Bacillus pumilus of sheep origin and application thereof

Abstract

The application discloses a Brevibacillus sp. of sheep origin and application thereof, and relates to the technical field of biology. The Brevibacillus sp. is named Brevibacillus sp. Gx001, is isolated from sheep vaginal secretion, and its 16s rDNA sequence is shown as SEQ ID NO:1, and its preservation number is CGMCC No.33355. The Brevibacillus sp. Gx001 of the application can promote the development of poultry intestinal tract and immune organs, and then promote growth and weight gain, can be applied to poultry feed as a feed additive, has significant economic benefit potential, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a sheep-derived Bacillus pumilus and its applications. Background Technology

[0002] Bacillus pumilus ( Bacillus pumilus *Bacillus simulans*, belonging to the phylum Firmicutes and the genus *Bacillus*, is a short, rod-shaped, motile, facultative anaerobic, Gram-positive bacterium. This bacterium can form spores, exhibits strong resistance to adverse conditions, and maintains a high survival rate and metabolic activity even in complex environments. It is also easy to preserve and contains high levels of bioactive components. Researchers have successfully isolated *Bacillus simulans* from soil, plant rhizosphere, animal feces, and animal gastrointestinal contents, and its applications in various fields have been extensively studied.

[0003] Regarding the application of Bacillus pumilus, studies have found that it can effectively prevent and control pests and diseases, improve crop yield and quality, and reduce the use of chemical pesticides. It can be used as a bio-fertilizer and bio-pesticide in crop production. Furthermore, studies have found that concentrated fermentation broth of Bacillus pumilus has antibacterial properties, and some strains of Bacillus pumilus are hemolytic and can be used to treat thrombosis. In addition, in livestock farming, adding Bacillus pumilus to animal feed can improve animal growth performance, enhance immune function, reduce harmful bacterial populations, and increase beneficial bacterial populations such as lactic acid bacteria. For example, the Bacillus pumilus disclosed in patent CN114736834 A, when added to poultry feed, can reduce tissue and organ damage caused by oxidative stress in poultry. Similarly, the Bacillus pumilus disclosed in patent 104263683A can promote the digestion and absorption of nutrients in feed, enhance animal immune function, increase daily weight gain, and reduce the feed conversion ratio.

[0004] Although Bacillus pumilus has a wide range of applications, different strains have different biological characteristics. Systematic identification and characteristic studies of strains from specific hosts are of great significance for continuously expanding the collection and research scope of Bacillus pumilus strains, broadening its application range, and improving its utilization efficiency. Summary of the Invention

[0005] To address the above shortcomings, this invention provides a sheep-derived Bacillus pumilus and its application in the preparation of feed additives. The specific technical solution is as follows: This invention discloses a sheep-derived *Bacillus pumilus* isolated from sheep vaginal secretions. Its 16S rDNA sequence is shown in SEQ ID NO: 1. The bacterium is named *Bacillus pumilus* Gx001 and was deposited on January 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It is classified and named *Bacillus pumilus* (…). Bacillus pumilus (The accession number is CGMCC No. 33355).

[0006] The microbiological characteristics of Bacillus pumilus Gx001 of this invention are as follows: Gram-positive, rod-shaped, with blunt rounded ends, mostly existing alone, short and straight, with obvious spores, and no capsule.

[0007] The present invention also provides the application of the aforementioned Bacillus pumilus in the preparation of feed additives.

[0008] Preferably, the feed additive is a poultry feed additive.

[0009] Preferably, the feed additive is a liquid microbial agent, and the viable count of the feed additive is 1×10⁻⁶. 6 ~1×10 7 CFU / ml.

[0010] The present invention also provides poultry feed containing the aforementioned Bacillus pumilus Gx001.

[0011] Preferably, the poultry is chicken.

[0012] Compared with the prior art, the beneficial effects of the present invention are: The Bacillus pumilus Gx001 strain screened in this invention can promote the development of the intestinal and immune organs in poultry, thereby promoting poultry weight gain. Experiments showed that chicken flocks fed with this strain exhibited significantly better results than the control group in terms of total weight gain, weight gain rate, and average daily weight gain. The feed conversion ratio was also significantly lower in the control group without this strain. Furthermore, it did not produce any toxic side effects on the liver, kidneys, or other vital organs of the chickens, demonstrating good safety. Simultaneously, this strain has a clear origin and well-defined biological characteristics, enriching the resources of Bacillus pumilus strains applicable to livestock farming. It can be used as a feed additive in poultry feed, and its application is simple, with low production costs, demonstrating good potential for widespread application and economic benefits. Attached Figure Description

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 The colony morphology of Bacillus pumilus Gx001 of the present invention grown on LB solid medium; Figure 2 The colony morphology of Bacillus pumilus Gx001 of the present invention grown on blood agar plates; Figure 3 Staining and microscopic examination of Bacillus pumilus Gx001 of this invention; Figure 4 The results of 16S rDNA PCR amplification of Bacillus pumilus Gx001 in this invention are shown. Figure 5 This is a phylogenetic tree of the 16S rRNA sequence of Bacillus pumilus Gx001 in this invention. Detailed Implementation

[0015] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0016] Example 1: Isolation and Identification of Bacillus pumilus Gx001 1.1 Source of experimental bacterial samples Secretions were collected from the vaginas of sheep raised at a farm in Liucheng County, Liuzhou City, Guangxi Zhuang Autonomous Region, and immediately sent to the laboratory after being placed into 50mL sterile centrifuge tubes.

[0017] 1.2 Reagents Gram staining solution, batch number 250804, Beijing Luqiao Technology Co., Ltd.; Blood agar plates, batch number 250909, Beijing Luqiao Technology Co., Ltd.; Cedarwood oil, batch number 110501025, Shanghai Yiyang Instrument Co., Ltd.; LB liquid medium and LB solid medium (dry powder), batch number L1010, Beijing Solarbio Science & Technology Co., Ltd. Bacterial biochemical reaction kit, batch number L1015, Guangdong Huankai Biotechnology Co., Ltd.; Bacterial genomic DNA extraction kit, batch number A1015A, Tiangen Biotech (Beijing) Co., Ltd.

[0018] 1.3 Screening and culture of target bacteria In a biosafety cabinet, secretions collected from the sheep vagina were inoculated onto blood agar plates and incubated at 37°C for 24 hours. The culture was then observed. Single colonies were continuously purified on blood agar plates using the streak plating method until they appeared. These single colonies were then inoculated onto blood agar plates and incubated at 37°C for 24 hours. After incubation, the bacterial colony morphology, size, edge, and transparency were observed. The bacterial strain was identified using Bergey's Manual of Bacteriological Identification, and a single colony was selected for Gram staining and microscopic examination. The target strain was named Gx001.

[0019] 1.4 Biochemical Reactions A single colony of the pure culture isolate Gx001 was prepared into a homogeneous bacterial suspension with a McFarland turbidity of 1.0. 2 ml of this suspension was added to a biochemical identification strip (with 7 reaction wells containing substrate; wells 1-7 are for lysozyme, lysozyme control, mannitol, glucose, starch, nitrate, and VP, respectively). The same single colony was inoculated into 3% hydrogen peroxide solution, and then inoculated into semi-solid agar via stab inoculation with the cap fully closed. Simmons citrate biochemical tubes were also inoculated with the cap partially closed, and gelatin biochemical tubes were inoculated with the cap fully closed. The inoculated semi-solid agar was incubated at 30℃±1℃, while the identification strips and Simmons citrate and gelatin biochemical tubes were incubated at 36℃±1℃. Immediately after inoculation with 3% hydrogen peroxide solution, the presence of bubbles was observed.

[0020] 1.5 Bacterial 16S rDNA PCR amplification and sequencing Single colonies of the isolated strain Gx001 were picked and inoculated into sterilized LB liquid medium, placed in a biochemical incubator at 37°C for 24 h, and then stored at 4°C for later use.

[0021] Bacterial DNA was extracted using the column adsorption method of Tiangen's kit. The extracted DNA of the isolated strain Gx001 was sent to Nanning Guotuo Biotechnology Co., Ltd. to amplify its 16S rDNA sequence using the strain's DNA as a PCR template. Primers were synthesized by Nanning Guotuo Biotechnology Co., Ltd., as shown in Table 1.

[0022] Table 1 Primer sequence information After amplification, PCR products were tested using 1.0% agarose gel electrophoresis. Successful PCR products were sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. The results were then submitted to NCBI for BLAST comparison. Phylogenetic trees were constructed using MEGA 11.0 software for strains with homology higher than 99% and other representative Bacillus strains.

[0023] 2 Results 2.1 Colony morphology The isolated strain Gx001 was inoculated into LB solid medium and serum agar medium and incubated at 37°C. Figure 1 As can be seen, after culturing this strain on LB solid medium for 24 h, single colonies formed visible, round, pale yellow, opaque colonies. Colonies grown on blood agar medium for 24 h were round or oval, pale yellow, with a frosted glass-like texture on the surface, and β-hemolysis was observed around the colonies. Figure 2 After 36 hours of culture, colonies were picked and placed in 0.9% physiological saline. A precipitate formed, which was difficult to dissolve (this is a preliminary step for bacterial microscopy). Staining and microscopic examination are as follows... Figure 3 As shown, this strain is a Gram-positive bacterium, rod-shaped, with blunt, rounded ends, mostly existing singly, short and straight, with obvious spores, and no capsule.

[0024] 2.2 Biochemical reaction identification results The biochemical identification results are shown in Table 2 below. Based on the criteria for biochemical identification results and the bacterial biochemical identification code book, the isolated strain Gx001 was identified as Bacillus.

[0025] Table 2. Biochemical test results of isolated strain Gx001 Note: "+" indicates a positive result, meaning a corresponding color reaction occurs; "-" indicates a negative result, meaning no color reaction occurs or the color is absent.

[0026] 2.3 16S rDNA Sequence Analysis The amplification results of 16S rDNA PCR of isolated strain Gx001 are as follows: Figure 4 As shown, a clear band is visible at 750 bp. Sequencing by Guangzhou Qingke Biotechnology Co., Ltd. yielded its 16S rDNA sequence, as shown in SEQ ID NO: 1, with a confirmed length of 1383 bp.

[0027] After obtaining the sequencing results, BLAST comparison using the NCBI database revealed that the isolated strain Gx001 had a 100% similarity to Bacillus pumilus. This study, based on the 16S rDNA gene sequence of strain Gx001, downloaded the Bacillus genus (Bacillus) from the NCBI GenBank database. Bacillus Representative reference sequences from different species were used to construct phylogenetic trees using the neighbor-joining method. Bootstrap sequences were repeated 1000 times, and bootstrap values ​​were labeled at branch nodes. A scale bar of 0.01 represents 1% sequence difference. Phylogenetic trees of strains based on 16S rDNA sequences. Figure 5 The results showed that the isolated strain Gx001 was most similar to Bacillus pumilus KT321684.1 and KC692175.1. Based on morphological observation, biochemical reaction test and sequence analysis, the isolated strain Gx001 was identified as Bacillus pumilus.

[0028] Example 2: Pathogenicity of Bacillus pumilus Gx001 in mice Sixteen healthy 4-week-old Kunming mice, weighing 20-25 grams, half male and half female, were selected. Pathogenicity experiments were conducted for 7 days using different infection routes. The mice were allowed free access to food throughout the experiment. The mice's condition was observed daily, and data were recorded.

[0029] Gavage group: Five mice were selected as the experimental group, and three mice were selected as the blank control group. From day 1 to 4, the experimental group mice were gavaged daily with 200 µL of a solution containing 8 × 10⁻⁶ mg / L of glucose. 7 Mice were administered a CFU / mL solution of Bacillus pumilus Gx001, while the control group was fed 200 µL of purified water daily. After 5-7 days, both groups were stopped from oral feeding and their condition was observed. All mice survived, exhibited normal mental state, behavior, appetite, and feces, with no significant differences between the experimental and control groups. No obvious clinical symptoms were observed in the mice during the experiment.

[0030] Injection group: Six mice were selected as the experimental group, and two mice were selected as the blank control group. On the first day, the experimental group mice received an intraperitoneal injection of 200 µL of a concentration of 8 × 10⁻⁶. 7 Mice were injected with CFU / mL of Bacillus pumilus Gx001 bacterial suspension, while the control group mice received no injections. Mice were observed for 2-7 days. All mice survived, exhibited normal mental state, behavior, appetite, and feces; there were no significant differences between the experimental and control groups. No obvious clinical symptoms were observed in the mice during the experiment.

[0031] The above experiments comprehensively evaluated the safety of Bacillus pumilus Gx001 from two routes of infection, clarifying the optimal bacterial concentration for this bacterium at 8 × 10⁻⁶. 7 CFU / ml did not cause gastrointestinal infection, bloodstream infection, or wound infection; the preliminary assessment was that the bacterial concentration was 8 × 10⁻⁶. 7 Short Bacillus strains at cfu / ml are non-pathogenic.

[0032] Example 3: Application and Effect of Bacillus pumilus Gx001 in Chicken Feed Additives Twenty healthy 14-day-old Sanhuang chickens from the same batch, all female, with an average weight of 323.34 grams, were selected. Four experimental groups were set up. Groups 1, 2, and 3 were fed with Bacillus subtilis Gx001 (bacterium 7) screened in Example 1, at a concentration of 1×10⁻⁶. 6 CFU / ml, 1×10 7 CFU / ml, 1×10 8 CFU / ml; Group 4 served as the control group, fed 10 mL of PBS solution. Each group contained 5 chickens, all fed standard feed (initially 310 g / day, increasing by 50 g every 7 days) and unlimited drinking water during the experiment. Probiotics were administered by mixing 10 ml of probiotic solution with 10 g of feed and then mixing it into the daily diet. The experiment lasted from April 24, 2026 to May 14, 2026, a total of 21 days. Weight changes in the flock were recorded regularly; the results are shown in Table 3.

[0033] Among them, the initial weight and final weight are the average values, the total weight gain = final weight - initial weight, the average daily weight gain = total weight gain / 20 days, the weight gain rate = (total weight gain / initial weight) × 100%, the weight gain compared to the control group = total weight gain of each group - weight gain of the control group, and the relative improvement rate = weight gain compared to the control group / total weight gain of the control group.

[0034] Table 3. Weight changes of chickens in each group Table 3 shows: Total weight gain improvement: Total weight gain increased by 20.8% (from 2083.07g in the control group to an average of 2515.80g across the three groups). Group 2 contained Bacillus 7 (1×10⁻⁶). 6 The best performance was observed in CFU / ml, with a weight gain of 851.21g more than the control group, representing a relative increase of 40.86%; Group 3 (1×10⁻⁶ CFU / ml) showed the best performance. 8 The group with the lowest CFU / ml weight gain was only 55.95g more than the control group, with a relative improvement rate of only 2.69%. The order of total weight gain improvement among the groups was: Group 2 > Group 1 > Group 3.

[0035] Daily weight gain: The average daily weight gain increased by 20.8% (from 104.15g in the control group to an average of 125.79g across the three groups). The daily weight gain in each group was significantly higher than that in the control group (104.15g / day). Group 2 had the highest daily weight gain (146.71g / day), which was 1.4 times that of the control group. The ranking of daily weight gain among the groups was: Group 2 > Group 1 > Group 3 > Control Group.

[0036] Comparison of weight gain rates: The weight gain rate increased by 17.9% (from 124.96% in the control group to an average of 147.36% across the three groups). The highest increase was recorded in Group 2 at 177.22%.

[0037] It is evident that, compared to the control group, the total weight gain, average daily weight gain, and weight gain rate of the Sanhuang chickens were all improved after feeding probiotics (groups 1 to 3). Among all the probiotics mentioned above, the concentration of 1×10⁻⁶ was the highest. 7 The CFU / ml concentration of Bacillus pumilus Gx001 (group 2) showed the best growth-promoting effect, with all growth indicators ranking first among all groups. At this concentration, the weight gain rate of Sanhuang chickens in this experiment reached 1.42 times that of the control group, and the total weight gain was 40.86% higher than that of the control group. This indicates that adding this concentration of Bacillus pumilus Gx001 to chicken feed can effectively promote the growth of Sanhuang chickens and can be used as a chicken feed additive, showing good prospects for industrial application.

[0038] To investigate the effects of the present invention *Bacillus pumilus* Gx001 on chicken organs, three chickens were selected from each of the above groups and fed for another 21 days. Two chickens from each group, close to their average weight, were then dissected, and the weights of organs such as the heart, liver, and spleen were measured (see table below for 10). 6-1 It is one of the two chickens selected in group 1, 10 6-2 It's another one; 10 7-1 It is one of the two chickens selected from the two groups, 10 7-2 It's another one; 10 8-1 It is one of the two chickens selected from the three groups, 10 8-2 (This refers to another chicken; Control-1 is one of the two chickens selected from the four groups, and Control-2 is the other chicken). The organ index was calculated as organ weight / live body weight. The weight of the duodenum, jejunum, ileum, cecum, etc., was measured, and the relative intestinal weight index was calculated as intestinal weight / body weight × 100%. The length of the duodenum, jejunum, ileum, cecum, etc., was measured, and the relative intestinal length index (cm / kg) was calculated as intestinal length (cm) / body weight (kg). The results are shown in Tables 4 and 5. Table 4 Organ Index of Chickens in Each Group Table 5. Intestinal length of chickens in each group Table 4 shows: ① The heart index ranged from 0.0038 to 0.0055, with minimal differences between groups. The mean value for the control group was 0.0048. The heart index of patients fed with Bacillus pumilus Gx001 fluctuated slightly around that of the control group, indicating that the bacterium did not cause abnormal cardiac hyperplasia / atrophy and had no negative impact on cardiac development. ② No group showed a significant increase in the liver index, indicating that feeding with the bacterium did not induce hepatomegaly or liver damage. ③ The spleen index (a core immune indicator) was 0.0009 in the control group, the lowest among all groups. This bacterium enhanced spleen development and immune potential better than the control group. This indicates that there was no pathological enlargement of the heart, liver, spleen, lungs, and immune organs such as the thymus and bursa of Fabricius in all groups, indicating that feeding is safe and that Bacillus pumilus Gx001 promotes stronger development of peripheral immune organs.

[0039] Intestinal length reflects the digestive and absorptive surface area. The jejunum is the main site of nutrient absorption, and the longer the jejunum, the stronger its absorption potential. Table 5 shows that: ① The maximum duodenal length of *Bacillus pumilus* Gx001 individuals was 34.3 cm, the highest in the entire experiment, indicating that this bacterium can promote duodenal development. ② *Bacillus pumilus* Gx001 can significantly increase the length of the jejunum (a key absorption segment). ③ The maximum ileal length of *Bacillus pumilus* Gx001 individuals was 26.5 cm, and the overall average ileal length was higher than that of the control group, indicating that this bacterium has an advantage in the development of the posterior small intestine (the ileum is associated with the absorption of trace elements and vitamins). ④ The cecum is responsible for the fermentation of crude fiber. The cecal length ranges of each group were similar, with no significant differences between groups.

[0040] To investigate the ability of the Bacillus pumilus Gx001 of this invention to decompose cellulose, the applicant determined the cellulase activity of Bacillus pumilus Gx001. The results showed that the β-glucosidase activity was 20.08 U / mL and the endoglucanase activity was 11.18 U / mL. Both key enzymes showed high activity, proving that the Bacillus pumilus of this invention can completely secrete a complex enzyme system for cellulose degradation, possessing the complete ability to decompose cellulose, and can rapidly degrade feeds with high cellulose content, promoting consumption and absorption.

[0041] In summary, feeding chickens with the Bacillus subtilis Gx001 of this invention resulted in optimal spleen index and ileum length, as well as enhanced peripheral immunity and trace element absorption. In contrast, the control group exhibited the lowest growth, intestinal tract development, and immune organ development, demonstrating that adding the Bacillus subtilis Gx001 of this invention can improve broiler growth and intestinal and immune organ development.

[0042] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A type of sheep-derived Bacillus pumilus ( Bacillus pumilus ), characterized in that, Its name is Bacillus pumilus Gx001, and its accession number is CGMCC No.33355.

2. The *Bacillus pumilus* according to claim 1, characterized in that, The 16S rDNA sequence of the Bacillus pumilus Gx001 is shown in SEQ ID NO:

1.

3. The *Bacillus pumilus* according to claim 1, characterized in that, The short-lived Bacillus Gx001 was isolated from sheep vaginal secretions.

4. The use of Bacillus pumilus as described in claim 1 in the preparation of feed additives.

5. The application according to claim 4, characterized in that, The feed additive is a poultry feed additive.

6. The application according to claim 5, characterized in that, The feed additive is a liquid microbial agent, and the viable bacteria count of the feed additive is 1×10⁻⁶. 6 ~1×10 7 CFU / ml.

7. A poultry feed containing the Bacillus pumilus Gx001 as described in claim 1.

8. The poultry feed according to claim 7, characterized in that, The poultry in question is chicken.

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