Bian chicken source bacillus licheniformis and application thereof
By screening and developing Bacillus licheniformis YYBJ-06-01 from chickens, the problem of maintaining intestinal health and promoting production performance in animals without the use of antibiotics has been solved, achieving the effect of a safe and residue-free feed additive, and improving the immunity and growth performance of animals.
Patent Information
- Application Number
- CN202511580542.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-31
- Publication Date
- 2025-12-16
AI Technical Summary
How can we develop a safe, residue-free, and drug-resistant feed additive that maintains animal gut health, promotes production performance, and has good adaptability and colonization ability for different animal gut environments without the use of antibiotics?
A strain of Bacillus licheniformis YYBJ-06-01, derived from chickens, was screened and developed. It was named Bacillus licheniformis and can grow in acidic environments and low bile salt conditions. It has antibacterial and growth-promoting properties and can be used in feed to improve animal immunity and growth performance.
Bacillus licheniformis YYBJ-06-01 significantly enhanced animal immunity, increased serum immunoglobulin IgG levels, reduced inflammatory factor levels, enhanced antioxidant function, and effectively inhibited pathogens such as Escherichia coli and Salmonella, promoting chick growth and improving flock uniformity.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of microbial technology, and particularly relates to a licheniformis from a border chicken and application thereof. BACKGROUND
[0002] The overuse of antibiotics has led to problems such as antibiotic residues, increased bacterial drug resistance and reduced environmental carrying capacity in the farming industry. In order to solve this problem, many countries and regions have begun to take measures to restrict the use of antibiotics in the farming industry. However, after prohibiting the addition of antibiotics in feed, the farming industry will face major challenges, namely, maintaining the intestinal health of animals, promoting their production performance, and developing safe, residue-free and drug-resistant feed additives, which become important tasks for the farming industry, without the use of antibiotics.
[0003] Probiotic preparations are a new type of safe, green, efficient, residue-free and pollution-free feed additive, which has a series of effects such as promoting animal growth, preventing diseases, improving immune function, and regulating the balance of intestinal flora of host animals, and is widely used as a microecological feed additive in the livestock industry to replace the use of antibiotics. Therefore, the development of microecological preparations with antibiotic-reducing and antibiotic-replacing effects in the field of livestock and poultry breeding is in line with the development and has broad application prospects. However, the key problem in the research of probiotics is the development and screening of excellent strains.
[0004] Licheniformis has the effects of promoting animal growth and metabolism, enhancing immune capacity, and inhibiting inflammation of the body, and therefore, Bacillus plays a positive role in aspects such as nutrient absorption, disease prevention and treatment, livestock breeding and food safety. However, the application effect is not consistent in actual animal production. Due to the different intestinal environments of different animals, the adaptation ability of probiotics from different sources to the intestinal tract of animals also has differences, which affects their colonization of the intestinal tract and the exertion of effects. Probiotics from the same animal intestinal tract can overcome the adverse factors in the digestive tract and the adverse effects of other microbial communities, rapidly colonize and rapidly exert effects, and the superior properties of homologous probiotics have been highly valued. Border chickens belong to a dual-purpose breed with large egg weight and good meat quality, have strong adaptability to the environment, can resist low temperature, and are mainly distributed in high-latitude regions of China, and are also one of the chicken breeds listed in the genetic resources protection list of livestock and poultry in China. Therefore, the screening of border chicken homologous probiotics provides a theoretical basis and strain selection for the development and preparation of border chicken source probiotics, and has a wide application prospect. SUMMARY
[0005] The present application aims to provide a licheniformis from a border chicken to solve the problems in the background art.
[0006] To achieve the above object, the present application provides the following technical scheme: A Bacillus licheniformis from Bianji, named Bacillus licheniformis YYBJ-06-01, preserved in China Center for Type Culture Collection, with the preservation number CCTCC No. M 20252095, the preservation time September 23, 2025, and the preservation address: China Center for Type Culture Collection (Wuhan University), 299 Banyi Road, Wuchang District, Wuhan, Hubei Province, China.
[0007] Preferably, the Bacillus licheniformis from Bianji can grow in an acidic environment with pH = 3.5-6.5.
[0008] Preferably, the Bacillus licheniformis from Bianji can grow in a culture medium with a bile salt mass concentration of 0.1%-0.2%.
[0009] Another object of the present application is to provide the use of the above-mentioned Bacillus licheniformis from Bianji or its bacterial liquid in the preparation of an antibacterial agent.
[0010] Preferably, the antibacterial agent is used to inhibit at least one of Escherichia coli, Salmonella and Staphylococcus aureus.
[0011] Another object of the present application is to provide the use of the above-mentioned Bacillus licheniformis from Bianji or its bacterial liquid in the preparation of a feed.
[0012] Preferably, the feed is used to improve the immunity of animals, inhibit the oxidative stress of animals and promote the growth of animals.
[0013] Preferably, the animal is poultry.
[0014] Preferably, the animal is chicken.
[0015] Preferably, the animal is Bianji.
[0016] The present application maintains the intestinal health of animals and promotes the production performance of animals without using antibiotics, and develops a safe, non-residual and non-drug-resistant feed additive. Specifically, the present application provides a B. licheniformis YYBJ-06-01 from a side chicken, which is a homologous probiotic and can effectively colonize the intestinal tract of the side chicken, effectively promote the growth of chicks and improve the uniformity of the group. The B. licheniformis YYBJ-06-01 from the side chicken can significantly enhance the immunity of the body, increase the content of serum immunoglobulin IgG, and reduce the level of inflammatory factors. At the same time, the B. licheniformis YYBJ-06-01 from the side chicken can effectively increase the activity of serum antioxidant enzymes, reduce the content of malondialdehyde, and enhance the antioxidant function of the body. In addition, the B. licheniformis YYBJ-06-01 from the side chicken has a clear inhibitory effect on common pathogenic bacteria such as Escherichia coli and Salmonella, and is sensitive to most common antibiotics, and has high biological safety, which provides excellent strain resources for the development of green feed additives to replace antibiotics. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 It is a genetic evolution tree constructed based on the 16S rRNA gene sequence of B. licheniformis YYBJ-06-01.
[0018] Figure 2 It is the growth of B. licheniformis YYBJ-06-01 under different acidic conditions.
[0019] Figure 3 It is the result graph of the bile salt tolerance experiment of B. licheniformis YYBJ-06-01.
[0020] Figure 4 It is a result graph of the effect of B. licheniformis YYBJ-06-01 on the growth of chicks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
[0021] Figure 5 It is a result graph of the effect of B. licheniformis YYBJ-06-01 on the content of serum GSH-Px of Youyu side chickens (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
[0022] Figure 6 It is a result graph of the effect of B. licheniformis YYBJ-06-01 on the content of serum MDA of Youyu side chickens (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
[0023] Figure 7 It is a result graph of the effect of B. licheniformis YYBJ-06-01 on the content of serum IgG of Youyu side chickens (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
[0024] Figure 8 The results of the influence of Bacillus licheniformis YYBJ-06-01 on the content of IL-1β in the serum of Youyu border chicken are shown in the figure (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). DETAILED DESCRIPTION
[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work are within the protection scope of the present application.
[0026] The following examples are the implementation cases of the technical solutions of the present application in practical application, but are not limited thereto. The materials and reagents involved therein are commercially available products if not specifically stated. The experimental methods used are conventional methods if not specifically stated.
[0027] Example 1: The present example provides a method for isolating and identifying Bacillus licheniformis, which specifically comprises: I. Isolation of the strain: 1 g of the intestinal contents of the border chicken was collected aseptically, and then serially diluted with phosphate buffered saline (PBS). 100 μL of each of the 10 -4 , 10 -5 , 10 -6 dilutions were spread on Bacillus selective medium plates, and incubated at 37°C for 24 h. After the incubation, the colony morphology was observed, and a yellow single colony was picked and streaked on an LB plate for purification. After repeated purification for 3 times, the strain was transferred to an LB slant for culture and preservation. One of the strains was selected and named as YYBJ-06-01, and then subjected to the next step of identification and research.
[0028] II. 16S rRNA Gene Sequence Analysis: Following the instructions of the bacterial genome extraction kit, total bacterial DNA was extracted and amplified by PCR using universal primers 27F and 1492R for the 16S rRNA gene. The PCR reaction volume was 50 μL: 2 μL total DNA from strain YYBJ-06-01, 25 μL PCR mix, 1 μL each of forward and reverse primers, and ddH2O added to a total volume of 50 μL. The PCR amplification program was: 95℃ pre-denaturation for 2 min; 94℃ denaturation for 20 s, 55℃ annealing for 20 s, 72℃ extension for 40 s, for a total of 30 cycles; and a final extension at 72℃ for 10 min. After identification by 1% agarose gel electrophoresis, the PCR amplification products were purified using a gel recovery kit and sequenced by Sino-American Taihe Biotechnology (Beijing) Co., Ltd. The sequencing results were compared with the 16S rRNA gene by selecting sequences with high similarity from the NCBI database (https: / / pubmed.ncbi.nlm.nih.gov / ) using online BLAST. Phylogenetic trees were constructed using Mega7.0 software with the neighuor-joining (NJ) algorithm to determine the evolutionary relationship of the isolated strains.
[0029] Following the above method, the isolated strain YYBJ-06-01 was subjected to PCR amplification and sequencing (sequence shown in SEQ ID NO:1 of the sequence listing). Multiple sequences with high similarity to the 16S rRNA gene sequence of strain YYBJ-06-01 were searched using the online BLAST function of the NCBI database. Clustal W sequence homology analysis showed that the 16S rRNA gene sequence of strain YYBJ-06-01 had the highest homology (99.59%) with the 16S rRNA gene sequence of Bacillus licheniformis strain DSM13 (accession number: NR_118996.1). Phylogenetic analysis of the 16S rRNA genes of the 13 Bacillus licheniformis strains was performed using the maximum nearest neighbor (NJ) method. Figure 1 As shown, the results indicate that the 16S rRNA gene sequence of strain YYBJ-06-01 is homologous to Bacillus licheniformis strains TF-2 and DSM-13, forming an evolutionary branch. This strain is named Bacillus licheniformis YYBJ-06-01 and is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC No. M 20252095, deposited on September 23, 2025, at the China Center for Type Culture Collection (Wuhan University), No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province.
[0030] Example 2: Determination of the acid and bile salt tolerance of Bacillus licheniformis YYBJ-06-01, specifically including: S1, preparation of bacterial liquid: inoculate the above-activated Bacillus licheniformis YYBJ-06-01 strain into LB liquid medium at an inoculation amount of 5%, cultivate at 37°C for 24h, centrifuge at 3500r / min for 15min, then suspend the precipitated bacterial bodies with sterile normal saline, adjust the bacterial body concentration to 10 8 CFU / mL, and store in a 4°C refrigerator for standby use.
[0031] S2, effect of different pH values on strain survival: aseptically take the activated Bacillus licheniformis YYBJ-06-01 strain liquid and inoculate into LB liquid medium with different initial pH (2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5) at an inoculation amount of 1%, set 3 replicates for each pH, and cultivate at 37°C on a shaking table, then measure the OD600 value at 0, 3, 9, 12h, respectively. After the measurement, calculate the average value from the results of the 3 parallel tests, draw a curve according to the calculation results, and the results are shown in Figure 2 .
[0032] S3, effect of different bile salt concentrations on the survival of YYBJ-06-01: inoculate the bacterial suspension cultured for 24h into LB liquid medium with bile salt mass concentrations of 0, 0.1%, 0.2%, 0.3%, 0.4% at an inoculation amount of 1.0%, cultivate at 37°C, and measure the OD600 value of the bacterial suspension at 0 and 9h, respectively, with 3 replicates for each group. Finally, draw a statistical chart with the average value as the standard, as shown in Figure 3 .
[0033] From Figure 2 it can be seen that the number of Bacillus licheniformis YYBJ-06-01 bacterial bodies increases with the increase of pH, and the bacterial bodies cannot grow in the culture medium with pH of 2 and 3; the growth is slow in the culture medium with pH of 3.5; when the pH is increased to 4 or higher, the growth rate of the bacterial bodies is significantly accelerated, and the growth of the bacterial bodies is best in the culture medium with pH of 6.5. By comparison, it can be concluded that the growth state of the strain is obviously inhibited in the environment with pH of 3, and the strain has certain tolerance to the acidic environment with pH of 4, and the growth state of the strain is best in the environment with pH≥5.0. From Figure 3 it can be seen that Bacillus licheniformis YYBJ-06-01 is subjected to bile salt tolerance test, the bacterial bodies grow normally in the bile salt-free medium; the growth is relatively good in the medium containing 0.1% and 0.2% bile salt; and the bacterial bodies almost cannot grow when the bile salt concentration exceeds 0.3%.
[0034] Example 3: Study on the bacteriostatic ability of Bacillus licheniformis YYBJ-06-01, specifically as follows: The antibacterial performance of the strain was determined by Oxford cup agar diffusion method. After 24 h of activation culture, the YYBJ-06-01 bacterial liquid was centrifuged at 4°C and 10000 r / min for 10 min, and the supernatant was taken for standby. 200 μL of fresh bacterial liquid of Escherichia coli, Salmonella and Staphylococcus aureus was evenly coated on LB solid medium. After the bacterial liquid was completely absorbed, an Oxford cup was placed on each plate, 200 μL of supernatant was added into each Oxford cup, and the plate was placed vertically in a constant temperature incubator at 37°C. After 24 h, whether an antibacterial ring was formed was observed, and the diameter of the antibacterial ring was accurately measured. The test was repeated three times. As shown in Table 1, the antibacterial degree is indicated by the size of the antibacterial ring, “+++” indicates 24-30 mm, “++” indicates 16-23 mm, “+” indicates 9-15 mm, and “-” indicates no antibacterial ring.
[0035] Table 1 Antibacterial properties of fermentation supernatant of strain YYBJ-06-01 on common pathogens
[0036] As can be seen from Table 1, the fermentation supernatant of Bacillus licheniformis YYBJ-06-01 has good inhibitory effect on Escherichia coli, Salmonella and Staphylococcus aureus, and the diameters of the antibacterial rings are about 16 mm, 12 mm and 13 mm, respectively.
[0037] Example 4: Study on the sensitivity of Bacillus licheniformis YYBJ-06-01 to common antibiotics, as follows: The antibiotic sensitivity was determined by paper disc diffusion method. Specifically, 200 μL of activated culture of the isolated strain after 24 h was evenly coated on LB solid medium, and the paper disc diffusion method was used to determine the drug resistance of the strain to 10 kinds of antibiotics including tetracycline, chloramphenicol, gentamicin, compound sulfamethoxazole, penicillin, ampicillin, erythromycin, ceftriaxone, lincomycin and ciprofloxacin. The diameter of the antibacterial ring greater than 1 mm of the diameter of the drug sensitive disc (6 mm) was considered to have antibacterial property, and the diameter of the antibacterial ring less than or equal to 1 mm was considered to have no antibacterial property. As shown in Table 2, the antibacterial degree is indicated by the size of the antibacterial ring, “+++” indicates 24-30 mm, “++” indicates 16-23 mm, “+” indicates 7-15 mm, and “-” indicates no antibacterial ring.
[0038] Table 2 Results of antibiotic sensitivity experiment of strain YYBJ-06-01
[0039] Table 2 shows that the resistance of Bacillus licheniformis YYBJ-06-01 to 10 antibiotics was determined by the disk diffusion method. The results showed that Bacillus licheniformis YYBJ-06-01 was sensitive to tetracycline, chloramphenicol, gentamicin, trimethoprim-sulfamethoxazole, penicillin, ampicillin, erythromycin, ceftriaxone, and ciprofloxacin, but not sensitive to lincomycin.
[0040] Example 5: Experiment on the effects of Bacillus licheniformis YYBJ-06-01 on the growth performance, immunity, and antioxidant function of Youyubian chicken, as detailed below: I. Materials and Methods: Experimental Design and Experimental Diets: The experiment employed a randomized controlled trial design, selecting 80 one-day-old chicks of similar weight and dividing them into 4 groups. The control group was fed a basal diet; the experimental groups consisted of two subgroups, fed with Bacillus licheniformis YYBJ-06-01 (1×10⁻⁶ viable count) supplemented with 500 mg / kg (low-dose group) and 1000 mg / kg (high-dose group) respectively. 9 The antibiotic group was fed a diet supplemented with 30 mg / kg florfenicol. They had free access to food and water. At the end of the experiment, they were fasted for 5 hours but allowed free access to water. Blood was then collected from 6 animals in each group, and the serum was separated and stored at -20°C for the determination of antioxidant indicators, serum immunoglobulin levels, and cytokines.
[0041] The serum antioxidant indicators were measured as follows: malondialdehyde (MDA) content and glutathione peroxidase (GSH-Px) activity in the serum of border chickens were measured. The kits were products of Nanjing Jiancheng Bioengineering Institute.
[0042] The immunological indicators were measured as follows: On day 40, 6 chickens were randomly selected from each replicate, and the serum immunoglobulin G (IgG) (Shanghai Future Industrial Co., Ltd.) and IL-1β (Wuhan Jilide Biotechnology Co., Ltd.) levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit according to the kit instructions for the corresponding indicators.
[0043] II. Experimental Results: The effects of each group on the growth performance of Youyubian chickens were observed by comparing the fecal shape and growth status of chicks in the experimental and control groups. It was found that neither high nor low doses of Bacillus licheniformis YYBJ-06-01 caused diarrhea or poor growth in the chicks, and their digestive organs were not damaged. Figure 4As shown, analysis of the weight gain of 42-day-old chicks revealed that chicks fed with Bacillus licheniformis YYBJ-06-01 showed significantly different growth compared to the control group. Furthermore, the chicks within the group exhibited better uniformity in growth. The body weight of the low-dose group was not significantly different from that of the antibiotic group (P>0.05), indicating that Bacillus licheniformis YYBJ-06-01 possesses a certain growth-promoting ability.
[0044] The effect of Bacillus licheniformis on antioxidant indices in the serum of Youyubian chickens, such as Figures 5-6 As shown, compared with the control group, serum GSH-Px levels in the low-dose and high-dose groups of border chickens were significantly increased (P < 0.01, P < 0.0001), while there was no significant difference between the low-dose group and the antibiotic group (P > 0.05). Serum MDA levels were significantly decreased compared with the control group, but there was no difference between the low-dose and high-dose groups and the antibiotic group (P > 0.05). These results indicate that Bacillus licheniformis YYBJ-06-01 can effectively increase serum GSH-Px levels and decrease serum MDA levels, thereby enhancing the antioxidant capacity of border chickens.
[0045] The effects of Bacillus licheniformis YYBJ-06-01 on serum immune indicators in Youyubian chickens, such as Figures 7-8 As shown, compared with the control group, the addition of 500 mg / kg and 1000 mg / kg Bacillus licheniformis YYBJ-06-01 to the diet increased the serum IgG level in border chickens (P<0.01, P<0.001) and decreased the serum IL-1β level (P<0.01, P<0.001). There was no significant difference between the low-dose group and the antibiotic group, indicating that Bacillus licheniformis YYBJ-06-01 can improve the immune function of border chickens and reduce the level of inflammatory factors in the body to a certain extent.
[0046] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.
Claims
1. A type of Bacillus licheniformis derived from chickens, characterized in that, It was named Bacillus licheniformis YYBJ-06-01 and is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M20252095.
2. The *Bacillus licheniformis* strain from chickens according to claim 1, characterized in that, The *Bacillus licheniformis* strain from the border chickens can grow in an acidic environment with a pH of 3.5-6.
5.
3. The *Bacillus licheniformis* strain from chickens according to claim 1, characterized in that, The *Bacillus licheniformis* strain from the border chickens can grow in a culture medium with a bile salt concentration of 0.1%-0.2%.
4. The use of *Bacillus licheniformis* or its bacterial culture as described in any one of claims 1-3 in the preparation of an antibacterial agent.
5. The application according to claim 4, characterized in that, The antibacterial agent is used to inhibit at least one of Escherichia coli, Salmonella, and Staphylococcus aureus.
6. The use of Bacillus licheniformis or its bacterial solution from chickens as described in any one of claims 1-3 in the preparation of feed.
7. The application according to claim 6, characterized in that, The feed is used to improve animal immunity, inhibit oxidative stress, and promote animal growth.
8. The application according to claim 7, characterized in that, The animals described are poultry.
9. The application according to claim 8, characterized in that, The animal in question is a chicken.
10. The application according to claim 9, characterized in that, The animal in question is a pheasant.