Clostridium butyricum as well as culture method and application thereof

Abstract

The invention discloses clostridium butyricum as well as a culture method and application thereof, belongs to the field of feed additives, and aims to solve the problems that a controllable fattening period is shortened and the efficacy of the clostridium butyricum cannot be completely exerted due to the fact that a wide range of clostridium butyricum needs a relatively long adaptation period for an endogenous intestinal environment of an animal. The clostridium butyricum from the North Naa Biotechnology Co., Ltd. Is cultured by simulating an animal endogenous environment in vitro, and after the clostridium butyricum which completely adapts to animal endogenous is obtained, the screened animal endogenous clostridium butyricum is coated by adopting a coating technology, so that the clostridium butyricum is not damaged by a strong acid environment in an animal stomach bag; the clostridium butyricum strain has the advantages that the clostridium butyricum strain can be used for preparing animal endogenous clostridium butyricum, a large quantity of animal endogenous clostridium butyricum can reach intestinal tracts and be released, and the animal endogenous clostridium butyricum can quickly adapt to animal intestinal environment due to higher adaptability of the strains, and can quickly form efficient colonization and functional expression under the condition of almost no adaptation period. The feed additive can be used as a biological feed additive.

Description

Technical Field

[0001] The present invention relates to the field of animal microecological preparations, and in particular to Clostridium butyricum and a culture method and application thereof, so as to enhance the application effect of Clostridium butyricum. Background Art

[0002] Clostridium butyricum (CB) is an obligate anaerobic, Gram-positive bacillus of the genus Clostridium, Bacillaceae, found as spores in anaerobic environments. C. butyricum can produce lactic and butyric acids in anaerobic environments, combining the advantages of both lactic acid bacteria and bacilli. However, C. butyricum typically has an optimal growth temperature of 25°C and a pH of 7.0, which are incompatible with the 40°C and slightly acidic environment of the animal gastrointestinal tract. Furthermore, compared to the hindgut, where C. butyricum is heavily colonized, the small intestine is relatively aerobic and rapidly empties, making it less conducive to the growth of C. butyricum, allowing it to survive and germinate only briefly. Finally, this intestinal segment is the first section of the intestine that C. butyricum must pass through, resulting in significant loss of C. butyricum. Summary of the Invention

[0003] In response to the problems existing in the prior art, the present invention provides a Clostridium butyricum and its culture method and application, which simulates the endogenous environment of an animal in vitro, screens Clostridium butyricum, and allows Clostridium butyricum that is more suitable for the animal's internal environment to be directly provided to the animals.

[0004] The present invention is achieved by providing a Clostridium butyricum (Clostridium butyricum), wherein the Clostridium butyricum is Clostridium butyricum DZ-X-001, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) with a deposit number of CGMCC No. 34115 and a deposit date of April 7, 2025.

[0005] The Clostridium butyricum is endogenous Clostridium butyricum in encapsulated animals.

[0006] A method for culturing endogenous Clostridium butyricum in an encapsulated animal comprises the following steps: (1) Using sterile water as a carrier, prepare an animal endogenous culture medium containing the following components: Tris-HCl buffer 50-100 mM, pH 5.5-7.5 NaCl 0.0006%-0.0018% KCl0.04%-0.08% CaCl2·2H2O0.015%-0.045% MgCl2·6H2O0.02%-0.06% Casein peptone 1%-2% Glucose 0.5%-1% Yeast powder 0.5%-1% β-glucan 0.025%-0.5% Pepsin 0.01%-0.05% Trypsin 0.02%-0.1% Amylase 0.02%-0.1% Lipase 0.005%-0.02% Lactic acid bacteria 1×10 8 -1×10 10 CFU / L Bifidobacterium 5×10 5 -1×10 11 CFU / L The percentages stated are mass-to-volume ratios; (2) Preparation of animal endogenous Clostridium butyricum fermentation broth The prepared animal endogenous culture medium is inoculated with a Clostridium butyricum fermentation broth with an OD of ≥1.0 at λ=600nm, cultured at a temperature of 40-45°C in an anaerobic environment for 2-3 days, the fermented bacterial broth is diluted and spread on a full nutrient medium plate, and vigorously growing Clostridium butyricum colonies are selected according to colony morphology by microscopic observation; the colonies picked by the inoculation loop are then dissolved in sterile water to prepare a bacterial suspension with an OD of ≥0.5 at λ=600nm, the bacterial suspension is inoculated again into the animal endogenous culture medium, and this step is repeated until the Clostridium butyricum fermentation broth cultured in the animal endogenous culture medium is diluted to 10 with sterile water. -6 At the second level, if the number of viable bacteria is greater than or equal to 100, it is considered that Clostridium butyricum has fully adapted to the endogenous environment, and the obtained bacterial liquid is animal endogenous Clostridium butyricum fermentation liquid: (3) Animal endogenous Clostridium butyricum coating (3-1) Preparation of culture medium mixed solution The culture medium mixture solution includes the following components in terms of volume percentage: The animal endogenous culture medium 30%-50% Whey protein liquid 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, passing it through a 20-mesh sieve and storing it for later use, adding it to boiling water at a mass volume ratio of 10%, soaking it for 15 minutes, and filtering it through filter paper to obtain the tea extract; (3-2) Preparation of coating wall material solution and protective agent According to the mass volume ratio, the coating wall material solution includes the following components: 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% Glycerin 0.05%-0.15% Resistant starch 7.5%-8.5% The balance is distilled water; According to the mass volume ratio, the protective agent includes the following components: Microalgae protein 9.5%-10.5% Monosodium glutamate 1.5%-2.5% Trehalose 2.5%-3.5% Glycerin 2.5%-3.5% The balance is sterile water; (3-3) Animal endogenous Clostridium butyricum coating Step 1: Adjust the pH value of the prepared culture medium mixed solution to 6.5±0.2; Step 2: Add the fermentation broth of endogenous Clostridium butyricum to the mixed solution, grow the endogenous Clostridium butyricum to be inoculated to the logarithmic growth phase, add 10% of the coating wall material solution and 10% of the protective agent by volume of the mixed culture medium solution, and fully mix to complete the coating of the endogenous Clostridium butyricum by the wall material solution and the protective agent; Step 3: Turn on the main switch of the spray dryer and set the following settings on the operation 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 ° C; wait for at least 30 minutes to allow the internal conditions of the spray dryer to reach the preset values; insert the feed liquid tube into the mixed liquid prepared in the second step, and after the mixed liquid is completely extracted, obtain the dried encapsulated animal endogenous Clostridium butyricum in the collector. The obtained encapsulated animal endogenous Clostridium butyricum has a preservation number of CGMCC No. 34115.

[0007] Furthermore, the lactic acid bacteria is Lactobacillus plantarum.

[0008] Furthermore, in the first step of step (3-3), the pH value of the solution is adjusted with 0.1 mol / L sodium hydroxide solution.

[0009] Furthermore, in step (3-2), the coating wall material solution is prepared, mixed and then sterilized by high pressure.

[0010] Furthermore, in step (3-2), the protective agent is prepared by a uniform hydration method, with a rotation speed range of 800-1200 r / min and a uniform stirring time of 20-30 min at room temperature.

[0011] The use of the above-mentioned Clostridium butyricum in the preparation of feed additives.

[0012] Furthermore, the added amount is 50g-1000g of encapsulated animal endogenous Clostridium butyricum per ton of feed.

[0013] The present invention has the following advantages and technical effects: by simulating the gastrointestinal environment of animals, an endogenous Clostridium butyricum culture model is established, which allows it to proliferate in large quantities. After completing metabolic adaptation and expression, the Clostridium butyricum is collected and applied to animal breeding, thereby achieving rapid matching of the Clostridium butyricum with the animal intestinal environment; according to the structural characteristics of the animal gastrointestinal tract, a Clostridium butyricum coating process is formulated to improve the stress resistance of the Clostridium butyricum in the animal gastrointestinal environment, avoid its loss in the stomach and the front part of the intestine, maximize its exposure to the colon, enhance its use effect, and improve economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a graph showing the count of viable bacteria in simulated gastric fluid.

[0015] The Clostridium butyricum is Clostridium butyricum DZ-X-001, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC), with the deposit number CGMCC No. 34115. The deposit location is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit time is April 7, 2025. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0017] The following are examples of the encapsulated animal endogenous Clostridium butyricum and its culture method and application.

[0018] 1. Preparation of Encapsulated Animal Endogenous Clostridium butyricum (CGMCC NO. 34115) 1.1 Fermentation screening of endogenous Clostridium butyricum in animals Take a 5L conical 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 potassium hydrogen phosphate, and 0.5g of magnesium sulfate. Set the culture temperature to 38°C, anaerobic environment, and fermentation broth pH = 2.5. Add all the lyophilized Clostridium butyricum powder (BNCC 379338) purchased from Beina Biotechnology Co., Ltd. to the culture medium for fermentation. When the fermentation broth test reading is greater than or equal to 1.0 under the conditions of the UV spectrophotometer set to detect OD = 600nm, the culture is considered complete, and the Clostridium butyricum fermentation broth is obtained. Move to a 4°C refrigerator for storage and set aside.

[0019] Prepare animal endogenous culture medium according to Table 1, using sterile water as the carrier, and prepare 1 L.

[0020] Table 1 Animal endogenous culture medium

[0021] After preparing the animal endogenous culture medium as shown in the table above, inoculate the prepared Clostridium butyricum (BNCC 379338) fermentation broth into 1 L of the animal endogenous culture medium. Set the culture temperature to 40-45°C in a strictly anaerobic environment for 2-3 days. Dilute and spread the cultured bacterial broth on a complete nutrient medium plate. Observe under a microscope and select actively growing Clostridium butyricum colonies based on their morphology using an inoculation loop. Dissolve them in 10 ml of sterile water and measure the OD at 600 nm to ensure it is ≥0.5.

[0022] Then the bacterial suspension with OD≥0.5 was inoculated into 1L of animal endogenous culture medium, and the experimental steps were repeated. The number of viable bacterial colonies was counted after each plate coating until the butyric acid fermentation liquid cultured in the animal endogenous culture medium was diluted to 10 with sterile water. -6 At the second level, the number of viable bacteria detected is greater than or equal to 100, which means that the common Clostridium butyricum has fully adapted to the endogenous environment and the Clostridium butyricum that is not suitable for the environment has been eliminated. Therefore, the bacterial liquid obtained at this time is the animal endogenous Clostridium butyricum fermentation liquid.

[0023] 1.2 Animal endogenous Clostridium butyricum coating 1.2.1 Solution preparation (1) Preparation of whey protein solution: whey protein, sucrose and distilled water were used to prepare a whey protein solution with a mass volume ratio of 6%, wherein the mass ratio of whey protein powder to sucrose was 1:1 (i.e., 100 mL of sample included 6 g of whey protein powder + 6 g of sucrose).

[0024] (2) Preparation of tea extract: Dry and crush the unfermented green tea and pass it through a 20-mesh sieve for storage. Add it to boiling water at a mass volume ratio of 10% and soak it for 15 minutes. Filter it through filter paper to obtain the tea extract.

[0025] (3) Preparation of coating wall material solution: Use 1 L of distilled water to prepare the coating wall material solution according to the contents in Table 2, mix well and sterilize under high pressure.

[0026] (4) Preparation of protective agent: Prepare protective agent according to Table 3 with 1 L of deionized water. After adding the protective agent, set the rotor speed to 800-1200 r / min and stir at a constant speed for 20-30 minutes at room temperature.

[0027] Table 2 Preparation of coating wall material solution (unit: % g / ml)

[0028] Table 3 Preparation of protective agent (unit: % g / ml)

[0029] 1.2.2 Preparation of Encapsulated Animal Endogenous Clostridium butyricum (CGMCC No. 34115) Step 1: Mix the culture medium mixture solution thoroughly according to Table 4.

[0030] Step 2: Use 0.1 mol / L sodium hydroxide solution to adjust the pH value of the solution to 6.5 ± 0.2.

[0031] Step 3: Inoculate the animal's endogenous Clostridium butyricum fermentation broth.

[0032] Step 4: When the endogenous Clostridium butyricum to be inoculated into the animal grows to the logarithmic growth phase (fermentation for about 16 hours), add the coating wall material solution and protective agent respectively, each accounting for 10% of the volume ratio of the mixed culture medium solution, and complete the coating of the endogenous Clostridium butyricum by the wall material and protective agent through thorough mixing.

[0033] Step 5: Set the spray drying conditions on the spray dryer as follows: inlet temperature 100-160°C, flow rate 80-150 mL / h, pump peristaltic speed 10-30 r / min, pressure 1.0-2.0×10 4 Pa, fan frequency 15-25 Hz, needle setting 3-5, outlet temperature 40-80 ℃.

[0034] Step 6: After turning on the machine, start the spray dryer and preheat for more than 30 minutes to reach the preset temperature.

[0035] Table 4 Preparation of culture medium mixed solution (unit: %)

[0036] 2 Application Cases 2.1 Tolerance Verification 2.1.1 Preparation of porcine simulated gastric and small intestinal fluid (1) Preparation of simulated porcine gastric juice: weigh 2 g of NaCl, 3.2 g of pepsin, 7 mL of 36.5% concentrated HCl, add water to 1000 mL, mix well, and prepare artificial gastric juice.

[0037] (2) Preparation of simulated porcine small intestinal fluid: Weigh 6.8 g of KH2PO4 and dissolve it in 500 mL of water. Adjust the pH to 6.8 with 0.1 mol / L NaOH solution. Weigh 10 g of pancreatic enzyme and dissolve it in water. Mix the two solutions and add 3 g of porcine bile salt. Dilute with water to 1000 mL to prepare artificial small intestinal fluid.

[0038] 2.1.2 Test on the ability of bacterial powder to tolerate gastrointestinal fluid environment Clostridium butyricum (No. BNCC379338) purchased from Beina Biotechnology Co., Ltd. was prepared according to Table 5. 1 g of the prepared bacterial powder was taken from each of the four groups and placed in four test tubes containing 8 mL of simulated gastric fluid at a constant temperature of 37°C. The mixture was incubated at 60 r·min. -1 After mixing, the viable bacteria in the simulated gastric fluid were counted by gradient dilution plate pouring method at 5 h. Figure 1 ) and calculate the viable bacterial rate. Simulated gastric fluid was replaced with simulated intestinal fluid and treated in the same manner for 6 h. The viable bacterial rate was calculated. Each treatment was repeated three times. The experimental results are expressed as "mean ± standard deviation" and statistically analyzed using Duncan's method with SPSS 23.0 software. P <0.05 indicates significant difference. P <0.01 indicates extremely significant differences.

[0039] Table 5 Different methods of preparing bacterial powder

[0040] 2.1.3 Results of bacterial powder tolerance to gastrointestinal fluid environment Clostridium butyricum primarily targets the pig colon. Since it takes 2-5 hours for the stomach of an adult pig to empty food, the survival rate of Clostridium butyricum was measured after 2 and 5 hours in simulated gastric fluid. Since food takes 4-6 hours to reach the colon after entering the small intestine, the survival rate of Clostridium butyricum was measured after 4 and 6 hours in simulated small intestinal fluid.

[0041] The test results are shown in Tables 6 and 7. The encapsulated animal endogenous Clostridium butyricum (CGMCC NO. 34115) can significantly improve its survival rate in gastric juice and small intestinal fluid ( P <0.01), so that the survival rate of encapsulated animal endogenous Clostridium butyricum (CGMCC NO. 34115) can reach more than 75% after 5 hours in gastric juice and 6 hours in small intestinal juice, which is beneficial to increase the release of the bacteria in the colon.

[0042] Table 6 Comparison of gastric juice tolerance (unit: %)

[0043] Note: The data in the same industry with different lowercase letters indicate significant differences (0.01< P <0.05), different capital letters indicate extremely significant differences ( P <0.01), the same lowercase letters or no letters indicate no significant difference ( P >0.05). Same as the table below.

[0044] Table 7 Comparison of small intestinal fluid tolerance (unit: %)

[0045] 2.2 Encapsulated endogenous Clostridium butyricum powder improves feed utilization 2.2.1 Feed Nutritional Composition Testing Program The feed group supplemented with coated endogenous Clostridium butyricum powder was used as the experimental group, the feed group supplemented with Clostridium butyricum powder (No.: BNCC379338) purchased from Beina Biotechnology Co., Ltd. was used as the control group, and the feed group without Clostridium butyricum powder was used as the blank control group. The Baiyun Mugang nutritional complete chicken feed purchased from the market was used as the test benchmark. After adding bacterial powder at a ratio of 1%, the feed was treated with wet heat at 37℃ and humidity of 45% for 3 days. Six replicates were set for each group to test the final nutritional composition of the feed.

[0046] Step 1: Dissolve the three groups of powder in sterile water and put them into a 50KD dialysis bag. Clamp both ends of the dialysis bag with clips and set aside.

[0047] Take one group of powders as an example: Step 2: Prepare 0.2 mol / L phosphate hypotonic buffer solution with pH=6.50.

[0048] 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 a rotor, set the speed to 1200 rpm, the temperature to 41°C, and the time to 4 hours.

[0049] Step 4: Prepare 0.2 mol / L phosphate hypotonic buffer solution, pH = 7.99.

[0050] Step 5: Remove the dialysis bag and place it in the phosphate hypotonic buffer prepared in step 4, then repeat the extraction procedure in step 3.

[0051] Step 6: Feed macromolecules, cellulose and other components cannot pass through the dialysis bag, so the hypotonic solution after the two extractions is collected and freeze-dried to obtain freeze-dried powder label 1-1, and the remaining material in the dialysis bag is freeze-dried to obtain freeze-dried powder label 1-2 for later use.

[0052] The other two groups were prepared with the same method, and 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.

[0053] 2.2.2 Test results of feed nutrients after bacterial powder treatment The freeze-dried powder was taken out, and the total protein and total sugar of each group of freeze-dried powder No. 1 were detected by Kjeldahl nitrogen determination method and sulfuric acid phenol test method, respectively. The total fat and crude fiber of each group of freeze-dried powder No. 2 were detected by Soxhlet extraction method and cellulose detector.

[0054] The test results were expressed as "mean ± standard deviation" and statistically analyzed using the Duncan's method using SPSS 23.0 software. P <0.05 indicates significant difference. P <0.01 indicates extremely significant differences.

[0055] A large number of literatures have shown that Clostridium butyricum has a significant effect on the total sugar and crude protein of feed. The results of this experiment are shown in Table 8. The total sugar and crude protein of group A, which was supplemented with coated endogenous Clostridium butyricum powder, were significantly higher than those of group B, which was supplemented with ordinary Clostridium butyricum powder ( P <0.05).

[0056] Table 8 Comparison of nutritional components of feed fermented with different bacterial solutions (unit: %)

[0057] 2.3 Effects on growth performance and plasma biochemical, antioxidant, and immune parameters in weaned piglets 2.3.1 Experimental animals and groups Two hundred healthy, similarly sized, weaned "Du Changda" hybrid piglets were randomly selected at 28 days of age and had an initial weight of (6.5 ± 0.5) kg. They were divided into four groups, each containing five pens (each pen counted as one replicate), with ten pigs per pen (five males and five females). The four groups were: Group A (negative control): fed a basal diet (without antibiotics); Group B (positive control): fed a basal diet supplemented with 20 mg / kg colistin sulfate, 50 mg / kg bacitracin zinc, and 70 mg / kg chlortetracycline; Group C: fed a basal diet supplemented with 500 mg / kg encapsulated endogenous Clostridium butyricum (CGMCC No. 34115); and Group D: fed a basal diet supplemented with 500 mg / kg of Clostridium butyricum purchased from Beina Biotechnology Co., Ltd. (No. BNCC379338).

[0058] 2.3.2 Test methods During the three-day pre-trial period, 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, the experimental pigs were individually weighed and then entered the main trial period, which lasted 16 days. All experimental pigs were managed under uniform conditions, with free access to food and water. A dedicated person was responsible for the feeding, management, and sanitation of the experimental pigs.

[0059] Table 9 Basic feed composition and nutrient levels (air-dry basis) Unit: % (except for those with marked units)

[0060] Note: (1) Vitamin premix provides 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.

[0061] (2) Mineral premix provides the following per kilogram of feed: Fe 120mg, Cu 100mg, Zn 100mg, Mn40mg, I 0.30mg, Se 0.30mg.

[0062] (3) Except for digestible energy and net energy, which are calculated values, all other nutrient levels are measured values.

[0063] Determination of growth performance indicators: On the 1st and 16th days of the main test period, the pigs were weighed after fasting for 16 hours (water was not allowed) in each pen, and the average daily gain (ADG) was calculated; during the test period, the feed consumption was counted in the pen, the average daily feed intake (ADFI) was calculated, and the feed-to-weight ratio (F / G) was calculated.

[0064] Diarrhea rate determination: During the test period, the fecal excretion of each piglet was observed at 08:00, 10:00, 14:00 and 16:00 every day, and the number of piglets with diarrhea and the number of days were recorded. The diarrhea rate was calculated for each pen. The calculation formula is: Diarrhea rate (%) = 100 × (number of piglets with diarrhea × number of days the test piglets had diarrhea) / (total number of test piglets × number of days in the test period).

[0065] At the end of the experiment, one male and one female piglet of near-average weight were selected from each pen. Approximately 10 mL of blood was collected from the anterior vena cava and placed in a centrifuge tube containing sodium heparin (20 IU / mL). The blood was centrifuged at 12,000 rpm for 15 minutes to prepare plasma. Plasma biochemical parameters (including plasma endotoxin and insulin-like growth factor-I (IGF-I) levels) were measured, as were plasma antioxidant parameters (including malondialdehyde (MDA), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC)). Immunological parameters (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) were measured.

[0066] Data statistical analysis: The experimental data were analyzed using SPSS 23.0 statistical software. P <0.01 is extremely significant, 0.01< P <0.05 was considered significant difference. P >0.05 was considered as the standard for judging insignificant difference, and the test results were expressed as "mean ± standard deviation".

[0067] 2.3.2 Test results As shown in Table 10, the ADG index and diarrhea rate of group C were significantly different from those of group A ( P <0.01, and significantly different from group D (0.01< P <0.05), and there was no significant difference with group B ( P >0.05); the ADFI index was significantly higher than that of group D. This indicates that the application of the encapsulated animal endogenous Clostridium butyricum (CGMCC No. 34115) provided by the present invention can significantly increase the average daily weight gain of piglets and reduce the diarrhea rate of piglets, compared with the Clostridium butyricum (No.: BNCC379338) provided by Beina Biotechnology Co., Ltd., and the effect is not significantly different from that of antibiotics.

[0068] Table 10 Effects on growth performance and diarrhea rate of weaned piglets

[0069] 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); T-AOC, IGF-Ⅰ, and IgG levels were significantly different from those in group A ( P <0.01, and the difference was significant compared with group D (0.01< P <0.05). Table 11 Effects on plasma biochemical indices, antioxidant indices and immune indices in weaned piglets

[0070] By simulating the animal's endogenous environment in vitro, we screened Clostridium butyricum (BNCC379338) from Beina Biotechnology Co., Ltd. Acidic conditions and the stimulation of magnesium sulfate resulted in high germination rates and rapid reproduction. Considering the pig's gastrointestinal temperature, the culture temperature was set at 40°C. Strict oxygen control during the fermentation process ensured that anaerobic respiration dominated, stimulating the growth of Clostridium butyricum and rapidly achieving high-volume metabolism of butyrate and other organic acids. A phosphate buffer prevented drastic changes in the system due to acid production, ensuring system stability. Based on these changes, we can identify Clostridium butyricum strains that are more suited to the specific animal environment and can be directly supplied to domestic animals in the future.

[0071] At the same time, considering the loss problem of the small intestine segment, trehalose in the present invention can strengthen the protective effect on the cell membrane, phenolic compounds can also be detoxified by forming a complex with bacterial surface proteins, and tannins can be combined with proteins by forming 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 membrane such as the surface proteins of the bacteria act as a medium for the bacteria to communicate with the environment. They can respond to environmental changes by regulating relevant signal pathways, or be buried in macromolecular compounds through covalent or non-covalent interactions, thereby reducing the damage to the cells. Therefore, the present invention adds unfermented green tea extract and whey protein with a high tannin content to the endogenous culture medium, ferments to form macromolecular polymers, and adds a protective agent at the same time, thereby improving the stress resistance of endogenous Clostridium butyricum in the pig gastrointestinal tract, controlling the release time of endogenous Clostridium butyricum into the gastrointestinal tract, and increasing the bacterial species release in the colon. Endogenous Clostridium butyricum loses wall material in the small intestine segment without losing effective bacteria. When it reaches the hindgut segment, the wall material is consumed, and a large amount of endogenous Clostridium butyricum is released, thereby improving colonization efficiency.

[0072] Based on this, the present invention aims to enhance the application effect of Clostridium butyricum in improving animal production performance by establishing an encapsulated animal endogenous Clostridium butyricum culture model based on the unique characteristics of the animal gastrointestinal tract structure and environment.

[0073] 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 in the scope of protection of the present invention.

Claims

1. A Clostridium butyricum, characterized in that The Clostridium butyricum is Clostridium butyricum DZ-X-001, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC), with the deposit number CGMCC No. 34115 and the deposit time: April 7, 2025.

2. The Clostridium butyricum according to claim 1, characterized in that The Clostridium butyricum is endogenous Clostridium butyricum in encapsulated animals.

3. A method for culturing endogenous Clostridium butyricum in an encapsulated animal, characterized in that: The following steps are involved: (1) Using sterile water as a carrier, prepare an animal endogenous culture medium containing the following components: Tris-HCl buffer 50-100 mM, pH 5.5-7.5 NaCl 0.0006%-0.0018% KCl0.04%-0.08% CaCl2·2H2O0.015%-0.045% MgCl2·6H2O0.02%-0.06% Casein peptone 1%-2% Glucose 0.5%-1% Yeast powder 0.5%-1% β-glucan 0.025%-0.5% Pepsin 0.01%-0.05% Trypsin 0.02%-0.1% Amylase 0.02%-0.1% Lipase 0.005%-0.02% Lactic acid bacteria 1×10 8 -1×10 10 CFU / L Bifidobacterium 5×10 5 -1×10 11 CFU / L The percentages stated are mass-to-volume ratios; (2) Preparation of animal endogenous Clostridium butyricum fermentation broth In the prepared animal endogenous culture medium, the fermentation liquid of Clostridium butyricum was added until the fermentation liquid of Clostridium butyricum cultured in the animal endogenous culture medium was diluted to 10 -6 At the second level, if the number of viable bacteria is greater than or equal to 100, it is considered that Clostridium butyricum has fully adapted to the endogenous environment of the animal, and the obtained bacterial liquid is the endogenous Clostridium butyricum fermentation liquid of the animal: (3) Animal endogenous Clostridium butyricum coating (3-1) Preparation of culture medium mixed solution The culture medium mixture solution includes the following components in terms of volume percentage: The animal endogenous culture medium 30%-50% Whey protein liquid 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, passing it through a 20-mesh sieve and storing it for later use, adding it to boiling water at a mass volume ratio of 10%, soaking it for 15 minutes, and filtering it through filter paper to obtain the tea extract; (3-2) Preparation of coating wall material solution and protective agent According to the mass volume ratio, the coating wall material solution includes the following components: 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% Glycerin 0.05%-0.15% Resistant starch 7.5%-8.5% The balance is distilled water; According to the mass volume ratio, the protective agent includes the following components: Microalgae protein 9.5%-10.5% Monosodium glutamate 1.5%-2.5% Trehalose 2.5%-3.5% Glycerin 2.5%-3.5% The balance is sterile water; (3-3) Animal endogenous Clostridium butyricum coating The animal endogenous Clostridium butyricum fermentation broth is added to the culture medium mixed solution mixture, the animal endogenous Clostridium butyricum to be inoculated is grown to the logarithmic growth phase, the coating wall material solution and the protective agent are respectively added, and the endogenous Clostridium butyricum is coated with the wall material solution and the protective agent to prepare a mixed solution, and the mixed solution is passed through a spray dryer to obtain coated animal endogenous Clostridium butyricum, which has a preservation number of CGMCC No. 34115.

4. The method for culturing endogenous Clostridium butyricum in an encapsulated animal according to claim 3, characterized in that: The step (2) is to prepare an animal endogenous Clostridium butyricum fermentation broth: inoculate the prepared animal endogenous culture medium with an OD of ≥1.0 at λ=600nm into the Clostridium butyricum fermentation broth, culture at a temperature of 40-45°C in an anaerobic environment for 2-3 days, dilute and spread the fermented bacterial broth on a full nutrient medium plate, observe under a microscope, and select the Clostridium butyricum colonies with vigorous growth according to the colony morphology; then dissolve the colonies picked by the inoculation loop in sterile water to prepare a bacterial suspension with an OD of ≥0.5 at λ=600nm, inoculate the bacterial suspension again into the animal endogenous culture medium, and repeat this step until the Clostridium butyricum fermentation broth cultured in the animal endogenous culture medium is diluted to 10 with sterile water. -6 At the second level, if the number of viable bacteria is greater than or equal to 100, it is considered that Clostridium butyricum has fully adapted to the endogenous environment of the animal, and the obtained bacterial liquid is the endogenous Clostridium butyricum fermentation liquid of the animal: The step (3-3) of coating the animal endogenous Clostridium butyricum comprises the following steps: Step 1: Adjust the pH value of the prepared culture medium mixed solution to 6.5±0.2; Step 2: Add the fermentation broth of endogenous Clostridium butyricum to the mixed solution, grow the endogenous Clostridium butyricum to be inoculated to the logarithmic growth phase, add 10% of the coating wall material solution and 10% of the protective agent by volume of the mixed culture medium solution, and fully mix to complete the coating of the endogenous Clostridium butyricum by the wall material solution and the protective agent; Step 3: Turn on the main switch of the spray dryer and set the following settings on the operation 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 ° C; wait for at least 30 minutes to allow the internal conditions of the spray dryer to reach the preset values; insert the feed liquid tube into the mixed liquid prepared in the second step, and after the mixed liquid is completely extracted, obtain the dried encapsulated animal endogenous Clostridium butyricum in the collector. The obtained encapsulated animal endogenous Clostridium butyricum has a preservation number of CGMCC No. 34115.

5. The method for culturing endogenous Clostridium butyricum in an encapsulated animal according to claim 3, characterized in that: In step (1), the lactic acid bacteria is Lactobacillus plantarum.

6. The method for culturing endogenous Clostridium butyricum in an encapsulated animal according to claim 4, 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.

7. The method for culturing endogenous Clostridium butyricum in an encapsulated animal according to claim 4, characterized in that: In step (3-2), the coating wall material solution is prepared, mixed and then sterilized by high pressure.

8. The method for culturing endogenous Clostridium butyricum in an encapsulated animal according to claim 4, characterized in that: In step (3-2), the protective agent is prepared, and a uniform speed hydration method is adopted, with a rotation speed range of 800-1200 r / min and a uniform stirring time of 20-30 min at room temperature.

9. Use of Clostridium butyricum according to claim 1 in the preparation of a feed additive.

10. The use of Clostridium butyricum in preparing a feed additive according to claim 9, characterized in that: The added amount is 50g-1000g of encapsulated animal endogenous Clostridium butyricum per ton of feed.

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