Clostridium butyricum DNCBCR-212 and application thereof

Abstract

The invention provides clostridium butyricum DNCBCR-212 and application thereof, and belongs to the technical field of microorganisms. The clostridium butyricum DNCBCR-212 provided by the invention is preserved in the China General Microbiological Culture Collection Center on March 24, 2023, and the preservation number of the clostridium butyricum DNCBCR-212 is CGMCC (China General Microbiological Culture Collection Center) No.26897. The clostridium butyricum DNCBCR-212 provided by the invention has the advantages that the clostridium butyricum DNCBCR-212 The clostridium butyricum DNCBCR-212 not only can increase the body weight, daily gain and average dry matter feed intake of the calves before weaning and improve the apparent digestibility of neutral detergent fibers, crude proteins and crude fat, but also can improve the oxidation resistance and immune function of the calves before weaning.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Clostridium butyricum DNCBCR-212 and its applications. Background Technology

[0002] Monensin, a feed antibiotic, is widely used in ruminant farming due to its growth-promoting and anti-inflammatory properties. However, excessive antibiotic use not only leads to an increase in drug-resistant strains and toxic side effects but also easily causes rumen flora imbalance due to antibiotic abuse. Conversely, banning antibiotics has resulted in serious problems such as decreased feed utilization and increased disease incidence. Therefore, finding feed additives that can both prevent inflammation and promote growth performance in livestock is crucial for implementing the phase-out of medicated feed additives and reducing the use of veterinary antibiotics.

[0003] Developing novel biological feed additives is a key strategy for replacing antibiotics in feed. While the abundant metabolites of Clostridium butyricum can enhance the immunity of ruminants, their impact on nutrient utilization is limited. The gastrointestinal microecological system of ruminants is complex and variable, influenced by factors such as diet, host, age, environment, and feeding management. Adding probiotics can regulate the stability of rumen microbiota and pH in ruminants. Numerous studies have shown that adding probiotics to dairy cow diets not only improves the rumen environment but also avoids antibiotic-like drug residue problems, while effectively improving production performance. Therefore, screening suitable strains can effectively improve feed utilization and growth performance in ruminants. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a Clostridium butyricum strain DNCBCR-212 and its application. The Clostridium butyricum strain DNCBCR-212 provided by this invention can effectively improve feed utilization, growth performance, digestibility, antioxidant function and immune function of pre-weaning calves.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A strain of Clostridium butyricum ( Clostridium butyricum DNCBCR-212, the Clostridium butyricum DNCBCR-212, was deposited on March 24, 2023 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 26897.

[0006] In this invention, the depositary address of the Clostridium butyricum is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides a microbial preparation comprising Clostridium butyricum DNCBCR-212 as described in the above technical solution.

[0008] In the present application, the concentration of Clostridium butyricum DNCBCR-212 is preferably 2 x 10 7 cfu / d~2 x 10 9 cfu / d.

[0009] The present application also provides a feed additive comprising the Clostridium butyricum DNCBCR-212 described in the above technical solution or the microbial preparation described in the above technical solution.

[0010] The present application also provides the application of the Clostridium butyricum DNCBCR-212, the microbial preparation or the feed additive described in the above technical solution in improving the feed utilization rate of pre-weaning calves.

[0011] In the present application, the improvement of the feed utilization rate of pre-weaning calves preferably refers to the increase of the apparent digestibility of neutral detergent fiber, crude protein and crude fat.

[0012] The present application also provides the application of the Clostridium butyricum DNCBCR-212, the microbial preparation or the feed additive described in the above technical solution in improving the growth performance and digestive performance of pre-weaning calves.

[0013] In the present application, the improvement of the growth performance and digestive performance of pre-weaning calves preferably refers to the increase of the body weight, daily weight gain and average dry matter intake of pre-weaning calves, and the increase of the apparent digestibility of neutral detergent fiber, crude protein and crude fat.

[0014] The present application also provides the application of the Clostridium butyricum DNCBCR-212, the microbial preparation or the feed additive described in the above technical solution in improving the antioxidant capacity of pre-weaning calves.

[0015] The present application also provides the application of the Clostridium butyricum DNCBCR-212, the microbial preparation or the feed additive described in the above technical solution in improving the immune function of pre-weaning calves.

[0016] Beneficial technical effects: the present application provides a Clostridium butyricum (C. Clostridium butyricum ) DNCBCR-212 and its application, which was preserved in the China General Microbiological Culture Collection Center on March 24, 2023, with the preservation number of CGMCC No. 26897. The Clostridium butyricum DNCBCR-212 not only can increase the body weight, daily weight gain and average dry matter intake of pre-weaning calves, but also can improve the apparent digestibility of neutral detergent fiber, crude protein and crude fat, and can improve the antioxidant capacity and immune function of pre-weaning calves. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1Phylogenetic tree of Clostridium butyricum strain DNCBCR-212 and other related strains. Figure 2 Blood plate test of DNCBCR-212; wherein, the left panel is Clostridium butyricum group, and the right panel is E. coli k99 control group. Figure 3 Effect of feeding Clostridium butyricum DNCBCR-212 on the composition of the rumen microflora of Angus calves; CON = control group, basal diet; CB212 = Clostridium butyricum group, basal diet + 2.0 x 10 9 CFU / kg. Figure 4 Effect of feeding Clostridium butyricum DNCBCR-212 on the composition of the rumen microflora of Angus calves. DETAILED DESCRIPTION

[0018] In order to better understand the present application, the content of the present application is further illustrated below in combination with examples, but the content of the present application is not limited only to the following examples. The materials, reagents and the like used in the examples and test examples of the present application can be obtained from commercial channels unless otherwise specified, and the methods used in the examples and test examples of the present application are conventional methods unless otherwise specified.

[0019] Example 1 Strain isolation and identification Clostridium butyricum DNCBCR-212 (hereinafter referred to as strain DNCBCR-212) is a new strain isolated and screened by the applicant from the rumen fluid of Holstein cows.

[0020] 1.1 Strain isolation and identification 1.1.1 Culture medium (1) Nutrient agar (NA) medium: peptone 5 g, beef extract 3 g, NaCl 5 g, agar 15 g, deionized water 1 L, pH 7.2 ± 0.2.

[0021] (2) Tryptic soy agar (TSA): casein tryptic digest 15 g, soybean powder tryptic digest 5 g, NaCl 5 g, agar 15 g, deionized water 1 L, pH 7.3 ± 0.2.

[0022] 1.1.2 Isolation (1) Sample pretreatment: 1.5 g of rumen was placed in a 50 mL sterile centrifuge tube, 10 mL of pre-cooled sterile physiological saline (4℃) was added, 5~10 sterile glass beads were put in, vortexed for 5 min to homogenize, and left for 5 min to precipitate large particles.

[0023] (2) Heat shock treatment: Take 1 mL of supernatant into 2 mL sterile EP tube, and place it in an 80°C constant temperature water bath (preheat to 80±0.5°C in advance), heat for 30 min (±30 s), and mix evenly every 10 min. Immediately cool in ice bath for 5 min to terminate the heat shock.

[0024] (3) Gradient dilution and coating: Take 100 μL of the suspension after heat shock into 900 μL of sterile normal saline (10 - ¹ Dilution), vortex for 10 s; sequentially dilute to 10 - ², 10 - ³, 10 -4 , 10 -5 (Each time, change the new gun head); take 100 μL of 10 - ³, 10 -4 , 10 -5 dilution solution, and drop it in the center of the NA plate; use a sterile L-shaped coating rod to evenly spread it (label the dilution degree on the plate), and stand for 10 min to absorb the liquid.

[0025] (4) Culture and primary screening: Invert the plate and place it in a 37°C incubator for 24±2 h.

[0026] Observe the colony characteristics: Typical morphology of Bacillus: dry and opaque, irregular edge, rough surface (such as Bacillus subtilis).

[0027] Mark the target colonies with a marker pen (at least 3 single colonies for each type of morphology).

[0028] (5) Purification culture Use a sterile inoculation loop to pick the target colonies, and divide them into zones on a TSA plate (four-zone method); after 37°C culture for 24 h, pick a single isolated colony from the third or fourth zone; repeat the streaking and purification once to ensure no contamination.

[0029] (6) Strain preservation Preserve it in a sterile glycerol freezing tube, and store it at -80°C.

[0030] 1.13 Identification Extract the 16S rDNA sequence of DNCBCR-212, and send it for detection. The specific sequence is shown in Seq_1. BLAST comparison of the obtained sequence shows that DNCBCR-212 is Clostridium butyricum (Clostridium butyricum ). The phylogenetic tree is shown in Figure 1 .

[0031] The optimal growth temperature range of strain DNCBCR-212 is 37°C-39°C, and the maximum viable cell count reaches 9.6×10 6At CFU / mL, the survival rate of *Clostridium butyricum* DNCBCR-212 was above 95% after 3 hours at pH 3 and pH 2. The 16S rDNA sequence (Seq_1) of DNCBCR-212 was compared with the 16S rDNA sequences of all identified bacteria in the NCBI database, revealing that strain DNCBCR-212 is similar to *Clostridium* species. Clostridium butyricum The 16S rDNA sequence of strain MT903083.1 showed the highest homology similarity (99.68%), ultimately identifying the bacterial species as Clostridium butyricum. Clostridium butyricum The applicant conducted whole-genome sequencing of the strain, obtained its genome, and deposited it with the China General Microbiological Culture Collection Center, with registration number CGMCC NO.26897.

[0032] 1.1 Antibacterial activity test The antibacterial properties of Clostridium butyricum DNCBCR-212 against Escherichia coli K99 and Staphylococcus aureus were determined using the Oxford cup method.

[0033] The antibacterial activity results of Clostridium butyricum DNCBCR-212 are shown in the figure. Figure 2 See Table 1. Experimental results show that Clostridium butyricum DNCBCR-212 has a certain inhibitory effect on Escherichia coli K99 and Staphylococcus aureus.

[0034] Table 1. Inhibition zone (mm) of DNCBCR-212 1.2 Enzyme production test The ability of Clostridium butyricum DNCBCR-212 to produce amylase and protease was determined using a diffusion method.

[0035] The enzyme production results of Clostridium butyricum DNCBCR-212 are shown in Table 2. The experimental results indicate that Clostridium butyricum DNCBCR-212 can produce proteases to hydrolyze proteins.

[0036] Table 2. Enzyme hydrolysis zone (mm) of DNCBCR-212 1.3 Antibiotic susceptibility studies This study tested the susceptibility of Clostridium butyricum DNCBCR-212 to 10 antibiotics and will be conducted in accordance with the NCCLS antibiotic susceptibility testing standards.

[0037] The antibiotic susceptibility results of Clostridium butyricum DNCBCR-212 are shown in Table 3. The results indicate that the bacterium is sensitive to ceftriaxone, cefotaxime, and tetracycline; moderately sensitive to erythromycin, ciprofloxacin, and streptomycin; and resistant to penicillin, ampicillin, gentamicin, and trimethoprim-sulfamethoxazole.

[0038] Table 3. Antibiotic susceptibility of DNCBCR-212 (mm) Note: "R" represents drug resistance; "I" represents moderate sensitivity; "S" represents sensitivity.

[0039] 1.4 Safety Study According to the standard method for detecting hemolysis characteristics on blood agar plates, after amplification of the experimental strains on solid culture medium, single colonies (E. coli K99 positive control) were aseptically picked and streaked onto blood agar plates. After incubation at 37℃ for 48 hours, hemolysis was observed. The criteria for determining the type of hemolysis were: α-hemolysis showed a grass-green transparent ring around the colony; β-hemolysis showed a clearly defined, completely transparent ring; and γ-hemolysis showed no change in the characteristics of the culture medium. This detection system can effectively distinguish the differences in hemolysis characteristics among different strains.

[0040] Safety test results of Clostridium butyricum DNCBCR-212 showed that no hemolysis was observed in the Clostridium butyricum group on blood agar plates, while β-hemolysis was observed in the Escherichia coli k99 control group.

[0041] The DNCBCR-212 strain is suitable for the body temperature of ruminants, has strong acid resistance, strong inhibitory ability against Escherichia coli, can produce proteases to hydrolyze proteins, and has not shown hemolysis.

[0042] 1.5 Preparation of Clostridium butyricum preparation: (1) Cultivation of primary seed culture: The preserved Clostridium butyricum DNSYK-1 was inoculated into 2L RCM liquid culture medium, anaerobic, 37℃, and statically cultured for 24h to obtain fermentation broth; (2) Centrifugation: Centrifuge the fermentation broth at 4000 r / min, remove the supernatant, and obtain bacterial sludge; (3) Coating: After washing twice with physiological saline, the bacterial sludge was shaken with an equal volume of 1wt% skim milk for 5 minutes to obtain the coating solution; (4) Freeze-drying: The coating liquid is placed in a freeze-drying tray and freeze-dried for 48 hours to obtain freeze-dried bacterial powder.

[0043] The final lyophilized powder prepared according to the method of this embodiment contains 2 × 10⁻⁶ cells. 9 cfu / g.

[0044] Example 2 Animal Experiment Materials and Methods This study was conducted from April to July 2024 at a ranch in Zadoi County, Qinghai Province, China. The Animal Care and Use Committee of Northeast Agricultural University reviewed and approved all procedures used in this study.

[0045] 2.1 Experimental design and sample collection A total of 16 Angus calves (mean ± SD; body weight = 27.76 ± 2.83) at 21 days of age participated in this randomized clinical trial. All calves were immediately separated from their dams at birth, weighed, and transferred to individual pens, bedded with straw that was renewed every 24 h. Calves were considered to have successfully received their first feeding if they ingested at least 4 L of treated high-quality colostrum (whiteness ≥ 22%, equivalent to immunoglobulin ≥ 50 mg / mL) within 4 h of birth, according to the routine protocol for calf rearing on the farm. Two liters of colostrum were administered at 12 and 24 h.

[0046] Body weight was measured on the morning of day 21, and a randomization sequence was generated using Excel (Microsoft 365) software based on body weight, dividing the animals into two groups with 8 replicates each: control (CON) and C. butyricum DNCBCR-212 (CB; 2 x 10 7 Solid feed was provided after MR feeding in the morning every day, and the feed composition is shown in Table 4. C. butyricum was sprayed on a small amount of creep feed, and the remaining creep feed was supplemented after the calves had finished eating. To ensure that the calves could freely eat the amount, at least 200 g / d of solid feed remained every day. The calves were added to the study before weaning (from day 21 to day 77). Gradual weaning began on day 78 after the end of the trial. One calf in the CON group died on day 35 without treatment.

[0047] Table 4 Nutrient levels of concentrate (dry matter basis, %) Individual body weight was assessed every 4 weeks during the experimental period (from day 21 to day 77). All body weight measurements were performed in the morning before feeding using a digital scale (sensitivity = 0.1 kg) that had been previously checked, certified, and validated. The average daily gain was determined for both groups of animals based on body weight measurements. The feed conversion ratio was determined by the ratio of the average daily feed intake to the average daily gain.

[0048] Blood samples and rectal fecal samples were collected every 4 weeks. Blood samples were centrifuged at 3000 x g for 10 min, and 1 mL of serum / plasma was aliquoted and frozen (-20 °C) for further processing. Another 1 mL of whole blood was kept in a 4 °C incubator and sent to the laboratory within 2 h for biochemical monitoring using a fully automated biochemical analyzer. Fecal samples were manually collected from the rectum of each calf approximately 4 h after morning feeding. Five grams of fresh fecal samples were transferred to sterile cryotubes and stored frozen (-80 °C). Additionally, approximately 2 g of fresh feces were placed in plastic tubes, acidified with 2 mL of 25% phosphoric acid, and diluted with 6 mL of distilled water. The supernatant was stored frozen at -20 °C after centrifugation at 2500 x g for 20 min at 4 °C.

[0049] Fecal scores were performed in the morning according to Osorio’s criteria (Osorio et al., 2012). Fecal shape was scored as 1 for good, 2 for soft (pudding-like), 3 for wet (mush-like), and 4 for liquid (splashing). Diarrhea was defined as fecal scores of 3 and 4, and the diarrhea rate was the number of calves with diarrhea divided by the total number of calves. Drug treatments were recorded daily, and calves were treated with drugs when diarrhea, fever, and pneumonia occurred.

[0050] 2.2 Sample analysis Feed and fecal samples were collected weekly for analysis and nutritional components were collected according to previously described and validated methods, including DM, ash, OM, CP, starch, and crude fat (AOAC, 1990). Neutral detergent fiber concentrations were determined according to Van Soest and Robertson (1979), and ADF and ADL were calculated according to the procedures described by Goering and Van Soest (1970) (Forage fiber analysis (apparatus reagents, 388 procedures and some applications). Agriculture Handbook. US Department of Agriculture, Washington, DC).

[0051] Biochemical parameters were measured using a fully automated biochemical analyzer. Glucose, total protein, and immunoglobulin concentrations were measured using commercial assay kits (Huaying Biotechnology Institute, Beijing, China). Oxidative biomarkers glutathione peroxidase, superoxide dismutase (SOD), MPO, malondialdehyde (MDA), and total antioxidant capacity were analyzed according to the kit (Huaying Biotechnology Institute, Beijing, China) instructions. Insulin-like growth factor-1 was measured using an ELISA kit.

[0052] 2.3 Statistical analysis All data were analyzed using the GLM procedure of SPSS 27.0 software (SPSS Inc., Chicago, IL, USA). Duncan’s multiple range test was used to determine significant differences between means, P <0.05 was considered significant, and data were visualized using the GraphPad Prism 9.5 software package (GraphPad Inc., La Jolla, CA, USA).

[0053] 2.4 Results 2.4.1 Effect of Clostridium butyricum DNCBCR-212 on growth performance and digestibility in calves The initial body weight of the two groups of calves were 47.0 kg and 46.5 kg, respectively P > 0.05), the addition of Clostridium butyricum DNCBCR-212 in the diet effectively increased the body weight and daily gain of 77-day-old calves (Table 5). The addition of Clostridium butyricum DNCBCR-212 in the diet increased the average dry matter intake, but had no significant effect on feed efficiency P > 0.05; Table 5). The nutritional intake data are shown in Table 6. Compared with the CON group, the CB group effectively increased the apparent digestibility of NDF, crude protein and crude fat P

[0054] Table 5 Effect of CB group (n=8) and CON group (n=7) on the growth performance of pre-weaning calves Note: Microorganisms were directly fed with solid diet as carrier throughout the 56-day experimental period, the same below; P < 0.05 indicates significant difference, P < 0.01 indicates extremely significant difference, different letters indicate significant difference, the same below.

[0055] Table 6 Effect of CB group (n=8) and CON group (n=7) on the nutrient digestion of pre-weaning calves 2.4.2 Effect of Clostridium butyricum DNCBCR-212 on the blood of calves The results of the effect of Clostridium butyricum DNCBCR-212 on the blood of calves are shown in Table 7. As can be seen from Table 7, feeding Clostridium butyricum DNCBCR-212 had limited effect on the cell composition and cell structure in the blood, only showing that the mean corpuscular hemoglobin concentration (MCV) of the CB group decreased P < 0.05). But for the CON group, the CB group significantly reduced the activity of T-SOD and the content of MPO in the blood, and increased the content of bovine insulin growth factor P < 0.05), but had no significant effect on the content of MDA and GSH-px P > 0.05).

[0056] Table 7 Effect of CB group (n=8) and CON group (n=7) on the blood indexes of pre-weaning calves 2.5 Discussion ​Pre-weaning is a critical period for calf growth, but it is vulnerable to disease, which has a long-term impact on subsequent production performance. Therefore, it is essential to implement strategies to ensure the health status and nutritional level of pre-weaning calves. Early intake of probiotics can affect rumen fermentation status and rumen microbial flora, and also increase the content of beneficial bacteria in the intestinal tract, reducing the incidence of calf disease (Wang et al., 2022b, Wu et al., 2022).

[0057] Pre-weaning is a critical period for calf muscle and bone development, and the average daily gain of calves at this time is positively correlated with subsequent production performance. Previous studies have shown that Clostridium butyricum can increase the weight gain of pre-weaning Holstein calves and alleviate the adverse effects of heat stress in goats, but does not significantly change the digestibility and growth performance of fattening goats (Li et al., 2021, Xue et al., 2022, Zhang et al., 2022b). In this study, Clostridium butyricum DNCBCR-212 effectively increased the daily gain of pre-weaning calves, and this difference may be related to the period and dose of feeding probiotics (Wang et al., 2023). Clostridium butyricum DNCBCR-212 may have better growth-promoting effects on young ruminants in harsh environments or under bacterial invasion. Feeding Clostridium butyricum significantly increased the feed intake of Holstein cows and improved daily gain and final body weight, but had no significant effect on feed efficiency (Li et al., 2021). Therefore, we explored the effect of Clostridium butyricum on digestibility, and feeding Clostridium butyricum DNCBCR-212 effectively improved the utilization of NDF, protein, and fat in pre-weaning calves. IGF-1 secreted by the liver plays a crucial role in growth regulation, development, and metabolism. In this study, Clostridium butyricum DNCBCR-212 significantly increased the content of IGF-1 in the blood, but had no significant effect on blood glucose and ketones. Therefore, Clostridium butyricum DNCBCR-212 can increase the daily gain of calves by improving nutrient digestibility and growth hormones in the body.

[0058] The number of blood cells, morphological distribution and oxidative biomarkers in blood can be used as indicators to determine the blood condition, animal physiology and health status (Salem et al., 2019). After Babesia infection, the RBCs, HB, PCV, MCHC content of sick calves decreased, while the MCV increased significantly (Salem et al., 2019). In this study, the MCV in the blood of the CB group decreased, but there was no significant difference in other blood routine indexes, indicating that feeding Clostridium butyricum DNCBCR-212 did not affect the blood composition of pre-weaning calves. The addition of sodium butyrate in the diet significantly increased the serum T-SOD activity of calves, reduced the serum MDA concentration, and improved the antioxidant enzyme activity to enhance the antioxidant capacity of calves (Wang et al., 2022a). But feeding Clostridium butyricum significantly reduced the T-SOD activity, but had no effect on serum MDA concentration, it can be speculated that feeding Clostridium butyricum DNCBCR-212 reduced the antioxidant enzyme activity of the body, but had no effect on oxidative indicators. MPO is a key component of the cytotoxic response of neutrophils to invading pathogens, and is also an oxidative marker (<10.1074@mcp.ra120.002305.pdf>). The significant decrease in MPO content in the CB group again verified that Clostridium butyricum improved the antioxidant capacity and immune function of pre-weaning calves.

[0059] Example 3 Effect of Clostridium butyricum DNCBCR-212 on rumen and rectal microbial flora of Angus calves To further investigate the effects of Clostridium butyricum DNCBCR-212 on the rumen and rectal microbiota of Angus calves, 16S rDNA sequencing analysis was performed on rumen fluid samples (rumen fluid was collected before and after the experiment in section 2.2) and feces (same as section 2.2). In this experiment, the relative abundance of all microbial communities needed to be >0.1%, and the same microbial community with more than half of the biological replicates in each treatment group was considered a valid microbial community and participated in the analysis of microbiota composition. Therefore, the sequencing results showed that 548,229 and 823,673 valid sequences were obtained in the rumen fluid and fecal samples, respectively. Shannon and Simpson dilution curves, as one of the important indicators in α-diversity analysis, showed that the machine could effectively detect all samples in the CON and CB groups, which means that the sequencing depth was sufficient to accurately describe the composition of the rumen and rectal microbiota. Venn diagrams revealed 2174 shared strains in the rectal microbiota of the CON and CB212 groups, with 2585 strains unique to the CB212 group. Venn diagrams also showed 3656 shared strains in the rumen microbiota at the genus level, with 16952 strains unique to the CB212 group. This indicates a high degree of similarity in microorganisms across different treatment groups. β-diversity analysis results are commonly represented using principal component analysis (PCA) and nonmetric multidimensional scaling analysis (NMDS). PCA results showed significant differences in rumen microbial richness and diversity both among and within groups. P <0.05), while there was no significant difference in rectal microbiota ( P >0.10). NMDS analysis showed that the composition of the rectal microbiota was representative (0.10 < 0.10). P <0.20). Furthermore, the composition of the rumen microbiota is also quite representative ( P =0.10).

[0060] Ten phylum-level microorganisms were identified in the fecal samples. Figure 3As shown, sequencing analysis of the rectal microbiota revealed that Firmicutes, Bacteroidetes, and Tenericulates were the dominant phyla in the calf rectum. The genus-level analysis results are shown in Table 8. The dominant bacteria in the calf rectum were 5-7N15, *Lactobacillus*, *Oscillospira*, and *Blautia*. Among these, supplementation with *Clostridium butyricum* significantly increased the relative abundance of *Treponema*. P <0.05). However, there were no significant changes in other genus-level microorganisms in the fecal samples ( P >0.10).

[0061] Table 8. Effects of Clostridium butyricum DNCBCR-212 on the horizontal rectal microbial composition of Angus calves. Note: * indicates that the difference between different treatments is statistically significant. P <0.05).

[0062] Ten phylum-level microorganisms were identified in the rumen fluid samples. The dominant phylum-level bacterial groups included... Figure 4 As shown, the phyla are Firmicutes, Bacteroidetes, and Spirochaetes, respectively. Genus-level analysis (Table 9) showed that *Prevotella*, *Ruminococcus*, and *Succiniclasticum* were the dominant bacterial groups in the rumen microbiota. This experiment, through feeding *Clostridium butyricum*, significantly altered the rumen microbiota structure of calves. As shown in Table 9, feeding *Clostridium butyricum* significantly reduced the relative abundance of *Prevotella* in the rumen of calves. P <0.05). Notably, compared to CON, the relative abundance of *Succiniclasticum* was significantly increased in group CB212. P <0.05. Furthermore, this study paid special attention to Clostridium, but no significant changes were observed in Clostridium within the rumen. P >0.10).

[0063] Table 9. Effects of Clostridium butyricum DNCBCR-212 on the rumen microbial composition of Angus calves. Note: * indicates that the difference between different treatments is statistically significant.P <0.05), 2 SEM, total standard error of the mean Table 10 Effects of Clostridium butyricum DNCBCR-212 on rumen fermentation of Angus calves In summary, the present study provides unique insights into the effects of Clostridium butyricum DNCBCR-212 on pre-weaning calf growth performance, digestibility, digestive tract development, and microbial flora and their interactions. Feeding Clostridium butyricum DNCBCR-212 increased the content of isovaleric acid in the rumen and the content of butyric acid in the intestine (Table 10) and changed the composition of the microbial flora. It significantly improved the digestibility of NDF, CP, and EE and the secretion of IGF-1 in pre-weaning calves, increased daily weight gain and weaning weight. The addition of Clostridium butyricum to the diet reduced the activity of antioxidant enzymes, as indicated by a decrease in T-SOD content, but the content of MPO decreased, and the antioxidant capacity was enhanced. Clostridium butyricum improved the health status of pre-weaning calves, as indicated by a decrease in MPO content, fecal score, and the number of drug treatments. Therefore, feeding Clostridium butyricum can improve the growth performance and health status of pre-weaning calves.

[0064] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A Clostridium butyricum (C. butyricum) strain DNCBCR-212, characterized in that, Clostridium butyricum ) DNCBCR-212, which is characterized in that, The Clostridium butyricum DNCBCR-212 was preserved in the China General Microbiological Culture Collection Center on March 24, 2023, and the preservation number is CGMCC No. 26897.

2. A microbial preparation, characterized in that, The Clostridium butyricum DNCBCR-212 as claimed in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The Clostridium butyricum DNCBCR-212 has a concentration of 2 x 10 7 cfu / d ~ 2 x 10 9 cfu / d.

4. A feed additive, characterized in that, The Clostridium butyricum DNCBCR-212 as claimed in claim 1 or the microbial preparation as claimed in any one of claims 2-3.

5. The Clostridium butyricum DNCBCR-212 as claimed in claim 1, the microbial preparation as claimed in any one of claims 2-3, or the feed additive as claimed in claim 4 in improving the feed utilization rate of pre-weaned calves.

6. Use according to claim 5, characterized in that, The improvement of the feed utilization rate of pre-weaned calves is to improve the apparent digestibility of neutral detergent fiber, crude protein and crude fat.

7. The Clostridium butyricum DNCBCR-212 as claimed in claim 1, the microbial preparation as claimed in any one of claims 2-3, or the feed additive as claimed in claim 4 in improving the growth performance and digestive performance of pre-weaned calves.

8. Use according to claim 7, characterized in that, The improvement of the growth performance and digestive performance of pre-weaned calves is to increase the body weight, daily weight gain and average dry matter intake of pre-weaned calves, and improve the apparent digestibility of neutral detergent fiber, crude protein and crude fat.

9. The Clostridium butyricum DNCBCR-212 as claimed in claim 1, the microbial preparation as claimed in any one of claims 2-3, or the feed additive as claimed in claim 4 in improving the antioxidant capacity of pre-weaned calves.

10. The Clostridium butyricum DNCBCR-212 as claimed in claim 1, the microbial preparation as claimed in any one of claims 2-3, or the feed additive as claimed in claim 4 in improving the immune function of pre-weaned calves.

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