fermentation method of the bacillus fermentation broth is as follows:

[0008] The seed liquid of Bacillus is inoculated into a 100L fermentation tank filled with 50L of liquid fermentation medium at a 10% inoculation amount, the dissolved oxygen is controlled at 30%-40%, and the fermentation is cultured for 12-18h.

[0009] Furthermore, the Bacillus is Bacillus licheniformis or Bacillus subtilis.

[0010] Furthermore, the Bacillus subtilis is Bacillus subtilis CGMCC NO.25499.

[0011] Furthermore, the number of viable bacteria in the Bacillus fermentation broth is 2*10 9 CFU / mL~5*10 9 CFU / mL.

[0012] The present invention also provides the use of the microecological preparation in preparing medicine or feed for inhibiting Clostridium perfringens and / or Staphylococcus aureus.

[0013] The invention also provides application of the microecological preparation in preparing bacillus powder.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The microecological preparation provided by the present invention has the characteristics of efficient antibacterial activity, no cytotoxicity, no hemolysis, high stability, etc. to Gram-positive bacteria, and can kill microorganisms including common clinically resistant strains, bacteria, etc. The microecological preparation prepared by the method of the present invention has excellent antibacterial properties and good stability. Utilizing the high-density fermentation medium, fermentation process and preparation process provided by the present invention, the microecological preparation is easy to generate on a large scale and at low cost, and is expected to replace antibiotics to become a safe, green and efficient ideal antibacterial product. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the minimum inhibitory concentration (maximum dilution multiple) of microecological preparations against different pathogenic bacteria. DETAILED DESCRIPTION

[0017] 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.

[0018] Example 1 Preparation of microecological preparations

[0019] The present embodiment involves strains: Bacillus licheniformis, Bacillus subtilis CGMCC NO.13010, Bacillus velez CGMCC NO.27887 and Bacillus subtilis CGMCC NO.25499.

[0020] Among them, Bacillus licheniformis was purchased.

[0021] Bacillus subtilis CGMCC NO.13010 is a strain of our laboratory, named Bacillus subtilis 10#, which was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee on September 19, 2016, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, classified and named: Bacillus subtilis, and the deposit number is CGMCC No.13010. For details, see patent CN118667706A.

[0022] Bacillus velezensis CGMCC NO.27887 is a strain of our laboratory, named H16, and classified as Bacillus velezensis. It has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, referred to as CGMCC, on July 14, 2023. The address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 27887. For details, see patent CN117568210A.

[0023] Bacillus subtilis CGMCC NO.25499 is a strain of our laboratory, named Bacillus subtilis BS-ZPW, classified as Bacillus subtilis, and deposited on August 8, 2022 at the General Microbiology Center of China Culture Collection Administration, referred to as CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC NO.25499.

[0024] 1. Culture medium preparation:

[0025] (1) The seed culture medium is LB medium, and the composition and content are as follows: peptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L.

[0026] (2) The composition and content of the fermentation medium in the upper tank are as follows: peptone 8 g / L, soybean cake powder 40 g / L, glucose 5 g / L, corn starch 40 g / L, yeast extract powder 3 g / L, potassium phosphate trihydrate 2 g / L, magnesium sulfate 0.25 g / L, manganese sulfate 0.3 g / L, light calcium carbonate 3 g / L, sodium chloride 2 g / L, pH 7.0±0.2.

[0027] 2. High-density fermentation method

[0028] (1) Primary seed liquid culture

[0029] Take a single colony from each strain plate, inoculate it into LB liquid culture medium, and culture it at 32°C for 16-18 hours to obtain the first-level seed solution.

[0030] (2) Secondary seed liquid culture

[0031] The first-level seed solution cultured in step 1 was inoculated into LB liquid culture medium with an inoculation amount of 2% and cultured at 32° C. overnight to obtain the second-level seed solution.

[0032] (3) Liquid fermentation

[0033] After the fermentation medium was sterilized, cooling water was passed to keep the temperature at 32°C. The secondary seed liquid was inoculated with 10% inoculum into a 100L fermentation tank containing 50L of liquid fermentation medium. The dissolved oxygen was controlled at 30%-40%. The fermentation culture was carried out for 12-18h. The number of viable Bacillus licheniformis was 1.26*10 9 CFU / mL, the number of viable bacteria of Bacillus subtilis CGMCC NO.13010 is 1.08*10 9 CFU / mL, the number of viable bacteria of Bacillus Velezii CGMCC NO.27887 is 3.9*10 9 CFU / mL, the number of viable bacteria of Bacillus subtilis CGMCC NO.25499 is 2.68*10 9 The antibacterial activity of the fermentation broth (Clostridium perfringens and Staphylococcus aureus) was detected, and the results are shown in Table 1. It can be seen from the results that Bacillus subtilis CGMCC NO.25499 has the strongest antibacterial activity and the largest dilution multiple. The fermentation broth of Bacillus subtilis CGMCC NO.25499 was selected for further post-treatment.

[0034] Table 1 Comparison of antibacterial activity of fermentation broths of different strains

[0035]

[0036] 3. Spray-dried powder preparation

[0037] The formulation composition and proportion of the carrier are as follows (substance concentration in 1L fermentation broth):

[0038] Solution 1. 5% diatomaceous earth, 1% gum arabic, 0.1% white carbon, 0.5% manganese sulfate;

[0039] Solution 2. Diatomaceous earth 2.5%, gum arabic 1%, white carbon black 0.1%, manganese sulfate 0.5%;

[0040] Scheme 3. Yeast cell wall 5%, gum arabic 1%, white carbon 0.1%, manganese sulfate 0.5%;

[0041] Solution 4: Add 5% white powder, 1% gum arabic, 0.1% white carbon black, and 0.5% manganese sulfate;

[0042] Scheme 5: Add 5% yellow powder, 1% gum arabic, 0.1% white carbon black, and 0.5% manganese sulfate;

[0043] Solution 6. Light calcium carbonate 5%, gum arabic 1%, white carbon black 0.1%, manganese sulfate 0.5%;

[0044] Scheme 7. Corn starch 5%, maltodextrin 5%, white carbon black 0.1%, gum arabic 1%.

[0045] The fermentation liquid of Bacillus subtilis CGMCC NO.25499 was filtered through a 40-mesh filter, and different carriers were added to the filtrate. The inlet air temperature was 160°C, the outlet air temperature was 80°C, and the powder was spray-dried. Weigh 1g of spray-dried powder, add 19mL of sterile water, shake at 220rpm and 37°C for 0.5h, and obtain a 20-fold dilution to be tested, and test the antibacterial effect on Clostridium perfringens and Staphylococcus aureus.

[0046] The antibacterial results of different carrier formulas are shown in Table 2. " / " indicates that the viscosity is too high to be powdered. The results show that the antibacterial effect of formula 2 is the best. The minimum inhibitory concentration for pathogenic bacteria was determined under this formula. The results are shown in Figure 1 , probiotic preparations can dilute Clostridium perfringens by about 4000 times (250 μg / mL) and Staphylococcus aureus by 16000 times (62.5 μg / mL).

[0047] Table 2 Comparison of antibacterial activity of different spray-dried carriers

[0048] 1 2 3 4 5 6 7 Golden grape 15.38mm 15.96mm 13.59mm 12.54mm 13.88mm 12.94mm / Gas production 13.05mm 17.35mm 11.66mm 12.95mm 11.58mm 10.91mm /

[0049] Comparative test

[0050] The strains involved are: Lactobacillus plantarum CGMCC NO.12849, Lactobacillus acidophilus CGMCC No.5093, Bacillus Velez CGMCC NO.27887, Bacillus subtilis CGMCC NO.13010, and Bacillus subtilis CGMCC NO.25499.

[0051] 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.

[0052] 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.

[0053] Referring to Example 1, different strains of fermentation liquid were cultured (lactic acid bacteria culture medium was MRS culture medium) (see Table 3 for details), and the viable count of Lactobacillus plantarum CGMCC NO.12849 was 2.3*10 9 CFU / mL, the number of viable Lactobacillus acidophilus CGMCC No.5093 is 1.82*10 9 CFU / mL, the number of viable Bacillus licheniformis is 1.75*10 9 CFU / mL, the number of viable bacteria of Bacillus subtilis CGMCC NO.13010 is 1.5*10 9 CFU / mL, the number of viable bacteria of Bacillus subtilis CGMCC NO.25499 is 2.75*10 9 CFU / mL. The selected spray-drying carrier formula is 2.5% diatomaceous earth, 1% gum arabic, 0.1% white carbon black, and 0.5% manganese sulfate. The antibacterial effect is compared under the same spray-drying conditions.

[0054] As can be seen from Table 3, this carrier formula is not suitable for lactic acid bacteria series. For spore-forming bacteria, this carrier formula can be successfully spray-dried and powdered. Therefore, it is inferred that this carrier formula is suitable for spray-dried powders of spore-forming bacteria fermentation liquid. The dilution multiple depends only on the concentration of antibacterial substances that the strain itself can produce. It can be seen that Bacillus subtilis CGMCC NO.25499 produces the highest concentration of antibacterial substances under the same conditions and has the largest dilution multiple.

[0055] Table 3 Comparison of spray drying effects of different strains

[0056]

[0057] Example 2 Study on the stability of microecological preparations

[0058] The microecological preparation of this embodiment uses Bacillus subtilis CGMCC NO.25499, and the spray-dried carrier components are 2.5% diatomaceous earth, 1% gum arabic, 0.1% white carbon black, and 0.5% manganese sulfate. The preparation method is prepared according to Example 1.

[0059] 1. High temperature resistance

[0060] Weigh 2.56g of the microecological preparation of the present invention, dilute to 50mL, and divide into 5 portions, one of which is a control, and three portions are placed in 80, 90, and 100°C water baths for heating for 0.5h; the last portion is placed in an autoclave and treated at 120°C for 0.5h. After the sample is cooled, the antibacterial changes are detected. The results are shown in Table 4. Different temperature treatments significantly change the antibacterial activity of the microecological preparation, indicating that the antimicrobial peptide is resistant to high temperatures.

[0061] Table 4 Comparison of antibacterial activity at different temperatures

[0062] CK 80 90 100 120 Golden grape 15.38mm 15.96mm 15.25mm 15.15mm 15.22mm Gas production 17.9mm 17.96mm 17.35mm 17.08mm 17.3mm

[0063] 2. pH resistance

[0064] The microecological preparation of the present invention (diluted 100 times) 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, and the antibacterial activity was determined with the untreated spray-dried powder as a 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 microecological preparation.

[0065] Table 5 Comparison of antibacterial activity at different pH treatments

[0066] Golden grape Gas production CK 14.13mm 14.86mm 2 13.20mm 14.02mm 4 12.68mm 14.6mm 6 13.89mm 15.25mm 8 14.19mm 15.06mm 10 12.35mm 15.23mm

[0067] 3. Simulated gastrointestinal tolerance test

[0068] 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.

[0069] 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.

[0070] The microecological preparation of the present invention (diluted 100 times) was enzymatically 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 microecological preparation can tolerate the gastrointestinal tract.

[0071] Table 6 Simulated gastrointestinal tolerance results

[0072] CK Pepsin Trypsin Golden grape 14.13mm 13.83mm 14.52mm Gas production 16.05mm 16.52mm 15.94mm

[0073] 4.37℃ accelerated test

[0074] The microecological preparation of the present invention (diluted 100 times) was placed in an aluminum foil bag, sealed and packaged, and placed in a 37°C incubator for 1 month, and the antibacterial activity was tested every 1 week. The results are shown in Table 7, and it can be seen that the antibacterial activity of the microecological preparation of the present invention was not significantly changed at 37°C for 1 month, indicating that the microecological preparation of the present invention is relatively stable.

[0075] Table 7 Accelerated test results

[0076] CK One week Week 2 Three weeks Four weeks Golden grape 12.87mm 12.50mm 12.46mm 12.30mm 12.51mm Gas production 16.35mm 16.92mm 16.63mm 17.02mm 16.28mm

[0077] 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 microecological preparation, characterized in that: The microecological preparation is prepared by spray drying after adding a carrier to the bacillus fermentation liquid to form a powder, and the carrier is prepared by one of gum arabic, white carbon black, manganese sulfate, diatomaceous earth, light calcium carbonate, yeast cell wall and Jiayi powder.

2. The microecological preparation according to claim 1, characterized in that: The carrier is made of diatomaceous earth, gum arabic, white carbon black and manganese sulfate.

3. The microecological preparation according to claim 2, characterized in that: To each liter of Bacillus fermentation liquid, 1-5% diatomaceous earth, 1-10% gum arabic, 0.1-0.5% white carbon black and 0.1-1% manganese sulfate are added.

4. The microecological preparation according to claim 3, characterized in that: To each liter of Bacillus fermentation broth, 2.5% diatomaceous earth, 1% gum arabic, 0.1% white carbon black and 0.5% manganese sulfate were added.

5. The microecological preparation according to any one of claims 1 to 4, characterized in that: The fermentation method of the bacillus fermentation liquid is as follows: The seed liquid of Bacillus is inoculated into a fermentation tank filled with liquid fermentation medium at an inoculation rate of 10%, the dissolved oxygen is controlled at 30%-40%, and the fermentation is cultured for 12-18 hours.

6. The microecological preparation according to any one of claims 1 to 4, characterized in that: The bacillus is bacillus licheniformis or bacillus subtilis.

7. The microecological preparation according to claim 6, characterized in that: The Bacillus subtilis is Bacillus subtilis CGMCC NO.25499.

8. The microecological preparation according to any one of claims 1 to 4, characterized in that: The number of viable bacteria in the Bacillus fermentation broth is 2*10 9 CFU / mL~5*10 9 CFU / mL.

9. Use of the probiotic preparation according to any one of claims 1 to 4 in the preparation of medicines or feeds for inhibiting Clostridium perfringens and / or Staphylococcus aureus.

10. Use of the probiotic preparation according to any one of claims 1 to 4 in the preparation of Bacillus powder.

Images