A probiotic composition with enhanced antibacterial effect

Abstract

The invention discloses a probiotic composition with enhanced antibacterial effect, belongs to the technical field of microorganisms, and is characterized in that the composition comprises bacillus subtilis, whose preservation number is CGMCC NO.20922 and whose preservation unit is the General Microbiological Center of China National Committee for the Collection of Microorganisms, and bacillus licheniformis, whose preservation number is CGMCC NO.24440 and whose preservation unit is the General Microbiological Center of China National Committee for the Collection of Microorganisms. The composition is used for feed addition in the process of animal feeding and for antibacterial.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a probiotic composition with enhanced antibacterial effect. Background Art

[0002] In large-scale farming, the use of feed additives is a very common means. In order to reduce the use of antibiotics in the farming process, microbial agents are widely used. Usually, Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans and other agents are used to varying degrees. However, in terms of antibacterial and anti-inflammatory effects, they do not meet the needs of the farming industry. Summary of the invention

[0003] The object of the present invention is to provide a probiotic composition with enhanced antibacterial effect to solve the problems raised in the above background technology.

[0004] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0005] A probiotic composition with enhanced antibacterial effect, characterized in that it includes Bacillus subtilis, whose preservation number is CGMCC NO.20922, the preservation unit is the General Microbiology Center of China National Microbiological Culture Collection Administration, the unit code is CGMCC, and the preservation date is 2020-10-21, and Bacillus licheniformis, whose preservation number is CGMCC NO.24440, the preservation unit is the General Microbiology Center of China National Microbiological Culture Collection Administration, the unit code is CGMCC, and the preservation date is 2022-02-28.

[0006] Furthermore, the ratios of Bacillus subtilis and Bacillus licheniformis are 5:5, 6:4, and 7:3.

[0007] Furthermore, Bacillus subtilis and Bacillus licheniformis are used in animal breeding.

[0008] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0009] Bacillus subtilis and Bacillus licheniformis have good inhibitory effects on different types of Clostridium perfringens. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0011] Figure 1 It is the antibacterial effect of single strains and combinations of Bacillus subtilis and Bacillus licheniformis. DETAILED DESCRIPTION

[0012] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0013] It should be noted that if no specific conditions are specified in the following examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0014] 1. Experimental Materials

[0015] 1.1 Strains

[0016] Bacillus subtilis and Bacillus licheniformis isolated in the laboratory.

[0017] Pathogens:

[0018] 1: Escherichia coli K88 (ATCC25922), 2: Salmonella (ATCC14028), 3: Clostridium perfringens type A (CVCC52), 4: Clostridium perfringens type A (CVCC1142), 5: Clostridium perfringens type C (CVCC1158), 6: Clostridium perfringens type A (CVCC2027), 7: Staphylococcus aureus (ATCC6538P).

[0019] 1.2 Dilution

[0020] Diluent: 0.9% sodium chloride aqueous solution. After preparation, dispense 9 ml of the diluent into a 15×150 test tube and sterilize at 121℃ for 30 minutes.

[0021] 1.3 Culture medium (each 1000 ml culture medium contains)

[0022] Probiotic activation medium: 10g tryptone, 5g yeast extract, 5g sodium chloride, 1.5g agar powder. Natural pH, sterilized at 121℃ for 30min.

[0023] NA medium: peptone 10g, beef extract 3g, NaCl 5g, adjust pH to 7.2-7.4, sterilize at 121℃ for 30min. For solid medium, add 1.5g agar powder.

[0024] Probiotic antibacterial medium: 10g tryptone, 5g yeast extract, 5g glucose, 5g beef extract, 0.15g anhydrous calcium chloride, 0.1g manganese sulfate monohydrate, 2.5g sodium chloride, 0.5g L-cysteine ​​hydrochloride, 0.5g tomato powder, adjust pH to 5.5, and sterilize at 121°C for 30min.

[0025] Antibacterial plate: The composition is the same as the probiotic antibacterial medium, with an additional 25 g of agar added, and sterilized at 121°C for 30 min.

[0026] 1.4 Experimental instruments

[0027] Temperature-controlled shaker, autoclave, pipette, vortexer, pH meter, constant temperature incubator, clean bench, PCR instrument, electrophoresis instrument, centrifuge, etc.

[0028] 2. Experimental methods

[0029] 2.1 Detection of the inhibitory effect of Bacillus licheniformis on Clostridium perfringens

[0030] (1) Activate 20 strains of Bacillus licheniformis isolated in the laboratory earlier, pick a single colony and culture it in 5 ml of probiotic antibacterial liquid culture medium with shaking, culture at 37°C and 200 rpm for 24 h, and measure the OD600 of the culture medium. -1 Dilute 2ml to 5ml probiotic antibacterial liquid culture medium and shake culture at 37℃, 200rpm for 24h. To ensure the consistency of inoculation volume, the inoculation volume of each strain can be adjusted according to the OD600 measurement results. The inoculation volume of the strain with the lowest OD600 value is 2ml, and the remaining strains are inoculated in proportion.

[0031] (2) Pick a single colony of activated Clostridium perfringens CVCC1142 and culture it anaerobically in 5 ml of NA liquid culture medium at 37°C and 200 rpm for 24 h.

[0032] (3) Take 200µl of the Clostridium perfringens liquid and apply it to the antibacterial plate until it is dry. After standing for 10 minutes, place an Oxford cup on the plate, add 200µl of Bacillus licheniformis liquid to the Oxford cup, and place it upright in an incubator at 37℃ for anaerobic culture for about 18 hours. Then use tweezers to take out the Oxford cup vertically and measure the diameter of the transparent circle. Measure vertically in two directions and the average value is the diameter of the antibacterial zone.

[0033] (4) Select strains with better antibacterial effects for subsequent experiments.

[0034] 2.2 Screening of lichens with antibacterial synergistic effects after combination with Bacillus subtilis

[0035] (1) Activate the Bacillus subtilis isolated in the previous laboratory, pick a single colony and culture it in 5 ml of probiotic antibacterial liquid culture medium with shaking, and culture it at 37°C and 200 rpm for 24 h.

[0036] (2) Pick a single colony of the screened Bacillus licheniformis and place it in 5 ml of probiotic antibacterial liquid culture medium for shaking culture at 37°C and 200 rpm for 24 h.

[0037] (3) Inoculate probiotic culture solution for 10 -1 Dilute 2 ml into 5 ml probiotic antibacterial liquid medium and culture at 37°C, 200 rpm for 24 hours. Experimental group: Inoculate Bacillus licheniformis and Bacillus subtilis at a ratio of 1:1 for mixed culture. Control group: Inoculate Bacillus licheniformis and Bacillus subtilis at the same total inoculation amount as the experimental group for single culture.

[0038] (4) Pick out single colonies of activated Clostridium perfringens, Escherichia coli, Salmonella, and Staphylococcus aureus and culture them anaerobically in 5 ml of NA liquid culture medium at 37°C and 200 rpm for 24 h.

[0039] (5) The Oxford cup method was used to conduct antibacterial experiments on Clostridium perfringens, Escherichia coli, Salmonella, and Staphylococcus aureus. The culture fluids of Escherichia coli, Salmonella, and Staphylococcus aureus were diluted 100 times before coating.

[0040] (6) Compare the antibacterial effects of single strains and mixed strains, and select the Bacillus licheniformis strain with the best antibacterial effect of the mixed strain greater than that of the single strain for subsequent experiments.

[0041] 2.3 Screening of the optimal ratio of Bacillus subtilis and Bacillus licheniformis combination

[0042] (1) Activate the screened Bacillus licheniformis and Bacillus subtilis, pick a single colony and culture it in 5 ml of probiotic antibacterial liquid culture medium with shaking at 37°C and 200 rpm for 24 h.

[0043] (2) Inoculate the culture medium for 10 -1 2 ml of the dilution solution was added to 5 ml of probiotic antibacterial liquid culture medium and cultured at 37°C and 200 rpm for 24 h. Bacillus licheniformis and Bacillus subtilis were inoculated at a ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 for mixed culture.

[0044] (3) Pick out activated single colonies of Clostridium perfringens, Escherichia coli, Salmonella, and Staphylococcus aureus and culture them anaerobically in 5 ml of NA liquid culture medium at 37°C and 200 rpm for 24 h.

[0045] (4) The Oxford cup method was used to conduct antibacterial experiments on Clostridium perfringens, Escherichia coli, Salmonella, and Staphylococcus aureus. The culture fluids of Escherichia coli, Salmonella, and Staphylococcus aureus were diluted 100 times before coating.

[0046] (5) Compare the antibacterial effects of mixtures of different proportions and select the proportion with the best antibacterial effect for subsequent experiments.

[0047] 2.4 Combination of Bacillus subtilis and Bacillus licheniformis for antibacterial activity

[0048] (1) Activate Bacillus subtilis and Bacillus licheniformis, pick a single colony and culture it in 5 ml of probiotic antibacterial liquid culture medium with shaking at 37°C and 200 rpm for 24 h.

[0049] (2) Inoculate the culture medium for 10 -12 ml of the dilution solution was added to 5 ml of probiotic antibacterial liquid culture medium and cultured at 37°C and 200 rpm for 24 hours. Experimental group: Bacillus licheniformis and Bacillus subtilis were inoculated at a ratio of 1:1 for mixed culture. Control group: Bacillus licheniformis and Bacillus subtilis were inoculated at the same total inoculation amount as the experimental group for single culture.

[0050] (3) Pick out activated single colonies of Clostridium perfringens, Escherichia coli, Salmonella, and Staphylococcus aureus and culture them anaerobically in 5 ml of NA liquid culture medium at 37°C and 200 rpm for 24 h.

[0051] (4) The Oxford cup method was used to conduct antibacterial experiments on Clostridium perfringens, Escherichia coli, Salmonella, and Staphylococcus aureus. The culture fluids of Escherichia coli, Salmonella, and Staphylococcus aureus were diluted 100 times before coating.

[0052] (5) Compare the antibacterial effects of a single strain and a mixed strain to verify whether Bacillus licheniformis has a synergistic effect on the antibacterial effect of Bacillus subtilis.

[0053] 2.5 Identification of 16S rRNA genes of Bacillus subtilis and Bacillus licheniformis

[0054] (1) Use Lysis Buffer (TaKaRa) to extract DNA from the strain according to the instructions, and the obtained DNA is directly used as a template for PCR amplification.

[0055] (2) The DNA template was amplified using universal primers 27F / 1492R, and the amplified product was sent to a sequencing company for sequencing.

[0056] (3) After splicing, the sequences were compared in the NCBI database.

[0057] 3. Experimental results

[0058] 3.1 Detection of the inhibitory effect of Bacillus licheniformis on Clostridium perfringens

[0059] The inhibition results of 20 strains of Bacillus licheniformis on Clostridium perfringens (CVCC1142) are shown in Table 1 (the diameter of the Oxford cup is 7.8 mm):

[0060] Table 1 Inhibitory effects of 20 strains of Bacillus licheniformis on Clostridium perfringens (CVCC1142)

[0061]

[0062] Five strains with obvious antibacterial effect on Clostridium perfringens were screened out from 20 strains of Bacillus licheniformis and were numbered ZBL654-658.

[0063] 3.2 Screening of lichens with antibacterial synergistic effects after combining Bacillus licheniformis ZBL654-658 with Bacillus subtilis

[0064] The antibacterial results of the combination of Bacillus licheniformis ZBL654-658 and Bacillus subtilis are shown in Table 2-6 (the diameter of the Oxford cup is 7.8 mm):

[0065] Table 2 Diameter of inhibition zone after combination of Bacillus licheniformis ZBL654 and Bacillus subtilis (mm)

[0066]

[0067] Table 3 Diameter of inhibition zone after combination of Bacillus licheniformis ZBL655 and Bacillus subtilis (mm)

[0068]

[0069] Table 4 Diameter of inhibition zone after combination of Bacillus licheniformis ZBL655 and Bacillus subtilis (mm)

[0070]

[0071] Table 5 Diameter of inhibition zone after combination of Bacillus licheniformis ZBL655 and Bacillus subtilis (mm)

[0072]

[0073] Table 6 Diameters of inhibition zones of Bacillus licheniformis and Bacillus subtilis after combination (mm)

[0074]

[0075] The antibacterial effects of five strains of Bacillus licheniformis (ZBL654-ZBS658) in combination with Bacillus subtilis were compared, and it was found that Bacillus licheniformis had the most obvious antibacterial synergistic effect on Bacillus subtilis.

[0076] 3.3 Screening of the optimal ratio of Bacillus subtilis and Bacillus subtilis

[0077] The antibacterial results of Bacillus licheniformis and Bacillus subtilis in different proportions are shown in Table 7 (the diameter of the Oxford cup is 7.8 mm):

[0078] Table 7 Diameters of inhibition zones of different pathogens after mixing Bacillus licheniformis and Bacillus subtilis in different proportions (mm)

[0079]

[0080] The antibacterial effects of the combinations of Bacillus subtilis and Bacillus licheniformis in different ratios were compared, and it was found that the antibacterial effect of the combination of Bacillus subtilis and Bacillus licheniformis was better when the ratio was 5:5, 6:4, and 7:3, among which the best antibacterial effect was achieved when the ratio was 5:5.

[0081] 3.4 Antibacterial results of the combination of Bacillus subtilis and Bacillus licheniformis

[0082] (1) The results of the inhibition zone diameter measurement after the combination of Bacillus subtilis and Bacillus licheniformis are shown in Table 8 (the diameter of the Oxford cup is 7.8 mm):

[0083] Table 8 Diameters of inhibition zones of single strains and combinations of Bacillus subtilis and Bacillus licheniformis (mm)

[0084]

[0085] The results of the combined antibacterial activity of Bacillus subtilis and Bacillus licheniformis showed that the combined antibacterial activity of Bacillus subtilis and Bacillus licheniformis was greater than that of the two single strains. The antibacterial results are shown in the photo. Figure 1 .

[0086] 3.5 16S rRNA gene sequences of Bacillus subtilis and Bacillus licheniformis

[0087] The 16S rRNA gene sequences of Bacillus subtilis and Bacillus licheniformis are as follows. They were compared in the NCBI database. The results showed that the homology between Bacillus subtilis and Bacillus subtilis was >99%, and the homology between Bacillus licheniformis and Bacillus licheniformis was >99%. Bacillus subtilis was determined to be Bacillus subtilis, and Bacillus licheniformis was determined to be Bacillus licheniformis.

[0088] 16S rRNA gene sequence of Bacillus subtilis:

[0089]

[0090] 16S rRNA gene sequence of Bacillus licheniformis:

[0091]

[0092] 4. Experimental conclusion

[0093] 1. Bacillus subtilis and Bacillus licheniformis have good inhibitory effects on different types of Clostridium perfringens, but their inhibitory effects on Escherichia coli, Salmonella and Staphylococcus aureus are weaker.

[0094] 2. When Bacillus subtilis is combined with Bacillus licheniformis, its inhibitory effect on Clostridium perfringens is stronger than that of either single strain, and its inhibitory effect on Escherichia coli, Salmonella, and Staphylococcus aureus is significantly enhanced.

[0095] In summary, when Bacillus subtilis and Bacillus licheniformis are combined in ratios of 5:5, 6:4, and 7:3, Bacillus licheniformis has a synergistic effect on the antibacterial activity of Bacillus subtilis, significantly improving the antibacterial intensity and antibacterial spectrum of Bacillus subtilis.

[0096] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0097] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A probiotic composition with enhanced antibacterial effect, characterized in that: Including Bacillus subtilis, whose deposit number is CGMCC NO. 20922, deposited by China National Microbiological Culture Collection Center, and Bacillus licheniformis, whose deposit number is CGMCC NO. 24440, deposited by China National Microbiological Culture Collection Center, The ratio of Bacillus subtilis to Bacillus licheniformis is 5:5 or 6:4 or 7:

3.

2. Use of the probiotic composition with enhanced antibacterial effect according to claim 1 in an animal breeding process, wherein the use is for non-disease diagnosis and treatment purposes.

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