Clostridium butyricum and culture method and application thereof

Abstract

The application discloses Clostridium butyricum and a culture method and application thereof, belongs to the field of feed additives, and aims at the problem that a wide range of Clostridium butyricum needs a long adaptation cycle to the endogenous intestinal environment of animals, thus causing the shortening of a controllable fattening period and the incomplete exertion of the efficacy of Clostridium butyricum. The Clostridium butyricum from Beina Biotechnology Co., Ltd. is cultured by adopting in-vitro simulation of the endogenous environment of animals, after the Clostridium butyricum completely adapted to the endogenous environment of animals is obtained, the endogenous Clostridium butyricum screened out is coated by adopting a coating technology, so that the endogenous Clostridium butyricum is not destroyed in the strong acid environment in the stomach of animals, and the endogenous Clostridium butyricum is ensured to reach the intestines in a large amount and be released. Since the strains are more adaptable, the endogenous Clostridium butyricum can rapidly adapt to the intestinal environment of animals, and in the case that there is almost no adaptation cycle, efficient colonization and functional expression are rapidly formed. The endogenous Clostridium butyricum can be used as a biological type of feed additive.

Description

Technical Field

[0001] This invention relates to the field of animal microecological preparations, specifically to a Clostridium butyricum strain, its culture method, and its application, thereby enhancing the efficacy of Clostridium butyricum. Background Technology

[0002] Clostridium butyricum (CB) is an obligate anaerobic, Gram-positive spore-forming bacillus belonging to the genus Clostridium in the family Bacillusceae. It exists widely in anaerobic environments in spore form. Under anaerobic conditions, CB can produce lactic acid and butyric acid, possessing the advantages of both lactic acid bacteria and spore-forming bacteria. However, the optimal growth temperature for CB is typically 25°C, and the optimal pH for growth is 7.0, which contradicts the 40°C and slightly acidic environment provided by the animal gastrointestinal tract. Furthermore, compared to the hindgut segment where CB colonizes extensively, the small intestine provides a relatively aerobic environment and is in a state of rapid emptying, which is unfavorable for CB growth, allowing it to survive and germinate only briefly. Finally, this segment of the intestine is the first part of the intestine that CB must pass through, resulting in a significant loss of CB during this process. Summary of the Invention

[0003] To address the problems existing in the prior art, this invention provides a Clostridium butyricum strain, its culture method, and its application. It simulates the endogenous environment of animals in vitro, screens Clostridium butyricum strains, and provides Clostridium butyricum strains that are more suitable for the animal's internal environment directly to the animals being raised.

[0004] The present invention is achieved as follows: a Clostridium butyricum, specifically Clostridium butyricum DZ-X-001, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34115, on April 7, 2025.

[0005] The Clostridium butyricum mentioned is an encapsulated, animal-derived Clostridium butyricum.

[0006] A method for culturing Clostridium butyricum encapsulated in animals includes the following steps:

[0007] (1) Using sterile water as a carrier, prepare an animal endogenous culture medium containing the following components.

[0008] 50-100 mM Tris-HCl buffer solution with pH 5.5-7.5

[0009] NaCl 0.0006%-0.0018%

[0010] KCl 0.04%-0.08%

[0011] CaCl2·2H2O 0.015%-0.045%

[0012] MgCl2·6H2O 0.02%-0.06%

[0013] Casein peptone 1%-2%

[0014] Glucose 0.5%-1%

[0015] Yeast powder 0.5%-1%

[0016] β-glucan 0.025%-0.5%

[0017] Pepsin 0.01%-0.05%

[0018] Trypsin 0.02%-0.1%

[0019] Amylase 0.02%-0.1%

[0020] Lipase 0.005%-0.02%

[0021] Lactic acid bacteria 1×10 8 -1×10 10 CFU / L

[0022] Bifidobacterium 5×10 5 -1×10 11 CFU / L

[0023] The percentage is a mass-to-volume ratio;

[0024] (2) Preparation of animal endogenous Clostridium butyricum fermentation broth

[0025] In the prepared animal endogenous culture medium, inoculate with Clostridium butyricum fermentation broth with OD ≥ 1.0 at λ = 600 nm. Incubate at 40-45℃ in an anaerobic environment for 2-3 days. Dilute and spread the fermented broth on a total nutrient substrate plate. Observe under a microscope and select vigorous Clostridium butyricum colonies according to their morphology. Then, dissolve the colonies picked by the inoculation loop in sterile water to prepare a bacterial suspension with OD ≥ 0.5 at λ = 600 nm. Inoculate the bacterial suspension again into the animal endogenous culture medium. Repeat this step until the Clostridium butyricum fermentation broth cultured in the animal endogenous culture medium is diluted with sterile water to a concentration of 10. -6 At the exponential level, if the viable count is greater than or equal to 100, it is considered that *Clostridium butyricum* has fully adapted to the endogenous environment, and the obtained bacterial solution is a fermentation broth of animal endogenous *Clostridium butyricum*.

[0026] (3) Animal endogenous Clostridium butyricum coating

[0027] (3-1) Preparation of culture medium mixture solution

[0028] The culture medium mixture comprises the following components by volume percentage:

[0029] The animal endogenous culture medium contains 30%-50%

[0030] Whey protein solution 10%-30%

[0031] Tea extract 20%-40%

[0032] The whey protein solution is prepared by using whey protein, sucrose, and distilled water to prepare a whey protein solution with a mass-to-volume ratio of 6%, wherein the mass ratio of whey protein to sucrose is 1:1.

[0033] The tea extract is prepared by drying and pulverizing unfermented green tea, passing it through a 20-mesh sieve, and storing it for later use. It is then added to boiling water at a mass-volume ratio of 10% and steeped for 15 minutes. After filtering through filter paper, the tea extract is obtained.

[0034] (3-2) Preparation of coating wall material solution and protective agent

[0035] According to the mass-volume ratio, the coating wall material solution includes the following components:

[0036] Whey protein 7%-9%

[0037] Chitosan 0.6%-0.8%

[0038] Stachyose 0.2%-0.4%

[0039] Seaweed polysaccharides 1.0%-1.3%

[0040] Carrageenan 0.15%-0.25%

[0041] Glycerin 0.05%-0.15%

[0042] Resistant starch 7.5%-8.5%

[0043] The remainder is distilled water;

[0044] According to the mass-volume ratio, the protective agent includes the following components:

[0045] Microalgae protein content: 9.5%-10.5%

[0046] Monosodium glutamate 1.5%-2.5%

[0047] Trehalose 2.5%-3.5%

[0048] Glycerin 2.5%-3.5%

[0049] The remainder is sterile water;

[0050] (3-3) Animal endogenous Clostridium butyricum coating

[0051] Step 1: Adjust the pH of the prepared culture medium mixture to 6.5 ± 0.2;

[0052] Step 2: Add the animal endogenous butyric acid Clostridium fermentation broth to the mixture. When the inoculated animal endogenous butyric acid Clostridium grows to the logarithmic growth phase, add the coating wall material solution and the protective agent at a volume ratio of 10% of the above mixed culture medium solution. During the thorough mixing process, the coating wall material solution and the protective agent complete the coating of the endogenous butyric acid Clostridium.

[0053] Step 3: Turn on the main switch of the spray dryer and set the following on the control panel: inlet temperature 100-160℃, flow rate 80-150mL / h, pump peristaltic speed 10-30r / min, pressure (1.0-2.0)×10 4 Pa, fan frequency 15-25 Hz, needle setting 3-5, outlet temperature 40-80 ℃; wait at least 30 minutes to allow the internal conditions of the spray dryer to reach the preset values; insert the liquid tube into the mixture obtained in the second step, and after all the mixture has been extracted, obtain the dried coated animal endogenous butyric acid Clostridium in the collector, and obtain the coated animal endogenous butyric acid Clostridium preservation number CGMCCNo.34115.

[0054] Furthermore, the lactic acid bacteria is *Lactobacillus plantarum*.

[0055] Furthermore, in step (3-3), the pH value of the solution is adjusted using a 0.1 mol / L sodium hydroxide solution.

[0056] Further, in step (3-2), the coating wall material solution is prepared, mixed, and then autoclaved.

[0057] Furthermore, in step (3-2), the preparation of the protective agent requires the use of a uniform hydration method, with the rotation speed set in the range of 800-1200 r / min and the uniform stirring time at room temperature being 20-30 min.

[0058] The above-mentioned application of Clostridium butyricum in the preparation of feed additives.

[0059] Furthermore, the addition amount is 50g-1000g of coated animal endogenous Clostridium butyricum per ton of feed.

[0060] The advantages and technical effects of this invention are as follows: By simulating the gastrointestinal environment of animals, an endogenous Clostridium butyricum culture model is established, allowing it to proliferate in large quantities. After completing metabolic adaptation and expression, it is collected and applied to animal feeding, achieving rapid matching between Clostridium butyricum and the animal's intestinal environment. Based on the structural characteristics of the animal's gastrointestinal tract, a Clostridium butyricum coating process is formulated to improve the resistance of Clostridium butyricum in the animal's gastrointestinal environment, avoid its loss in the stomach and the upper part of the intestine, maximize its exposure in the colon, enhance the effect of use, and improve economic benefits. Attached Figure Description

[0061] Figure 1 This is a diagram simulating the count of live bacteria in gastric juice.

[0062] The *Clostridium butyricum* strain described is *Clostridium butyricum* DZ-X-001, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34115, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, on April 7, 2025. Detailed Implementation

[0063] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0064] Examples of the coated animal endogenous Clostridium butyricum, its culture method, and its application of the present invention are as follows.

[0065] 1. Preparation of coated animal endogenous Clostridium butyricum (CGMCC NO. 34115)

[0066] 1.1 Screening of animal-derived endogenous Clostridium butyricum by fermentation

[0067] Take a 5L Erlenmeyer flask, add 1L of sterile water, then add 20g of glucose, 25g of peptone, 10g of yeast powder, 1g of potassium dihydrogen phosphate, 1g of dipotassium hydrogen phosphate, and 0.5g of magnesium sulfate. Set the culture temperature to 38℃, an anaerobic environment, and the pH of the fermentation broth to 2.5. Add all of the lyophilized Clostridium butyricum powder (BNCC 379338) purchased from Beina Biotechnology Co., Ltd. to the culture medium for fermentation. When the fermentation broth reading is greater than or equal to 1.0 under the condition of OD=600nm on a UV spectrophotometer, the culture is considered complete, and the Clostridium butyricum fermentation broth is obtained. Transfer it to a 4℃ refrigerator for later use.

[0068] Animal endogenous culture medium was prepared according to the contents of Table 1, using sterile water as a carrier, and 1L was prepared.

[0069] Table 1. Animal endogenous culture medium

[0070]

[0071] After preparing the animal endogenous culture medium as shown in the table above, inoculate 1L of the prepared Clostridium butyricum (BNCC 379338) fermentation broth into the animal endogenous culture medium. Set the culture temperature to 40~45℃ and maintain a strictly anaerobic environment for 2-3 days. Dilute and spread the cultured bacterial solution on a total nutrient substrate plate. Observe under a microscope and select vigorous Clostridium butyricum colonies according to their colony morphology using an inoculation loop. Dissolve the colonies in 10ml of sterile water and detect OD≥0.5 at a wavelength of 600nm.

[0072] The obtained bacterial suspension with OD ≥ 0.5 was then inoculated into 1 L of animal endogenous culture medium. This experimental procedure was repeated, and the number of viable colonies was counted after each plate plating, until the butyric acid fermentation broth cultured in the animal endogenous culture medium was diluted with sterile water to 10⁻⁶. -6 At the exponential level, if the number of viable bacteria is greater than or equal to 100, it is considered that ordinary Clostridium butyricum has fully adapted to the endogenous environment, and Clostridium butyricum that is not adapted to the environment has been eliminated. Therefore, the bacterial solution obtained at this time is the fermentation broth of animal endogenous Clostridium butyricum.

[0073] 1.2 Coating of animal endogenous Clostridium butyricum

[0074] 1.2.1 Solution Preparation

[0075] (1) Preparation of whey protein solution: Prepare a whey protein solution with a mass-volume ratio of 6% using whey protein, sucrose and distilled water, wherein the mass ratio of whey protein powder to sucrose is 1:1 (i.e., 100mL of sample contains 6g whey protein powder + 6g sucrose).

[0076] (2) Preparation of tea extract: Dry and crush unfermented green tea and store it through a 20-mesh sieve. Add 10% by mass-volume ratio to boiling water and soak for 15 minutes. Filter the tea extract through filter paper to obtain the tea extract.

[0077] (3) Preparation of coating wall material solution: Prepare wall material solution with 1L of distilled water according to Table 2, mix well and then autoclave.

[0078] (4) Preparation of protective agent: Prepare the protective agent with 1L of deionized water according to the contents of Table 3, add it to the rotor and set the speed to 800-1200r / min. Stir at room temperature for 20-30min.

[0079] Table 2 Preparation of coating wall material solution (Unit: % g / ml)

[0080]

[0081] Table 3. Preparation of Protective Agents (Unit: % g / ml)

[0082]

[0083] 1.2.2 Preparation of Coated Animal Endogenous Clostridium butyricum (CGMCC NO. 34115)

[0084] Step 1: Mix the culture medium mixture thoroughly according to Table 4.

[0085] Step 2: Adjust the pH of the solution to 6.5 ± 0.2 using 0.1 mol / L sodium hydroxide solution.

[0086] Step 3: Inoculate animals with endogenous Clostridium butyricum fermentation broth.

[0087] Step 4: Once the endogenous butyric acid bacteria inoculated into the animal have grown to the logarithmic growth phase (about 16 hours of fermentation), add the coating wall material solution and the protectant, each accounting for 10% of the volume of the mixed culture medium solution. By thoroughly mixing, the wall material and protectant will complete the coating of the endogenous butyric acid bacteria.

[0088] Step 5: Set the spray drying conditions on the spray dryer as follows: inlet temperature 100-160℃, flow rate 80-150mL / h, pump peristaltic speed 10-30r / min, and pressure 1.0-2.0×10⁻⁶. 4 Pa, fan frequency 15-25 Hz, needle setting 3-5, outlet temperature 40-80 ℃.

[0089] Step 6: After turning on the machine, start the spray dryer and preheat for at least 30 minutes to reach the preset temperature.

[0090] Table 4. Preparation of Culture Medium Mixtures (Unit: %)

[0091]

[0092] 2 Application Cases

[0093] 2.1 Tolerability verification

[0094] 2.1.1 Preparation of simulated porcine gastric and small intestinal fluids

[0095] (1) Preparation of simulated pig gastric juice: Weigh 2g of NaCl, 3.2g of pepsin, and 7mL of 36.5% concentrated HCl. Add water to 1000mL, mix well, and prepare artificial gastric juice.

[0096] (2) Preparation of simulated pig small intestinal fluid: Weigh 6.8g of KH2PO4, add 500mL of water to dissolve it, adjust the pH to 6.8 with 0.1mol / L NaOH solution, weigh 10g of pancreatic enzyme, add water to dissolve it, mix the two solutions, add 3g of pig bile salt, dilute with water to 1000mL, and prepare artificial small intestinal fluid.

[0097] 2.1.2 Test on the ability of bacterial powder to tolerate gastrointestinal fluid environment

[0098] Clostridium butyricum (product number: BNCC379338) purchased from Beina Biotechnology Co., Ltd. was used to prepare bacterial powder according to Table 5. At a constant temperature of 37℃, 1g of the prepared bacterial powder from each of the four groups was placed into four test tubes containing 8mL of simulated gastric fluid, and incubated at 60r·min. -1 After mixing for 5 hours, the viable bacteria in the simulated gastric fluid were counted using the gradient dilution plate pouring method. Figure 1 The viable bacterial rate was calculated. The viable bacterial rate was calculated after treating the simulated gastric fluid with simulated intestinal fluid for 6 hours using the same method. Each treatment was performed in triplicate. Experimental results are expressed as mean ± standard deviation and were statistically analyzed using Duncan's method with SPSS 23.0 software. P <0.05 indicates a significant difference. P <0.01 indicates a highly significant difference.

[0099] Table 5 Different methods of preparing bacterial powder

[0100]

[0101] 2.1.3 Results of bacterial powder tolerance to gastrointestinal fluid environment

[0102] Clostridium butyricum primarily affects the colon in pigs. The gastric emptying time for adult pigs is 2-5 hours; therefore, the survival rate of Clostridium butyricum in simulated gastric fluid was measured after 2 hours and 5 hours. Since food takes 4-6 hours to reach the colon after entering the small intestine in adult pigs, the survival rate of Clostridium butyricum in simulated small intestinal fluid was measured after 4 hours and 6 hours.

[0103] The experimental results are shown in Tables 6 and 7. The coated animal endogenous Clostridium butyricum (CGMCC NO. 34115) significantly improved its survival rate in gastric and small intestinal fluids. P <0.01), which allows the survival rate of coated animal endogenous Clostridium butyricum (CGMCC NO. 34115) to reach more than 75% after 5 hours in gastric juice and 6 hours in small intestinal juice, which is beneficial to increasing the release of the bacteria in the colon.

[0104] Table 6 Comparison of gastric juice tolerance (unit: %)

[0105]

[0106] Note: Different lowercase letters in the superscript of the same data indicate significant differences (0.01 < 0.01). P <0.05), different capital letters indicate extremely significant differences ( P <0.01), identical lowercase letters or no letters indicate no significant difference ( P >0.05). Same as the table below.

[0107] Table 7 Comparison of small intestinal fluid tolerance (unit: %)

[0108]

[0109] 2.2 Improvement of feed utilization by coated endogenous Clostridium butyricum powder

[0110] 2.2.1 Feed nutrient composition testing protocol

[0111] The feed group with added coated endogenous Clostridium butyricum powder was used as the experimental group, the feed group with added Clostridium butyricum powder (No.: BNCC379338) purchased from Beina Biotechnology Co., Ltd. was used as the control group, and the feed group without added Clostridium butyricum powder was used as the blank control group. Baiyun Mugang nutritional complete chicken feed purchased from the market was used as the test benchmark. After adding 1% of the powder, the feed was treated with humid heat at 37℃ and humidity at 45% for 3 days. Each group was set up with 6 replicates, and the final feed nutrient composition was tested.

[0112] Step 1: Dissolve the three powders in sterile water and put them into a 50KD dialysis bag. Secure both ends of the dialysis bag with clips and set aside.

[0113] Taking one of the powder formulations as an example:

[0114] Step 2: Prepare a 0.2 mol / L phosphate hypotonic buffer solution with pH=6.50.

[0115] Step 3: Take a beaker and add hypotonic phosphate buffer until the dialysis bag is fully soaked. Then place the beaker on a temperature-controlled magnetic stirrer, add the rotor, and set the speed to 1200 rpm, the temperature to 41°C, and the time to 4 hours.

[0116] Step 4: Prepare a 0.2 mol / L phosphate hypotonic buffer solution with pH=7.99.

[0117] Step 5: Remove the dialysis bag and place it in the phosphate hypotonic buffer solution prepared in step 4, then repeat the extraction steps in step 3.

[0118] Step 6: Since macromolecules and cellulose in the feed could not pass through the dialysis bag, the hypotonic solution after the two extractions was collected and freeze-dried to obtain freeze-dried powder No. 1-1. The residue in the dialysis bag was freeze-dried to obtain freeze-dried powder No. 1-2, which was then put into use.

[0119] The other two groups were prepared into freeze-dried powder using the same method. A total of 6 freeze-dried powders were obtained from the three groups, namely 1-1, 1-2, 2-1, 2-2, 3-1, and 3-2.

[0120] 2.2.2 Results of nutrient composition analysis of feed after microbial powder treatment

[0121] The lyophilized powder was removed, and the total protein and total sugar of each group of lyophilized powder sample 1 were determined by Kjeldahl nitrogen determination method and sulfuric acid phenol detection method, respectively. The total fat and crude fiber of each group of lyophilized powder sample 2 were determined by Soxhlet extraction method and cellulose analyzer.

[0122] The experimental results are expressed as mean ± standard deviation, and statistical analysis was performed using Duncan's method with SPSS 23.0 software. P <0.05 indicates a significant difference. P <0.01 indicates a highly significant difference.

[0123] Numerous studies have demonstrated that Clostridium butyricum significantly affects total sugar and crude protein in feed. The results of this experiment are shown in Table 8. The total sugar and crude protein levels in group A (with coated endogenous Clostridium butyricum powder) were significantly higher than those in group B (with ordinary Clostridium butyricum powder). P <0.05).

[0124] Table 8 Comparison of nutrient components in feeds fermented with different bacterial solutions (unit: %)

[0125]

[0126] 2.3 Effects on growth performance, plasma biochemistry, antioxidant and immune indicators in weaned piglets

[0127] 2.3.1 Experimental Animals and Grouping

[0128] Two hundred healthy Duroc-Landrace-Large White crossbred piglets of similar condition were randomly selected at 28 days of age, with an initial weight of (6.5±0.5) kg. They were divided into four groups based on similar weight and identical sex within each replicate, with five pens per group (each pen counted as one replicate), and ten pigs per pen (five males and five females). The four groups were: Group A (negative control group): fed a basal diet (without antibiotics); Group B (positive control group): fed a basal diet plus 20 mg / kg colistin sulfate, 50 mg / kg bacitracin zinc, and 70 mg / kg chlortetracycline; Group C: fed a basal diet plus 500 mg / kg coated endogenous Clostridium butyricum (CGMCC NO. 34115); and Group D: fed a basal diet plus 500 mg / kg Clostridium butyricum (BNCC379338) purchased from Beina Biotechnology Co., Ltd.

[0129] 2.3.2 Test Methods

[0130] The pre-trial period lasted 3 days, during which the experimental pigs were fed the corresponding experimental diet; the composition and nutritional level of the basal diet are shown in Table 9. At the end of the pre-trial period, each experimental pig was weighed and then entered the formal trial period, which lasted 16 days. All experimental pigs were managed under the same conditions, with free access to feed and water, and were managed and kept healthy by designated personnel.

[0131] Table 9. Composition and Nutritional Levels of Basal Feed (Air-dried Basal) Unit: % (unless otherwise indicated)

[0132]

[0133] Note: (1) The vitamin premix provides the following per kilogram of feed: VA 10500IU, VD3 1800IU, VE 8500IU, VK3 2.50mg, VB1 3.50mg, VB2 8.00mg, VB6 3.50mg, VB12 0.05mg, niacin 28.00mg, D-pantothenic acid 25.00mg, folic acid 0.50mg, biotin 0.50mg.

[0134] (2) The mineral premix provides the following per kilogram of feed: Fe 120mg, Cu 100mg, Zn 100mg, Mn 40mg, I 0.30mg, Se 0.30mg.

[0135] (3) Except for digestible energy and net energy, which are calculated values, all other nutrient levels are measured values.

[0136] Growth performance indicators were measured: On day 1 and day 16 of the trial period, each pen was weighed after fasting for 16 hours (water was not prohibited) and the average daily gain (ADG) was calculated. During the trial, feed consumption was recorded per pen and the average daily feed intake (ADFI) was calculated. The feed conversion ratio (F / G) was also calculated.

[0137] Diarrhea rate determination: During the trial period, the fecal excretion of each experimental piglet was observed at 08:00, 10:00, 14:00, and 16:00 daily. The number of piglets with diarrhea and the number of days were recorded, and the diarrhea rate was calculated per pen. The calculation formula is as follows:

[0138] Diarrhea rate (%) = 100 × (number of piglets with diarrhea × number of days of diarrhea in experimental piglets) / (total number of experimental piglets × number of days of the trial period).

[0139] After the experiment, one male and one female piglet close to average weight were selected from each pen, and approximately 10 mL of blood was collected from the anterior vena cava. The blood was placed in a centrifuge tube containing heparin sodium (20 IU / mL) and centrifuged at 12000 r / min for 15 min to prepare plasma. Plasma biochemical indicators (including plasma endotoxin and insulin-like growth factor-I (IGF-I) levels); plasma antioxidant indicators (including malondialdehyde (MDA), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC)); and plasma immune indicators (including interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA) levels).

[0140] Statistical analysis of data: The experimental data were analyzed using SPSS 23.0 statistical software. P <0.01 indicates a highly significant difference; 0.01 < P <0.05 indicates a significant difference. P >0.05 is the criterion for judging that the difference is not significant. The experimental results are expressed as "mean ± standard deviation".

[0141] 2.3.2 Test Results

[0142] As shown in Table 10, the ADG index and diarrhea rate in group C were significantly different from those in group A. P <0.01), significantly different from group D (0.01 < P <0.05), no significant difference from group B ( P >0.05); the ADFI index was significantly higher than that of group D. This indicates that the application of the coated animal endogenous Clostridium butyricum (CGMCC NO. 34115) provided by this invention can significantly improve the average daily weight gain of piglets and reduce the diarrhea rate of piglets compared with Clostridium butyricum (No. BNCC379338) provided by Beina Biotechnology Co., Ltd., and the difference in effect with antibiotics is not significant.

[0143] Table 10 Effects on growth performance and diarrhea rate of weaned piglets

[0144]

[0145] As shown in Table 11, the endotoxin content, SOD activity, GSH-Px activity, IgA content, IL-6 content, and IL-10 content in group C were significantly different from those in group A (<0.01). P <0.05); the levels of T-AOC, IGF-Ⅰ, and IgG were significantly different from those in group A. P <0.01), which is significantly different from group D (0.01 <P <0.05).

[0146] Table 11 Effects on plasma biochemical, antioxidant, and immune parameters in weaned piglets

[0147]

[0148] The endogenous environment of animals was simulated in vitro beforehand, and *Clostridium butyricum* (product number: BNCC379338) from Beina Biotechnology Co., Ltd. was screened. Under acidic conditions and with the stimulation of magnesium sulfate, *Clostridium butyricum* showed high budding rate and rapid reproduction. Considering the temperature environment in the pig's gastrointestinal tract, the culture temperature was set at 40℃. Strict oxygen control was implemented during fermentation to ensure that anaerobic respiration dominated, thereby stimulating the development of *Clostridium butyricum* and rapidly achieving the large-scale metabolism of butyric acid and other organic acids. Phosphate buffer ensured that the system did not undergo drastic changes due to acid production, guaranteeing system stability. Based on these modifications, *Clostridium butyricum* strains more suitable for the specific animal endogenous environment can be screened for direct supply to livestock.

[0149] Meanwhile, considering the loss in the small intestine, trehalose in this invention can enhance the protective effect on the cell membrane, and phenolic compounds can also detoxify by forming complexes with bacterial surface proteins. Tannins and proteins can form hydrogen bonds between the phenolic hydroxyl groups of tannins and the carbonyl groups of protein peptide bonds. When the external environment changes, the surface components of the bacterial cells, such as surface proteins, act as mediators for communication between the cells and the environment. They can respond to environmental changes by regulating relevant signaling pathways or by being buried in macromolecular compounds through covalent or non-covalent interactions, thereby reducing cell damage. Therefore, this invention improves the resistance of endogenous Clostridium butyricum in the porcine gastrointestinal tract by adding unfermented green tea extract with a high tannin content and whey protein to the endogenous culture medium, fermenting to form macromolecular polymers, and adding a protective agent. This controls the release time of endogenous Clostridium butyricum into the gastrointestinal tract, increases the release of bacteria in the colon, and ensures that the endogenous Clostridium butyricum loses its cell wall material in the small intestine without losing effective bacteria. By the time it reaches the hindgut, the cell wall material is consumed, and a large number of endogenous Clostridium butyricum are released, improving colonization efficiency.

[0150] Based on this, the present invention aims to improve the application effect of Clostridium butyricum in improving animal production performance by establishing a coated animal endogenous Clostridium butyricum culture model, taking into account the unique characteristics of animal gastrointestinal structure and environment.

[0151] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a coated Clostridium butyricum endogenous to an animal for the preparation of a feed additive, characterized in that, The coated Clostridium butyricum animal endogenous is obtained by coating Clostridium butyricum animal endogenous with preservation number CGMCC No. 34115, and the coating method of the coated Clostridium butyricum animal endogenous comprises the following steps: (3-1) Preparation of culture medium mixed solution The culture medium mixed solution comprises the following components in percentage by volume: The animal endogenous culture medium 30%-50% Whey protein solution 10%-30% Tea extract 20%-40% The whey protein solution is prepared by using whey protein, sucrose and distilled water to prepare a whey protein solution with a mass / volume ratio of 6%, wherein the mass ratio of whey protein to sucrose is 1:

1. The tea extract is prepared by drying and crushing unfermented green tea to pass through a 20-mesh sieve for storage, adding 10% of the tea to boiling water according to the mass / volume ratio, soaking for 15 minutes, filtering with filter paper to obtain the tea extract. (3-2) Preparation of coating wall material solution and protective agent The coating wall material solution comprises the following components in percentage by mass: Whey protein 7%-9% Chitosan 0.6%-0.8% Stachyose 0.2%-0.4% Seaweed polysaccharide 1.0%-1.3% Carrageenan 0.15%-0.25% Glycerol 0.05%-0.15% Resistant starch 7.5%-8.5% The balance is distilled water; The protective agent comprises the following components in percentage by mass: Microalgae protein 9.5%-10.5% Sodium glutamate 1.5%-2.5% Trehalose 2.5%-3.5% Glycerol 2.5%-3.5% The balance is sterile water; (3-3) Coating of Clostridium butyricum animal endogenous Add the Clostridium butyricum animal endogenous fermentation broth with preservation number CGMCC No. 34115 to the culture medium mixed solution, and when the inoculated Clostridium butyricum animal endogenous grows to the logarithmic growth phase, add the coating wall material solution and the protective agent respectively to coat the Clostridium butyricum animal endogenous with the wall material solution and the protective agent, and prepare a mixed solution. The mixed solution is subjected to spray drying to obtain the coated Clostridium butyricum animal endogenous.

2. Use of the coated Clostridium butyricum animal endogenous according to claim 1 for the preparation of a feed additive, characterized in that, The step (3-3) of coating Clostridium butyricum animal endogenous comprises the following steps: First step: adjust the pH value of the prepared culture medium mixed solution to 6.5±0.2; Second step: add the Clostridium butyricum animal endogenous fermentation broth to the culture medium mixed solution, and when the inoculated Clostridium butyricum animal endogenous grows to the logarithmic growth phase, add 10% of the coating wall material solution and 10% of the protective agent to the culture medium mixed solution respectively, and fully mix to coat the Clostridium butyricum animal endogenous with the wall material solution and the protective agent; Third step: open the total switch of the spray dryer, set the inlet temperature at 100-160℃, the flow rate at 80-150 mL / h, the pump peristaltic speed at 10-30 r / min, the pressure at (1.0-2.0) × 10 4 Pa, the fan frequency at 15-25 Hz, the needle setting at 3-5, and the outlet temperature at 40-80℃; wait for at least 30 min or more to allow the internal conditions of the spray dryer to reach the preset values; insert the liquid pipe into the mixed solution prepared in the second step, and obtain the dried coated animal endogenous Clostridium butyricum in the collector after the mixed solution is completely extracted.

3. Use of the coated Clostridium butyricum animal endogenous according to claim 2 for the preparation of a feed additive, characterized in that, In the first step of step (3-3), the pH value of the solution is adjusted with 0.1 mol / L sodium hydroxide solution.

4. The use of the coated Clostridium butyricum animal endogenous according to claim 1 in the preparation of a feed additive, characterized in that, In step (3-2), the coating wall material solution is prepared and sterilized by high pressure after being mixed uniformly.

5. The use of the coated Clostridium butyricum animal endogenous according to claim 1 in the preparation of a feed additive, characterized in that, In step (3-2), the protective agent is prepared by using the uniform hydration method, and the stirring speed is set to 800-1200 r / min at room temperature, and the uniform stirring time is 20-30 min.

6. The use of the coated Clostridium butyricum animal endogenous according to claim 1 in the preparation of a feed additive, characterized in that, The addition amount is 50g-1000g of the coated Clostridium butyricum animal endogenous per ton of feed.

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