Animal-derived pediococcus acidilactici and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to animal-derived pediococcus acidilactici and application thereof. The pediococcus acidilactici is classified and named as pediococcus acidilactici, the strain number of the pediococcus acidilactici is 12NN1, the pediococcus acidilactici is preserved in Guangdong Microbial Culture Collection Center on January 2, 2025, and the preservation number of the pediococcus acidilactici is GDMCC No: 65706. The strain is resistant to acid and cholate, has a remarkable inhibiting effect on growth and reproduction of pathogenic escherichia coli and staphylococcus aureus, is safe and non-toxic, does not have pathogenicity, and provides beneficial strain resources and biological materials for development of corresponding microbial preparations. Therefore, the compound can be used for preparing a biocontrol preparation for inhibiting the growth of pathogenic bacteria, preparing animal feed and preparing a preparation for preventing, improving and / or treating gastrointestinal tract pathogenic bacteria infection, so that the compound has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of animal-derived Pediococcus acidilactici and application thereof. Background Art

[0002] Lactic acid bacteria are the most dominant flora in the intestines of animals. They play an important role in the balance of gastrointestinal flora and animal immunity. They are gradually being valued and widely used as an important microecological preparation in the production of "antibiotic-free breeding" in animal husbandry. Pediococcus acidilactici is a common lactic acid bacteria belonging to the Lactobacillaceae family and the genus Pediococcus.

[0003] The current abuse of antibiotics has brought a series of problems and hidden dangers to animal farming and food safety, so it is urgent to find new green and safe substances or additives to replace antibiotics. Most of the lactic acid bacteria microecological preparations currently sold on the market are isolated and purified from soil or animal intestines. Studies have shown that microbial strains from animals are more ideal probiotics and are more adaptable to the gastrointestinal environment than other microbial preparations.

[0004] Therefore, it is of great significance to provide an animal-derived and more suitable microbial material for the development of microbial preparations. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a strain of animal-derived Pediococcus acidilactici in response to the urgent market demand for alternatives to reduced or banned antibiotics (antibiotics refer to antibiotics) in the prior art.

[0006] The technical problem that the present invention needs to solve is to provide the application of the above-mentioned Pediococcus acidilactici strain.

[0007] In order to solve the above technical problems, the present invention discloses an animal-derived Pediococcus acidilactici, with a strain number of 12NN1, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 2, 2025, with a deposit number of GDMCC No: 65706, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] In some embodiments of the present invention, the Pediococcus acidilactici is firstly isolated from 7 strains of lactic acid bacteria from 14 red panda feces, then 3 strains of Pediococcus acidilactici are obtained through screening and identification, and then the Pediococcus acidilactici is preferably obtained from the 3 strains of Pediococcus acidilactici.

[0009] Specifically, the nucleotide sequence of 16S rDNA of the Pediococcus acidilactici is shown as SEQ ID No.1.

[0010] Specifically, the Pediococcus acidilactici can tolerate high acid concentrations of pH 2.0 to 3.0 and bile salt concentrations of 0.1 to 0.3%, and has passed animal safety tests and is non-pathogenic.

[0011] The use of Pediococcus acidilactici in inhibiting the growth of pathogenic bacteria is also within the scope of protection of the present invention.

[0012] Specifically, the pathogenic bacteria include any one of pathogenic Escherichia coli and Staphylococcus aureus, or a combination of both.

[0013] In some embodiments of the present invention, the Escherichia coli is Escherichia coli AE17, and the Staphylococcus aureus is Staphylococcus aureus JSK-1.

[0014] Specifically, the bacterial liquid and / or supernatant of Pediococcus acidilactici is used to inhibit the growth of pathogenic bacteria.

[0015] In some embodiments of the present invention, Pediococcus acidilactici 12NN1 can effectively inhibit the growth of Escherichia coli, with a bacterial liquid inhibition zone of 16.62 mm and a supernatant inhibition zone of 16.23 mm; at the same time, it can effectively inhibit the growth of Staphylococcus aureus, with a bacterial liquid inhibition zone of 14.82 mm and a strain supernatant inhibition zone of 16.13 mm.

[0016] A biocontrol agent for inhibiting the growth of pathogenic bacteria is also within the scope of protection of the present invention.

[0017] Specifically, the biocontrol agent contains the Pediococcus acidilactici described in the present invention.

[0018] The use of Pediococcus acidilactici in the preparation of animal feed is also within the scope of protection of the present invention.

[0019] An animal feed, comprising the Pediococcus acidilactici described in the present invention.

[0020] Specifically, by preparing the Pediococcus acidilactici into animal feed, the nutritional value of the feed can be improved, the growth and development of animals can be promoted, the immunity of animals can be enhanced, and the occurrence of diseases can be reduced.

[0021] The use of the Pediococcus acidilactici in the preparation of a preparation for preventing, improving and / or treating gastrointestinal pathogen infection in animals is also within the scope of protection of the present invention.

[0022] A preparation for gastrointestinal pathogen infection, comprising the Pediococcus acidilactici.

[0023] Specifically, the lactic acid Pediococcus can effectively colonize in the intestines of animals, avoid gastrointestinal pathogen infection, promote intestinal peristalsis and other mechanisms, thereby improving animal indigestion, relieving constipation and other intestinal problems.

[0024] Beneficial effects:

[0025] The present invention isolates a strain of animal-derived Pediococcus acidilactici from red panda feces, which is classified and named Pediococcus acidilactici, with a strain number of 12NN1 and a preservation number of GDMCC No: 65706. The strain is significantly resistant to acid and bile salts, has a significant inhibitory effect on the growth and reproduction of pathogenic Escherichia coli, is safe and non-toxic, and is not pathogenic. In response to the market's urgent demand for alternatives to antibiotic reduction and prohibition, the strain provides beneficial bacterial resources and biological materials for the development of corresponding microbial preparations, can be used to prepare biocontrol preparations that inhibit the growth of pathogens, prepare animal feed, and prepare preparations for preventing, improving and / or treating gastrointestinal pathogen infections, and therefore has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will be further described in detail below with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more clear.

[0027] Figure 1 This is the colony streak morphology result of strain 12NN1.

[0028] Figure 2 This is a 4x microscopic examination result of strain 12NN1.

[0029] Figure 3 The 16S rDNA PCR amplification results of strain 12NN1, where M is DL2000 DNA Marker, 1 is strain 12NN1, and 2 is blank control.

[0030] Figure 4 The results of the inhibition zone experiment of the bacterial solution and supernatant of strain HL1 on Escherichia coli are shown in the figure below. The left figure is the bacterial solution of strain HL1, and the right figure is the supernatant of strain HL1.

[0031] Figure 5 The results of the inhibition zone experiment of the bacterial solution and supernatant of strain XW2 on Escherichia coli are shown in the figure below. The left figure is the bacterial solution of strain XW2, and the right figure is the supernatant of strain XW2.

[0032] Figure 6 The results of the inhibition zone experiment of the bacterial solution and supernatant of strain 12NN1 on Escherichia coli are shown in the figure below. The left figure is the bacterial solution of strain 12NN1, and the right figure is the supernatant of strain 12NN1.

[0033] Figure 7 The results of the antibacterial zone experiment of the bacterial liquid and supernatant of strain 12NN1 on Staphylococcus aureus are shown in the figure below. The left figure is the bacterial liquid of strain 12NN1, and the right figure is the supernatant of strain 12NN1. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below in conjunction with specific implementations, and the above and / or other advantages of the present invention will become more clear.

[0035] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0036] In the following examples, the MRS solid culture medium was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; the bacterial genomic DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the Gram staining kit was purchased from Nanjing Jiancheng Technology Co., Ltd.; and the PCR primers were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0037] Strain preservation information: A strain of animal-derived Pediococcus acidilactici, classified and named Pediococcus acidilactici, with strain number 12NN1, was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 2, 2025, with the preservation number GDMCC No: 65706.

[0038] Example 1: Isolation and purification of strains

[0039] On December 1, 2023, 14 fresh panda feces were collected and placed in sterile EP tubes, and the collected samples were immediately placed in a -80°C refrigerator for storage. Take 2g of fresh panda feces, add 10mL of sterile saline, and stir thoroughly to evenly distribute the sample in the saline. Take 100μL of the mixed sample liquid, dilute it 10 times with sterile water, inject it into the center of the surface of the MRS solid culture medium, and spread it evenly with a coating rod. The coated plate was placed in a 37°C constant temperature incubator for 24h, and the colony growth was observed and recorded. After the culture is completed, a typical single colony of lactic acid bacteria (round, smooth, neat edges, milky white) was picked from the plate using a sterile inoculation loop, inoculated onto a new MRS solid culture medium slope, and streaked for purification. Repeat the streaking purification 3 times, and finally 7 purified single colonies were obtained, which were round, slightly convex, with neat edges, and milky white. Figure 1 The colony streak morphology of 12NN1 is shown.

[0040] Example 2: Strain Identification

[0041] 1. Preliminary identification

[0042] The purified single colonies in Example 1 were subjected to Gram staining using a Gram staining kit, and the stained slides were placed under a microscope for observation. After Gram staining, the strains of the seven single colonies were observed under a microscope to be bluish purple, and the bacteria were arranged alone or in small groups. These seven single colonies were preliminarily determined to be Gram-positive cocci. Figure 2 The figure shows the results of Gram staining of 12NN1 under 4x microscopy.

[0043] 2. Biochemical identification

[0044] The direct inoculation method was used to pick the purified and isolated same colonies from the plates of 7 bacterial strains with an inoculation needle and inoculated them into biochemical identification tubes, which were then placed in a 37°C constant temperature incubator for culture. After 24 hours, the tubes were taken out to observe color changes, observe and analyze the bacteria's ability to decompose nutrients and the differences in metabolites, and record the data.

[0045] From the sugar fermentation test, we know that all 7 strains can ferment esculin, cellobiose, maltosalicylic acid and sucrose and produce acid but not gas, but cannot ferment sorbitol. The identification results were compared with the Instructions for Use of Microbiological Identification Tubes for Bacteria and the Bergey Manual of Bacterial Identification (Buchanan, 1984), and it was preliminarily determined that the strains used in the experiment were consistent with the biochemical characteristics of lactic acid bacteria, and the strains belonged to the genus Lactobacillus.

[0046] 3. Molecular Biology Identification

[0047] After the purified strains were activated, genomic DNA was extracted using a microbial DNA extraction kit, and the 16S rRNA gene of the strain was amplified by PCR using the template and universal primers. The PCR amplification products were imaged by agarose gel electrophoresis, and the amplification products were entrusted to Anhui General Biological Co., Ltd. for sequencing.

[0048] Among them, the PCR amplification primer pair is 16S-27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 16S-1492R: 5'-TACGGCTACCTTGTTACGACTT-3'.

[0049] PCR reaction system: 2×Taq Master Mix 10μL, upstream primer 1μL, downstream primer 1μL, DNA template 1μL, ddH2O 7μL, a total of 20μL. Sterile water was used instead of DNA template as a blank control.

[0050] PCR amplification program: The PCR reaction program was pre-denaturation at 94°C for 5 min, re-denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and 30 cycles of supplementary extension at 72°C for 10 min.

[0051] The 16S rDNA sequences of the strains were compared with the sequences in the NCBI database by BLAST. It was found that three strains (HL1, XW2, and 12NN1) had high homology with Pediococcus acidilactici. The results of agarose gel electrophoresis of the 16S rDNA gene PCR product of strain 12NN1 were as follows: Figure 3 As shown, clear bands can be seen by ultraviolet observation of the gel system, and there is an expected 1500 bp band between 1000 and 2000 bp. The nucleotide sequence of 16S rDNA of strain 12NN1 is shown in SEQ ID No.1.

[0052] Based on the above identification results, the present invention isolated 3 strains of Pediococcus acidilactici from red panda feces, namely Pediococcus acidilactici HL1, Pediococcus acidilactici XW2, and Pediococcus acidilactici 12NN1.

[0053] Example 3: Analysis of acid resistance of Pediococcus acidilactici

[0054] The pH value of MRS broth medium was adjusted to pH 2.0 by hydrochloric acid as the experimental group, and MRS broth medium was set as the control group. The three activated lactic acid Pediococcus were inoculated into MRS broth medium at a volume ratio of 1:100, and cultured at 37℃ for 24 hours. Then, the cultured bacterial solution was diluted 10 times and evenly spread on MRS solid medium. The number of live bacteria of the strains was determined respectively. After 24 hours of culture, the bacterial plate count was performed and the survival rate was calculated. The calculation formula of the survival rate is: survival rate % = N2 / N1×100%; where: N1 is the number of live bacteria in the acidity control group, and N2 is the number of live bacteria in the pH 2.0 experimental group after 24 hours of culture. The specific survival rate results are shown in Table 2. As can be seen from Table 2, the survival rate of strain HL1 under acidic conditions of pH 2.0 is 18.83%, the survival rate of XW2 is 18.18%, and the survival rate of 12NN1 is 3.13%.

[0055] Table 2 Survival rate of acid resistance test

[0056]

[0057] Furthermore, the pH value of MRS broth culture medium was set to pH 3.0 as the experimental group, and MRS broth culture medium was set as the control group. The survival rate of strain 12NN1 under the acidic condition of pH 3.0 was examined to be 85.94%.

[0058] Example 4: Analysis of Bile Salt Resistance of Pediococcus acidilactici

[0059] MRS broth medium containing 0.1%, 0.2%, and 0.3% pig bile salts were prepared respectively, and MRS broth medium without pig bile salts was used as a blank control group. 100 μL of activated lactic acid Pediococcus culture solution was inoculated into each of them and cultured for 3 hours, and then the cultured culture solution was diluted according to a volume ratio of 1:100, and 100 μL was applied to MRS broth medium containing different concentrations of pig bile salts. The steps of the blank control group without pig bile salts were the same, incubated at 37°C for 24 hours, and the viable bacteria were counted to evaluate the growth of lactic acid Pediococcus in the pig bile salt environment. The survival rate formula is as follows: survival rate % = N2' / N1'×100%. In the formula: N1' is the number of viable bacteria in the control group without pig bile salts, and N2' is the number of viable bacteria in the experimental group with 0.1%, 0.2%, or 0.3% pig bile salt concentrations.

[0060] As can be seen from Table 3, the three strains have a certain tolerance to different concentrations of bile salts. Compared with the other two strains of Pediococcus acidilactici, strain HL1 has the strongest tolerance at 0.1% bile salt concentration, strain 12NN1 has the strongest tolerance at 0.2% bile salt concentration, and strains XW2 and 12NN1 have similar tolerance at 0.3% bile salt concentration.

[0061] Table 3 Survival rate of bile salt tolerance experiment

[0062]

[0063] Example 5: Analysis of antibacterial activity of Pediococcus acidilactici

[0064] The activated three types of lactic acid Pediococcus were inoculated into MRS broth medium at a volume ratio of 1:100 and cultured for 24 hours. The cells and supernatant were separated by centrifugation at 6000×g for 10 minutes, and the supernatant was taken for use. An 8mm sterile puncher was used to punch holes in the center of the LB solid medium plate containing pathogenic Escherichia coli AE17 using the agar perforation diffusion method, and 100μL of freshly cultured lactic acid Pediococcus culture liquid and supernatant were added to the holes respectively. Note that the bacterial liquid and supernatant do not overflow from the hole to avoid affecting the observation of the inhibition zone. The inoculated plate was placed in a 37℃ incubator for 24 hours, and the formation of the inhibition zone was observed and recorded. The diameter of the inhibition zone was measured with a vernier caliper. The size of the inhibition zone can reflect the antibacterial activity and inhibitory effect of lactic acid Pediococcus on pathogenic bacteria.

[0065] The E. coli LB plate without inoculation of Pediococcus acidilactici was used as the blank control group, and its antibacterial zone diameter was 0 mm. The stock solutions and supernatants of Pediococcus acidilactici HL1, XW2, 12NN1 all showed different degrees of inhibition on E. coli. The results are shown in Figures 4 to 6 As shown in Table 4, Pediococcus acidilactici 12NN1 had the best antibacterial effect, with the bacterial liquid inhibition zone being 16.62 mm and the supernatant inhibition zone being 16.23 mm.

[0066] Table 4 Inhibition zone results of Pediococcus lactis antibacterial analysis

[0067]

[0068] Furthermore, the pathogenic bacteria was replaced with Staphylococcus aureus JSK-1, and the antibacterial effect of Pediococcus acidilactici 12NN1 was determined. The results showed that ( Figure 7 ), Pediococcus acidilactici 12NN1 can inhibit the growth of Staphylococcus aureus. The inhibition zone of the original solution is 14.82 mm, and the inhibition zone of the strain supernatant is 16.13 mm. The antibacterial effect of the supernatant is better than that of the bacterial solution.

[0069] Example 6: Animal safety test

[0070] Twenty healthy mice aged 7 weeks were randomly divided into 4 groups, 5 mice / group, namely HL1 test group, XW2 test group, 12NN1 test group and control group. The mice in the three test groups were gavaged with 0.5 mL / mouse per day, with a concentration of 1×10 8 CFU / mL suspension of Pediococcus acidilactici HL1, XW2, and 12NN1, and the mice in the control group were gavaged with sterile saline (0.5 mL / mouse) every day for 7 consecutive days. The mental state, food intake, drinking water, exercise, and hair growth of the four groups of mice were recorded during the experiment.

[0071] The results showed that during the experimental observation period, there was no death in the experimental group that was gavaged with lactic acid Pediococcus 12NN1 and the control group that was gavaged with normal saline, and there was no significant difference in the mental state, behavior, fur gloss, etc. of the two groups of mice. This shows that lactic acid Pediococcus 12NN1 is not pathogenic and is safe for animals. However, the experimental groups that were gavaged with lactic acid Pediococcus HL1 and lactic acid Pediococcus XW2 showed poor mental state, and one experimental mouse died respectively, indicating that strains HL1 and XW2 pose a safety threat to animals.

[0072] Combined with the experimental results of Examples 3 to 6, Pediococcus acidilactici 12NN1 has tolerance and antibacterial properties, and can survive in a strong acid environment of pH 2.0 to 3.0; at bile salt concentrations of 0.1%, 0.2%, and 0.3%, the survival rates are 81.61%, 33.87%, and 17.42%, respectively; it has a significant inhibitory effect on the growth of pathogenic Escherichia coli and Staphylococcus aureus. In addition, strain 12NN1 is not pathogenic and is safe for animals. Therefore, Pediococcus acidilactici 12NN1 was sent to a preservation institution for preservation, and the preservation information is: Pediococcus acidilactici, strain number 12NN1, preserved in Guangdong Provincial Microbiological Culture Collection Center on January 2, 2025, the preservation number is GDMCC No: 65706, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0073] The present invention provides a strain of animal-derived Pediococcus acidilactici and its application ideas and methods. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A strain of animal-derived Pediococcus acidilactici, classified and named as Pediococcus acidilactici, with strain number 12NN1, was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 2, 2025, with the deposit number GDMCC No: 65706.

2. The Pediococcus acidilactici according to claim 1, characterized in that The nucleotide sequence of 16S rDNA of the Pediococcus acidilactici is shown in SEQ ID No.

1.

3. Use of the Pediococcus acidilactici according to any one of claims 1 to 2 in inhibiting the growth of pathogenic bacteria.

4. The use according to claim 3, characterized in that: The pathogenic bacteria include any one of pathogenic Escherichia coli and Staphylococcus aureus or a combination of both.

5. The use according to claim 3, characterized in that: The bacterial liquid and / or supernatant of the Pediococcus acidilactici is used to inhibit the growth of pathogenic bacteria.

6. A biocontrol agent for inhibiting the growth of pathogenic bacteria, characterized in that: The biocontrol agent contains the Pediococcus acidilactici according to claim 1.

7. Use of the Pediococcus acidilactici according to claim 1 in preparing animal feed.

8. An animal feed, characterized in that: The animal feed contains the Pediococcus acidilactici according to claim 1.

9. Use of the Pediococcus acidilactici according to claim 1 in the preparation of a preparation for preventing, improving and / or treating gastrointestinal pathogen infection in animals.

10. A preparation for treating gastrointestinal pathogen infection, characterized in that: The gastrointestinal pathogen infection preparation contains the Pediococcus acidilactici according to claim 1.

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