Antibacterial and anti-inflammatory clostridium butyricum and application thereof

Abstract

The invention discloses antibacterial and anti-inflammatory clostridium butyricum and an application thereof. The clostridium butyricum CGMCC No.23377 is separated from chyme in digestive tracts of healthy broiler chickens, can produce cellulase, can inhibit various pathogenic enterobacteria, and can remove DPPH free radicals. The invention also provides a clostridium butyricum preparation prepared by using the strain. The invention further provides application of the clostridium butyricum and the clostridium butyricum preparation in the aspects of preventing ulcerative colitis and improving the production performance and chicken quality of broilers.

Description

Technical Field

[0001] The present invention relates to the application field of probiotics, in particular to an antibacterial and anti-inflammatory Clostridium butyricum and applications thereof. Background Art

[0002] Antibiotics have beneficial effects such as promoting growth, inhibiting pathogens, and preventing diseases, and have been widely used in livestock and poultry farming. However, the widespread use of antibiotics in animal production has led to the emergence of drug-resistant bacteria and the transfer of bacterial resistance, causing great harm to humans and animals. Finding alternatives to antibiotics that can promote growth, are non-toxic, have no residue, and have good antibacterial effects has become a research hotspot in recent years. In order to cope with the challenges that may arise from the removal of antibiotics and the influence of uncontrollable factors from the environment, finding alternatives to antibiotics that can stabilize the intestinal environment of animals is a feasible direction and a prerequisite for promoting the healthy and sustainable development of the livestock and poultry industry. In the complex livestock and poultry farming environment, factors such as pathogens, parasites, and nutritional deficiencies can cause damage to the animal intestine, reduce production performance, and affect economic benefits. Inflammation is a common manifestation of the host's innate defense against changes in the homeostasis of the tissue environment. Therefore, improving animal health from the perspective of preventing animal intestinal inflammation has become a focus of livestock and poultry farming.

[0003] Probiotics can enhance the host immune response, relieve oxidative stress, antagonize pathogens, and improve the composition of animal intestinal flora, thereby benefiting the healthy development of animals and further affecting their production performance. They are still a research hotspot for the development of new feed additives.

[0004] Clostridium butyricum, also known as Clostridium butyricum or Clostridium butyricum, is an anaerobic bacterium that produces butyric acid and belongs to the genus Clostridium in the family Bacillaceae. Clostridium butyricum often resides in the intestines of humans and animals and is extremely resistant to bile salts, acid, and high temperatures. This allows it to successfully pass through the small intestine and colonize the hindgut. It utilizes some incompletely digested carbohydrates to produce organic acids such as acetic acid and butyric acid, which helps protect the intestinal mucosal barrier function. Furthermore, butyric acid, as the primary energy source in the hindgut, is considered a potential treatment for a variety of diseases, including inflammatory bowel disease and antibiotic-associated diarrhea. It can also resist oxidative stress, thereby maintaining host health. As an intestinal commensal bacterium, Clostridium butyricum is widely accepted in the post-antibiotic era for its safety and efficacy, making it a potential candidate for alternatives to antibiotics.

[0005] Currently, there is a lack of Clostridium butyricum that has been systematically studied for its antimicrobial, intestinal health, immune, antioxidant, and anti-inflammatory properties. Therefore, screening Clostridium butyricum for its probiotic potential and exploring the use of Clostridium butyricum preparations to improve animal intestinal health and promote animal growth have important theoretical and practical significance. Summary of the Invention

[0006] The present invention aims to provide a Clostridium butyricum NCB having free radical scavenging activity and capable of inhibiting the growth of pathogenic bacteria, and its application in preventing ulcerative colitis and improving the production performance and muscle quality of broiler chickens.

[0007] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a strain of Clostridium butyricum NCB, which is isolated from the intestinal chyme of healthy broilers. After multiple probiotic performance tests, it was determined that the strain is an antibacterial and anti-inflammatory Clostridium butyricum, classified and named Clostridium butyricum, and has been preserved in the General Microbiology Center of China Culture Collection of Microorganisms, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, Collection Number CGMCC No. 23377, and Collection Date September 8, 2021.

[0008] The microbial characteristics of Clostridium butyricum CGMCC No. 23377 are as follows:

[0009] (1) The colony morphology is 0.8-2.6 mm in diameter, basically round, smooth, moist, with a raised center, and white or milky white in color;

[0010] (2) Microscopic examination of bacterial morphology: The bacteria are long rods or spindles, straight or slightly curved, with motile flagella around the peritrichous, endospores, ovoid spores, and subterminal; Gram staining is positive;

[0011] (3) Other characteristics: The bacteria are anaerobic and grow well in RCM containing fermentable carbohydrates and produce gas, which is manifested as a large number of bubbles appearing on the surface of the liquid during the fermentation process.

[0012] In a second aspect, the present invention provides a bacterial agent containing the Clostridium butyricum.

[0013] In a third aspect, the present invention provides a Clostridium butyricum preparation, which is in the form of a liquid or dry powder, and is prepared by the following method:

[0014] (1) inoculating the activated Clostridium butyricum into a seed culture medium, culturing at a constant temperature of 37° C. under anaerobic conditions for 24 to 32 hours, then inoculating the activated Clostridium butyricum into a fermentation medium at an inoculum rate of 2% to 10% (v / v), and culturing anaerobically for 48 to 72 hours to obtain a Clostridium butyricum fermentation broth, i.e., a Clostridium butyricum liquid;

[0015] The seed culture medium comprises: peptone 0.8-1.2%, beef extract 0.8-1.2%, yeast powder 0.3-0.6%, glucose 0.6-1.0%, (NH4)2SO4 0.1%, NaCl 0.5%, K2HPO4·H2O 0.4%, MnSO4·H2O 0.02%, MgSO4·7H2O 0.05%, CaCO3 0.2%, pH 7.0±0.1;

[0016] The fermentation medium comprises: 0.4-0.7% soy peptone, 0.3-0.7% tryptone, 0.1-0.4% yeast powder, 0.3-1.0% glucose, 0.8-1.2% corn flour, 0.1% (NH4)2SO4, 0.5% NaCl, 0.4% K2HPO4·H2O, 0.02% MnSO4·H2O, 0.05% MgSO4·7H2O, 0.2% CaCO3, 0.05-0.1% (v / v) defoaming agent, and a pH value of 7.0±0.1.

[0017] (2) mixing the Clostridium butyricum fermentation liquid and the carrier in a weight ratio of (0.6-1.0):1, then drying at low temperature and pulverizing to obtain Clostridium butyricum dry powder;

[0018] The carrier can be selected from one or more of bran, wheat bran, corn cob powder, glucose and corn gluten powder.

[0019] In a fourth aspect, the present invention provides the use of the Clostridium butyricum or its bacterial agent or the Clostridium butyricum preparation in the preparation of biological products for preventing animal ulcerative colitis, and for resisting oxidative stress induced by dextran sodium sulfate (DSS) and improving intestinal health.

[0020] Furthermore, the Clostridium butyricum and the Clostridium butyricum preparation are used to prevent animal ulcerative colitis, resist oxidative stress induced by dextran sodium sulfate (DSS), and improve intestinal health.

[0021] The present invention uses DSS to induce a C57BL / 6 mouse ulcerative colitis model. By observing the changes in mouse weight, diarrhea, bloody stools, disease activity index (DAI), and comparing the colon length and colon pathology scores of each treatment group after mouse dissection, and measuring relevant indicators in serum, colon tissue, and cecum, the preventive effect of Clostridium butyricum on DSS-induced ulcerative colitis mice is detected. The experimental results show that Clostridium butyricum treatment can improve the increase in DAI such as weight loss, diarrhea, and bloody stools in mice caused by DSS stimulation, and inhibit the shortening of colon length caused by DSS. Clostridium butyricum treatment can alleviate the damage to mouse colon tissue caused by DSS, including intestinal epithelial cell destruction, crypt destruction, intestinal gland expansion, and inflammatory cell infiltration; it can improve the increase in MPO activity caused by DSS; and it can significantly reduce the apoptosis of colon epithelial cells caused by DSS. Oral administration of Clostridium butyricum significantly increased serum total antioxidant capacity (T-AOC) and immunoglobulin G (IgG) levels in mice. It also significantly downregulated the DSS-induced increase in interleukin-1β (IL-1β), IL-6, and cyclooxygenase 2 (COX2) mRNA levels, significantly upregulated IL-10 mRNA levels, and significantly increased nuclear factor E2-related factor 2 (Nrf2) mRNA levels in the colon of mice with colitis. Clostridium butyricum mitigated the DSS-induced decrease in colonic goblet cell number and mucin (MUC) content. It also upregulated the DSS-induced expression of mucin-4 (MUC-4), occludin, and claudin-3 in colonic tissue and increased the expression of MUC-2 and zonula occludens 2 (ZO-2) in mice with colitis. Clostridium butyricum significantly mitigated the DSS-induced decrease in ZO-1 and claudin-1 levels. Clostridium butyricum increased the contents of cecal acetate, propionate, butyrate, and total short-chain fatty acids (SCFAs). Therefore, Clostridium butyricum has a good preventive effect on DSS-induced colitis in mice.

[0022] In a fifth aspect, the present invention provides the use of the Clostridium butyricum or its bacterial agent or the Clostridium butyricum preparation in improving the growth performance of broiler chickens.

[0023] In a sixth aspect, the present invention provides the use of the Clostridium butyricum or its bacterial agent or the Clostridium butyricum preparation in improving the intestinal morphology of broiler chickens (for non-disease treatment purposes).

[0024] Furthermore, the improvement of the broiler intestinal morphology is all or part of the following:

[0025] Adding Clostridium butyricum can improve the intestinal morphology of broiler chickens' jejunum: it significantly increases the villus height and V / C of the sky intestine of broiler chickens at 21 days and 42 days, and reduces the crypt depth of the sky intestine of broiler chickens at 21 days and 42 days.

[0026] In a seventh aspect, the present invention provides the use of the Clostridium butyricum or its bacterial agent or the Clostridium butyricum preparation in improving the immune function of broiler chickens (for non-disease treatment purposes).

[0027] Furthermore, the improving immune function of broiler chickens is all or part of the following:

[0028] (1) Adding Clostridium butyricum can significantly promote the development of immune organs in broiler chickens: significantly increasing the thymus index of broiler chickens at 21 and 42 days;

[0029] (2) Adding Clostridium butyricum can significantly improve the serum immunity of broiler chickens: it significantly increases the serum IgG content of broiler chickens at 21 days and 42 days.

[0030] In an eighth aspect, the present invention provides the use of the Clostridium butyricum or its bacterial agent or the Clostridium butyricum preparation in regulating the serum biochemical indicators of broiler chickens (for non-disease treatment purposes).

[0031] In a ninth aspect, the present invention provides the use of the Clostridium butyricum or its bacterial agent or the Clostridium butyricum preparation in improving the antioxidant capacity of broiler chickens (for non-disease treatment purposes).

[0032] Furthermore, the improvement of the antioxidant capacity of broiler chickens is all or part of the following:

[0033] (1) Adding Clostridium butyricum can improve the antioxidant capacity of broiler serum: it significantly increased the serum T-AOC and CAT levels of broiler chickens at 21 days and 42 days, and significantly reduced the serum malondialdehyde (MDA) content of broiler chickens at 21 days and 42 days;

[0034] (2) Adding Clostridium butyricum can improve the antioxidant capacity of broiler livers: it significantly increases the T-AOC and total superoxide dismutase (T-SOD), CAT, and GSH-Px contents in the livers of broiler chickens at 21 days and 42 days, and significantly reduces the MDA content in the livers of broiler chickens at 21 days and 42 days.

[0035] In a tenth aspect, the present invention provides the use of the Clostridium butyricum or its bacterial agent or the Clostridium butyricum preparation in improving the quality of chicken.

[0036] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0037] The present invention provides a Clostridium butyricum NCB isolated from healthy animals, which can secrete cellulase, inhibit intestinal pathogens, and scavenge DPPH free radicals, as well as a Clostridium butyricum preparation prepared using the strain. The present invention also provides the use of the Clostridium butyricum and the Clostridium butyricum preparation for preventing ulcerative colitis. The Clostridium butyricum can improve the weight loss, increased DAI and colon tissue damage of mice caused by DSS stimulation, improve the oxidative stress and increased inflammatory levels caused by DSS, improve the intestinal functional barrier damage caused by DSS, and is beneficial to preventing and maintaining the host's intestinal health, and increase the content of cecal acetic acid, propionic acid, butyric acid and SCFAs. The present invention further provides the use of the Clostridium butyricum and the Clostridium butyricum preparation in improving the production performance of broilers. The Clostridium butyricum preparation can improve the growth performance of broilers, improve intestinal morphology and immune function, and enhance the antioxidant capacity of animals. The Clostridium butyricum preparation of the present invention has good economic benefits and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The colony morphology and microstructure of Clostridium butyricum NCB in Example 1 of the present invention are shown.

[0039] Figure 2 This is the weight change of mice in Example 3 of the present invention.

[0040] Figure 3 It is the disease activity index (DAI) of mice in Example 3 of the present invention.

[0041] Figure 4 is the length of the mouse colon in Example 3 of the present invention.

[0042] Figure 5 The mouse colon tissue sections and pathological scores in Example 3 of the present invention are shown.

[0043] Figure 6 This is a staining image of mouse colon neutrophils in Example 3 of the present invention.

[0044] Figure 7 This is the TUNEL detection of mouse colon epithelial cells and its positive rate statistics in Example 3 of the present invention.

[0045] Figure 8 These are the serum T-AOC, SOD, and MDA concentrations of mice in Example 3 of the present invention.

[0046] Figure 9 is the concentration of immunoglobulin in mouse serum in Example 3 of the present invention.

[0047] Figure 10 The cytokine concentrations and mRNA levels of related genes in the mouse colon tissue in Example 3 of the present invention are shown.

[0048] Figure 11 3. PAS staining and MUC-2 immunohistochemistry of mouse colon tissue in Example 3 of the present invention.

[0049] Figure 12 The mRNA levels of the six barrier protein genes in the mouse colon in Example 3 of the present invention.

[0050] Figure 13 This is the immunohistochemical section of ZO-1, Occludin, and Claudin-1 in mouse colon and the statistical analysis of the positive areas thereof in Example 3 of the present invention.

[0051] Figure 14 This is a diagram showing the SCFA content and composition of the cecal contents of mice in Example 3 of the present invention.

[0052] In the figures, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. DETAILED DESCRIPTION

[0053] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0054] The percentage sign "%" involved in the present invention, unless otherwise specified, refers to mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0055] Example 1 Isolation, Screening and Functional Studies of Clostridium butyricum

[0056] 1. Isolation of strains

[0057] Sample collection: The samples for strain isolation came from chyme, fresh feces and soil samples of pigs, chickens, cattle and sheep.

[0058] Culture medium type:

[0059] (1) Reinforced Clostridium culture medium (RCM medium): peptone 1%, beef extract 1%, yeast extract 0.3%, glucose 0.5%, sodium chloride 0.5%, soluble starch 1%, sodium acetate 0.3%, cysteine ​​hydrochloride 0.05%, agar 2%, adjusted to pH 7.0 ± 0.1.

[0060] (2) Tryptone sulfite neomycin medium (TSN medium): tryptone 1.5%, sodium sulfite 1%, neomycin 0.005%, polymyxin 0.002%, yeast extract 1%, ferric citrate 0.05%, agar 2%, adjusted to pH 7.0 ± 0.1.

[0061] (3) LB (Luria-Bertani) medium: 1% tryptone, 0.5% NaCl, 1% yeast extract, 2% agar, adjusted to pH 7.0±0.1.

[0062] Single colonies were isolated from each sample, and 10 colonies that met the culture characteristics and colony morphology of Clostridium butyricum were selected for 16S rRNA gene sequence identification. The results were compared with the NCBI database by BLAST, and a new Clostridium butyricum strain, Clostridium butyricum NCB ( Figure 1 A), the 16S rRNA gene sequence of the strain is shown in SEQ ID NO: 1, which has 100% homology to the 16S rRNA gene sequence of Clostridium butyricum.

[0063] The 16S rRNA gene sequence of Clostridium beijerinckii is highly similar to that of Clostridium butyricum. To confirm that strain NCB is Clostridium butyricum, the present invention used specific Clostridium butyricum primers 21F: 5'-TCAATTAGAAGGCAGAGTACC-3' and 1023R: 5'-CTAAAACTGACTGTGGCATT-3' to amplify a 1005-bp C. butyricum-specific sequence. The sequencing result is shown in SEQ ID NO: 2, which shows 99% homology to the C. butyricum sequence. However, using specific primers 21F and 1023R, no corresponding fragment could be amplified from DNA of Clostridium beijerinckii. Based on these molecular identification results, strain NCB was ultimately confirmed to be Clostridium butyricum.

[0064] 2. Biological properties of Clostridium butyricum

[0065] The above-mentioned Clostridium butyricum NCB was inoculated into RCM medium and cultured anaerobically for 24 hours.

[0066] 2.1 SCFAs content determination

[0067] The supernatant of the Clostridium butyricum culture was collected and the contents of acetate, butyrate, and SCFAs were quantitatively determined by gas chromatography using crotonic acid as an internal standard. The Clostridium butyricum culture produced 3.03 g / L acetate, 1.37 g / L butyrate, and 4.45 g / L SCFAs.

[0068] 2.2 Antibacterial properties

[0069] The culture supernatant of the above-mentioned Clostridium butyricum strain was collected and its inhibitory rate against pathogens was tested in a 96-well plate. The antibacterial activity of the culture supernatant of Clostridium butyricum NCB against various pathogens was determined in a 96-well plate. The pathogens tested included Escherichia coli K88, Escherichia coli K99, Escherichia coli 987P, Escherichia coli CVCC1515, Salmonella CVCC519, Salmonella ATCC14028, Staphylococcus aureus ATCC43300, Staphylococcus aureus CVCC1882, Pseudomonas aeruginosa CGMCC1.10712, Listeria monocytogenes, and Clostridium perfringens CVCC 2030, totaling 11 pathogens. The culture supernatant of Clostridium butyricum can significantly inhibit Gram-negative bacteria Escherichia coli K88, Escherichia coli K99, Escherichia coli CVCC1515, Salmonella CVCC519, Salmonella ATCC14028, Pseudomonas aeruginosa CGMCC1.10712 and Gram-positive bacteria Clostridium perfringens CVCC 2030, with inhibition rates ranging from 72% to 92%. The inhibition rate against Staphylococcus aureus ATCC43300 also reached 68% (Table 1).

[0070] Table 1 Antibacterial effect of Clostridium butyricum NCB culture supernatant

[0071]

[0072]

[0073] Note: a,b,c,d,e Values ​​in the same column with different lowercase superscripts indicate significant differences (P<0.05).

[0074] 2.3 Cellulase activity

[0075] The culture supernatant of the above-mentioned Clostridium butyricum strain was taken and assayed using a cellulase assay kit. The cellulase activity produced by Clostridium butyricum was measured to be 13.26 U / mL.

[0076] 2.4 DPPH free radical scavenging test

[0077] Take 1 mL of the culture supernatant of the above-mentioned Clostridium butyricum strain and mix it evenly with 1 mL of 1.50 mmol / L 2,2-biphenyl-1-picrylhydrazyl (DPPH) anhydrous ethanol solution. Incubate in the dark at room temperature for 30 minutes and measure the absorbance at 517 nm. The blank group uses an equal volume of ethanol instead of DPPH solution, and the control group uses an equal volume of sterile water instead of Clostridium butyricum fermentation broth. VC serves as a positive control. The clearance rate is calculated as follows:

[0078] Clearance (%) = [1-(A1-A) / A0] × 100%

[0079] Where: A is the absorbance value of the blank group; A0 is the absorbance value of the control group; A1 is the absorbance value of the sample group.

[0080] The test results showed that the culture supernatant of Clostridium butyricum could effectively scavenge DPPH free radicals, and the scavenging rate was close to 100%, which was equivalent to the scavenging capacity of 0.125 g / L VC.

[0081] 3. Study on the stress resistance of Clostridium butyricum

[0082] The above-mentioned Clostridium butyricum NCB was inoculated into RCM medium and cultured anaerobically for 24 hours.

[0083] 3.1 Survival rate of Clostridium butyricum in artificial gastric and intestinal fluids

[0084] The fermentation broth of Clostridium butyricum was inoculated into RCM medium. After culturing for 24 h (vegetative cells) or 96 h (spore formation), it was inoculated into artificial gastric fluid at 10% (v / v). After culturing for 3 h, 100 μL was taken for gradient dilution, plate counting, and calculation of survival rate.

[0085] The fermentation broth of Clostridium butyricum was inoculated into RCM medium. After culturing for 24 h (vegetative cells) or 96 h (spores formed), it was inoculated into artificial intestinal fluid at 10% (v / v). After culturing for 4 h, 100 μL was taken for gradient dilution, plate counting, and calculation of survival rate.

[0086] The survival rate of vegetative cells of Clostridium butyricum in artificial gastric fluid was affected to a certain extent. After 3 hours of treatment, the survival rate of vegetative cells was 71.2%. The survival rate of spores of Clostridium butyricum in artificial gastric fluid was not affected; the survival rate of vegetative cells and spores of Clostridium butyricum in artificial intestinal fluid was not affected (Table 2).

[0087] Table 2 Survival rate of Clostridium butyricum in artificial stomach and intestinal fluid

[0088]

[0089]

[0090] Note: a,b Values ​​in the same row with different lowercase superscripts indicate significant differences (P<0.05).

[0091] 3.2 Bile salt tolerance of Clostridium butyricum

[0092] C. butyricum fermentation broth was inoculated into RCM medium and cultured for 24 hours (vegetative cells) or 96 hours (spore formation). The culture was then inoculated into RCM medium containing 0.1%, 0.3%, or 0.5% porcine bile salts. After 24 hours of anaerobic incubation at 37°C, samples were collected, plated, and counted for viability. 0.1% and 0.3% bile salts did not affect the survival of C. butyricum vegetative cells or spores, and 0.1% bile salts even slightly promoted the growth of C. butyricum. The viability of C. butyricum vegetative cells decreased to 23.4% in the presence of 0.5% bile salts, but the viability of spores was barely affected (Table 3).

[0093] Table 3 Tolerance of Clostridium butyricum to bile salts

[0094]

[0095] Note: a,b Values ​​in the same row with different lowercase superscripts indicate significant differences (P<0.05).

[0096] 3.3 Temperature tolerance of Clostridium butyricum

[0097] Clostridium butyricum was inoculated into RCM medium and cultured for 24 hours (vegetative cells) or 96 hours (spore formation). 10 mL of the culture medium was then placed in a water bath at 60°C, 80°C, and 100°C for 5 minutes, cooled, and serially diluted. Plate counts were performed, and the survival rate was calculated. High temperatures significantly reduced the survival rate of Clostridium butyricum vegetative cells, with the survival rate decreasing with increasing temperature. However, the survival rate of spores remained above 95% at temperatures between 60°C and 100°C (Table 4).

[0098] Table 4 Temperature tolerance of Clostridium butyricum

[0099]

[0100] Note: a,b Values ​​in the same row with different lowercase superscripts indicate significant differences (P<0.05).

[0101] Example 2 Preparation of Clostridium butyricum Preparation

[0102] The butyric bacterium is inoculated into a seed culture medium, placed in an anaerobic incubator at 37° C. and cultured for 24 to 32 hours, and then inoculated into a fermenter at an inoculum rate of 2% to 10%. N2 (or a mixed gas of N2 and H2) is introduced into the fermentation medium in the fermenter in advance to drive out the oxygen in the fermenter, and then the culture is anaerobically cultured at 37° C. for 48 to 72 hours to obtain a Clostridium butyricum NCB fermentation liquid.

[0103] The seed culture medium for Clostridium butyricum is: 1% peptone, 0.8% beef extract, 0.5% yeast powder, 1.0% glucose, 0.1% (NH4)2SO4, 0.5% NaCl, 0.4% K2HPO4·H2O, 0.02% MnSO4·H2O, 0.05% MgSO4·7H2O, 0.2% CaCO3, and pH value is 7.0±0.1.

[0104] The fermentation medium for Clostridium butyricum was composed of 0.5% soy peptone, 0.6% tryptone, 0.4% yeast extract, 0.8% glucose, 1% cornstarch, 0.2% (NH4)2SO4, 0.5% NaCl, 0.4% K2HPO4·H2O, 0.02% MnSO4·H2O, 0.03% MgSO4·7H2O, 0.2% CaCO3, and 0.1% (v / v) polyether polyol defoamer SGR-1810 (Shandong Xingrui Environmental Technology Co., Ltd.). The pH was adjusted to 7.0±0.1.

[0105] The fermentation broth of Clostridium butyricum is mixed uniformly with a carrier at a weight ratio of 0.8:1, and then dried and pulverized at low temperature to obtain Clostridium butyricum dry powder. The carrier can be one or more of bran, wheat bran, corn cob meal, glucose, and corn gluten meal. In this embodiment, the carrier used is a mixture of corn cob meal and glucose at a weight ratio of 1:1.

[0106] Example 3 Application of Clostridium butyricum in preventing ulcerative colitis in mice

[0107] 1. Materials and Methods

[0108] 1.1 Experimental Animals: Twenty-four 4-week-old C57BL / 6 male mice, with an initial body weight of 16-18 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0109] 1.2 Test reagents: DSS, molecular weight 36000-50000, purchased from MP Biochemical Company, USA.

[0110] 1.3 Test strain: Clostridium butyricum NCB. Select the preserved strain and culture it in liquid Clostridium butyricum RCM at 37℃ for about 24 hours. Then, streak it on solid RCM. After a single colony grows, pick a single colony and inoculate it into liquid RCM medium at 37℃ for about 24 hours. Then, count the viable bacteria until the number of bacteria reaches 10 8 CFU / mL, used for oral gavage of mice, with a gavage volume of 200 μL per mouse.

[0111] 1.4 Test methods

[0112] 1.4.1 Construction of ulcerative colitis mouse model

[0113] After 5 days of adaptive feeding, the experimental mice were gavaged with Clostridium butyricum NCB for three weeks, and then 2.0% DSS dissolved in ultrapure water replaced the normal drinking water of the mice, and the mice had free access for 7 consecutive days.

[0114] 1.4.2 Experimental treatment

[0115] Twenty-four mice were randomly divided into three treatment groups, with eight mice per group and four mice per cage. At the beginning of the experimental period, there was no difference in mean body weight between the groups. Mice were housed in a sterile room at 22-25°C with a 12-h light / dark cycle and free access to water and food. For the first three weeks, mice were gavaged with either normal saline or Clostridium butyricum culture solution. The experimental treatments were:

[0116] Control group: normal healthy mice, free access to drinking water throughout the whole process, and the gavage medium was normal saline.

[0117] DSS group: During the modeling period, mice were treated with 2.0% DSS dissolved in sterile water instead of normal drinking water to establish the disease model for 7 consecutive days. After the initiation of modeling, mice were weighed and fecal consistency, diarrhea, and blood in the stool were tested daily to assess the success of the model. Normal saline was used as the gavage medium.

[0118] Clostridium butyricum group: During the modeling period, mice were allowed to drink drinking water containing 2.0% DSS freely every day for 7 consecutive days. At the same time, probiotics were administered orally once a day, with each mouse receiving 200 μL of 10 8 CFU / mL of Clostridium butyricum fermentation broth was used, and the weight of the mice was measured, and the stool viscosity, diarrhea and bloody stool were tested.

[0119] After the fourth week, the mice in each treatment group were sacrificed and samples were collected.

[0120] 1.4.3 Disease Index (DAI): DAI was assessed using weight loss, stool consistency, and stool bleeding as indicators and recorded daily. The scores for each indicator are shown in Table 5, and the three indicators were then added together for calculation.

[0121] Table 5 Ulcerative colitis disease activity index score

[0122]

[0123] 1.4.4 Determination of serum biochemical indicators: T-AOC, superoxide dismutase (SOD), and MDA detection kits were used. For details, please refer to the instructions for the biochemical kits of Nanjing Jiancheng Bioengineering Institute.

[0124] 1.4.5 Serum enzyme-linked immunosorbent assay: A mouse-specific enzyme-linked immunosorbent assay (ELISA) kit was used to detect serum IgA, IgG, and IgM concentrations. For details, please refer to the instructions for the ELISA kit from Beijing Siliang Changyuan Co., Ltd.

[0125] 1.4.6 Colon Tissue Sectioning and Pathological Scoring: Colonic tissue was cleaned with sterile PBS buffer, fixed in 4% paraformaldehyde, and then embedded in paraffin. Sections were cut at a thickness of 5 μm. The cut tissue was mounted on glass slides and stained with a mixture of hematoxylin and eosin. Colonic tissue changes, including mucosal thickness, intestinal gland cells, goblet cells, epithelial cell destruction, crypt damage, and inflammatory cell infiltration, were observed under a microscope. Histopathological scores are shown in Table 6.

[0126] Table 6 Histopathological scores

[0127]

[0128] 1.4.7 Goblet Cell Quantification: Colonic tissue was cleaned with sterile PBS buffer, fixed in 4% paraformaldehyde, and then embedded in paraffin. Sections were made at a thickness of 5 μm. The sections were mounted on glass slides, stained with PAS mixed staining solution, and examined under a microscope for image analysis.

[0129] 1.4.8 TUNEL assay for apoptosis: After dewaxing, tissue sections were stained with TUNEL kit and observed and recorded under a fluorescence microscope.

[0130] 1.4.9 Immunohistochemistry: After dewaxing, the tissue sections were subjected to antigen retrieval, endogenous peroxidase was blocked, and serum was used for blocking. The sections were incubated with primary and secondary antibodies, respectively, and developed with DAB. The cell nuclei were counterstained, and the sections were dehydrated, mounted, and interpreted under a white light microscope.

[0131] 1.4.10 Analysis of the expression of cytokine- and oxidative stress-related genes and intestinal barrier-related genes in colon tissue: Colon tissue was collected and total RNA was extracted. The mRNA was then reverse transcribed into cDNA using a reverse transcription kit, and then quantitative PCR (qPCR) was performed using primers for related factors (Table 7).

[0132] Table 7 Fluorescence quantitative PCR primer sequences (5′-3′)

[0133]

[0134] 2. Test results

[0135] 2.1 Comparison of ulcerative colitis symptoms

[0136] During the DSS modeling period, the weight changes of mice in each group were shown in Figure 2 Compared with the control group, the weight of mice in the DSS group decreased significantly from the sixth day after DSS stimulation (P < 0.05), and the weight of mice in the Clostridium butyricum group was significantly higher than that in the DSS group from the fifth day (P < 0.05). At the same time, the mice were observed for diarrhea and bloody stools every day, and the scores were calculated according to the ulcerative colitis disease activity index assessment standard. Figure 3 As shown in the figure. Compared with the control group, mice in the DSS group began to experience diarrhea and occult blood in their stools on day three, with a significantly higher DAI than the control group (P < 0.05). On day five, mice in the DSS group developed loose stools with blood in their stools and were extremely lethargic. These results indicate that ulcerative colitis was successfully modeled in mice. Compared with the DSS group, pretreatment with Clostridium butyricum significantly ameliorated DSS-induced weight loss, diarrhea, and bloody stools from day three to day seven (P < 0.05).

[0137] 2.2 Analysis of colon length and pathological findings

[0138] The length of the colon of mice in each treatment group was measured after autopsy. Figure 4 As shown in the results, compared with the control group, the colon length of the DSS group was significantly shortened (P < 0.05), while pretreatment with Clostridium butyricum could significantly improve the colon shortening induced by DSS (P < 0.05). Figure 5 The results of colon tissue sections and pathological scores of mice in each treatment group were shown in Figure 2. Pretreatment with Clostridium butyricum alleviated the histopathological changes induced by DSS (P < 0.05), including loss of epithelial cells, loss of crypts, inflammatory cell infiltration, and decreased intestinal gland cells in colon tissue.

[0139] 2.3 Colonic MPO activity

[0140] Colonic MPO activity Figure 6 As shown in the data, MPO is considered to be a marker formed after neutrophil activation. Compared with the control group, DSS treatment resulted in a higher level of MPO positivity. There was a trend toward reduced colonic MPO activity in the Clostridium butyricum group (P=0.061).

[0141] 2.4 Colonocyte Apoptosis

[0142] Colon TUNEL test and its positive rate statistical results are as follows Figure 7 Compared with the control group, TUNEL-positive nuclei showed that DSS stimulation significantly increased colon epithelial cell apoptosis (P < 0.05), and pretreatment with Clostridium butyricum significantly attenuated this change (P < 0.05).

[0143] 2.5 Serum oxidative stress level and immunoglobulin content

[0144] Indicators related to serum oxidative stress levels in mice such as Figure 8 Compared with the control group, the serum T-AOC and SOD activities of mice in the DSS group were significantly decreased (P < 0.05), and Clostridium butyricum pretreatment significantly improved the serum T-AOC content of mice (P < 0.05). Figure 9 Compared with the control group, the serum IgG concentration of mice in the DSS group was significantly decreased (P < 0.05), and pretreatment with Clostridium butyricum significantly alleviated the DSS-induced decrease in serum IgG concentration in mice (P < 0.05). This suggests that Clostridium butyricum can alleviate the body's redox imbalance and reduced immune function.

[0145] 2.6 Expression of cytokines and oxidative stress-related genes in colonic tissue

[0146] The mRNA levels of inflammatory cytokines IL-1β, IL-4, IL-10, TNF-α, NF-κB, cyclooxygenase 2COX2, and oxidative stress-related factor Nrf2 in colon tissue were quantified by q-PCR. The mRNA levels of cytokines and oxidative stress-related genes in each group of mice are shown in Figure 10 Compared with the control group, DSS stimulation induced a significant increase in the transcriptional levels of IL-1β, IL-4, and IL-6 in the colonic tissues of mice (P < 0.05). Pretreatment with Clostridium butyricum significantly reduced the DSS-induced increase in IL-1β and IL-6 mRNA levels (P < 0.05). Compared with the DSS group, pretreatment with Clostridium butyricum significantly increased the mRNA level of IL-10 (P < 0.05). Compared with the control group, the transcriptional level of COX-2 in the DSS group was significantly increased (P < 0.05), while pretreatment with Clostridium butyricum significantly downregulated the transcriptional level of COX-2 (P < 0.05). Compared with the DSS group, pretreatment with Clostridium butyricum significantly increased the mRNA level of Nrf2 (P < 0.05). This suggests that Clostridium butyricum can alleviate the inflammatory effects of DSS by reducing the expression of pro-inflammatory factors and increasing the expression of anti-inflammatory factors, and can also alleviate the increased oxidative stress levels caused by DSS by promoting the upregulation of Nrf2 transcription.

[0147] 2.7 Analysis of intestinal barrier function

[0148] 2.7.1 Intestinal barrier integrity

[0149] The statistical results of PAS staining, MUC-2 immunohistochemistry, goblet cell count and MUC-2 positive rate of mice in each treatment group are shown in the table. Figure 11Compared with the control group, the number of goblet cells in the colonic epithelial cells in the DSS group was significantly decreased (P<0.05), while pretreatment with Clostridium butyricum significantly reduced the decrease in goblet cell number (P<0.05). Compared with the control group, the secretion of MUC-2 in the DSS group was significantly decreased (P<0.05), while pretreatment with Clostridium butyricum significantly increased the protein content of MUC-2 (P<0.05).

[0150] 2.7.2 Gene transcription of intestinal barrier proteins

[0151] The gene transcription results of six intestinal barrier proteins are shown in Figure 12 The results showed that compared with the control group, DSS stimulation caused a significant decrease in the mRNA levels of Muc-4, Occludin, Claudin-2, and Claudin-3 in the mouse colon (P<0.05), while pretreatment with Clostridium butyricum significantly upregulated the mRNA levels of Muc-4, Occludin, and Claudin-3 (P<0.05). Compared with the DSS group, the mRNA levels of Muc-2 and Zo-2 in the mouse colon in the Clostridium butyricum group were significantly increased (P<0.05).

[0152] 2.7.3 Immunohistochemical analysis of intestinal barrier proteins

[0153] The immunohistochemistry of ZO-1, Occludin, and Claudin-1 and the statistical analysis of their positive areas are shown in the figure. Figure 13 Compared with the control group, DSS-induced a significant decrease in the levels of ZO-1, Occludin, and Claudin-1 in the colonic tissues of mice (P<0.05). Pretreatment with Clostridium butyricum increased the levels of ZO-1 and Claudin-1 (P<0.05). These results suggest that pretreatment with Clostridium butyricum can effectively ameliorate DSS-induced intestinal barrier damage.

[0154] 2.8 Changes in cecal SCFA content and composition

[0155] The SCFA content and composition of the cecal contents of mice in each treatment group were as follows: Figure 14 Compared with the control group, the levels of acetate, propionate, butyrate, and total SCFA in the cecal contents of mice in the DSS group were significantly decreased (P<0.05). However, pretreatment with Clostridium butyricum significantly ameliorated the DSS-induced changes in the cecal SCFA metabolic profile, increasing the concentrations of acetate, propionate, butyrate (P<0.05), and total SCFA (P<0.05). These results suggest that pretreatment with Clostridium butyricum can regulate the DSS-induced disturbances in the intestinal SCFA metabolic profile.

[0156] Example 4 Application of Clostridium butyricum in Improving the Growth Performance of Broiler Chickens

[0157] 1. Materials and Methods

[0158] The experiment selected 300 Ross 308 broilers, half male and half female, and randomly divided them into 5 treatment groups, each with 6 replicates and 10 chickens in each replicate. The treatment groups were: basic diet (control group), basic diet + 1×10 8 CFU / kg Clostridium butyricum (low-dose group), basal diet + 5×10 8 CFU / kg Clostridium butyricum (medium dose group), basal diet + 1×10 9 The basal diet composition is shown in Table 8. The experimental period was 42 days. Feed consumption and weight gain of broilers in each replicate cage were recorded and calculated on days 21 and 42. One-way analysis of variance (ANOVA) was performed on the experimental data using SPSS 26.0. F-tests were used for factor significance, and multiple comparisons were performed using Tukey and Dunnett's methods. Results are expressed as mean values, and P < 0.05 was considered significant.

[0159] Table 8 Composition and nutritional components of broiler basal diet

[0160]

[0161]

[0162] 1 Each kilogram of feed provides: Iron, 111 mg; Copper, 10 mg; Manganese, 128 mg; Zinc, 142 mg; Vitamin A, 14,000 IU; Vitamin D3, 6,000 IU; Vitamin E, 70 mg; Vitamin K3, 4 mg; Vitamin B1, 7 mg; Vitamin B2, 13 mg; Vitamin B6, 13 mg; Vitamin B12, 29 μg; Choline, 1,835 mg; Folic acid, 3 mg; Niacin, 93 mg; Pantothenic acid, 27 mg.

[0163] 2. Test results

[0164] The experimental results are shown in Table 9. As shown in Table 9, supplementation with medium- and high-dose Clostridium butyricum significantly increased broiler weight on day 42 (P < 0.05). The ADFI of broilers in the low- and high-dose Clostridium butyricum supplementation groups was significantly lower than that in the control group (P < 0.05). The addition of medium-dose Clostridium butyricum to the diet significantly increased ADG in broilers from days 22 to 42 and throughout the entire experimental period (P < 0.05). These results demonstrate that Clostridium butyricum can promote the growth of broilers.

[0165] Table 9 Effects of Clostridium butyricum on growth performance of broiler chickens

[0166]

[0167]

[0168] Note: a,b The values ​​in the same row with different lowercase superscripts indicate significant differences (P<0.05);

[0169] BW: starting body weight, ADG: average daily gain, ADFI: average daily feed intake, FCR: feed conversion ratio, SEM: standard error of the mean.

[0170] Example 5 Application of Clostridium butyricum in Improving Broiler Intestinal Morphology

[0171] 1. Materials and Methods

[0172] The experiment selected 300 Ross 308 broilers, half male and half female, and randomly divided them into 5 treatment groups, with 6 replicates in each treatment. The treatment groups were: basic diet (control group), basic diet + 1×10 8 CFU / kg Clostridium butyricum (low-dose group), basal diet + 5×10 8 CFU / kg Clostridium butyricum (medium dose group), basal diet + 1×10 9 The basal diet composition is shown in Table 8. The experimental period was 42 days. Six chickens were randomly selected from each treatment group and slaughtered on days 21 and 42, respectively, to analyze the jejunal morphology of each treatment group. One-way analysis of variance was performed on the experimental data using SPSS 26.0. The F test for factor significance was used, and multiple comparisons were performed using Tukey and Dunnett's methods. Results are expressed as mean values, and P < 0.05 indicated a significant difference.

[0173] 2. Test results

[0174] The experimental results are shown in Table 10. As shown in Table 10, dietary supplementation with Clostridium butyricum significantly affected the intestinal morphology of broiler chickens 21 days after ingestion (P<0.05). Jejunal villus height in broiler chickens in the medium- and high-dose C. butyricum supplementation groups was significantly higher than that in the control and antibiotic groups (P<0.05). Jejunal crypt depth in broiler chickens in the C. butyricum supplementation groups was significantly lower than that in the control and antibiotic groups. Specifically, crypt depth in broiler chickens in the medium- and high-dose C. butyricum supplementation groups was significantly higher than that in the low-dose C. butyricum supplementation group (P<0.05). V / C values ​​in the C. butyricum supplementation groups were significantly higher than those in the control and antibiotic groups (P<0.05).

[0175] The addition of Clostridium butyricum to the diet significantly affected the intestinal morphology of broiler chickens at 42 days (P<0.05). The height of the jejunal villi in the broiler chickens in the C. butyricum supplementation groups was significantly higher than that in the control group (P<0.05), and the height of the jejunal villi in the broiler chickens in the medium-dose and high-dose C. butyricum supplementation groups was significantly lower than that in the antibiotic group (P<0.05). The depth of the jejunal crypts in the broiler chickens in the medium-dose C. butyricum supplementation group was significantly lower than that in the control and antibiotic groups (P<0.05), and the depth of the jejunal crypts in the broiler chickens in the low-dose C. butyricum supplementation group was significantly lower than that in the antibiotic group (P<0.05). The V / C ratios in the C. butyricum supplementation groups were significantly higher than those in the control and antibiotic groups (P<0.05).

[0176] Table 10 Effects of Clostridium butyricum on the morphological structure of jejunum in broiler chickens

[0177]

[0178] Note: a,b,c,d Values ​​in the same row with different lowercase superscripts indicate significant differences (P<0.05); V / C: villus height / crypt depth.

[0179] Example 6 Application of Clostridium butyricum in Improving Immune Function in Broiler Chickens

[0180] 1. Materials and Methods

[0181] The experiment selected 300 Ross 308 broilers, half male and half female, and randomly divided them into 5 treatment groups, with 6 replicates in each treatment. The treatment groups were: basic diet (control group), basic diet + 1×10 8 CFU / kg Clostridium butyricum (low-dose group), basal diet + 5×10 8 CFU / kg Clostridium butyricum (medium dose group), basal diet + 1×10 9 CFU / kg of Clostridium butyricum (high-dose group) and the antibiotic group (120 mg / kg chlortetracycline). The basal diet composition is shown in Table 8. The experimental period was 42 days. Six chickens were randomly selected from each treatment group for slaughter and sampling on days 21 and 42, respectively, and the immune function of broilers in each treatment group was analyzed. SPSS 26.0 was used to perform a one-way analysis of variance on the experimental data. The F test was used for factor significance, and the Tukey and Dunnett's method was used for multiple comparisons. The results are expressed as mean values, and P < 0.05 was considered significant.

[0182] 2. Test results

[0183] 2.1 Immune organs

[0184] The experimental results are shown in Table 11. As shown in Table 11, the thymus index of broilers in the diets supplemented with Clostridium butyricum was significantly higher at 21 days than in the control group (P < 0.05). The thymus index of the medium- and high-dose Clostridium butyricum supplemented groups was significantly higher at 21 days than in the antibiotic group (P < 0.05). The thymus index of the high-dose Clostridium butyricum supplemented group was significantly higher at 42 days than in the other groups (P < 0.05). These results indicate that Clostridium butyricum can promote the development of immune organs in broilers.

[0185] Table 11 Effects of Clostridium butyricum on immune organs of broiler chickens

[0186]

[0187]

[0188] Note: a,b,c Values ​​in the same row with different lowercase superscripts indicate significant differences (P<0.05).

[0189] 2.2 Serum immunoglobulin content

[0190] The test results are shown in Table 12. As shown in Table 12, the serum IgG levels of broiler chickens in the groups supplemented with Clostridium butyricum were significantly higher than those in the antibiotic group at 21 days (P < 0.05). The serum IgG levels of broiler chickens in the high-dose Clostridium butyricum supplementation group were significantly higher than those in the control group at 21 days (P < 0.05). The serum IgM levels of broiler chickens in the groups supplemented with Clostridium butyricum were significantly higher than those in the antibiotic group at 21 days (P < 0.05). The serum IgG levels of broiler chickens in the groups supplemented with Clostridium butyricum were significantly higher than those in the control and antibiotic groups at 42 days (P < 0.05). The serum IgM levels of broiler chickens in the low- and medium-dose Clostridium butyricum supplementation groups were significantly higher than those in the antibiotic group at 42 days (P < 0.05). These results indicate that Clostridium butyricum can improve the serum immune function of broiler chickens.

[0191] Table 12 Effect of Clostridium butyricum on serum immunoglobulin levels in broiler chickens

[0192]

[0193] Note: a,b,c The values ​​in the same row with different lowercase superscripts indicate significant differences (P<0.05);

[0194] IgA: Immunoglobulin A, IgG: Immunoglobulin G, IgM: Immunoglobulin M.

[0195] Example 7 Application of Clostridium butyricum Preparation in Improving Serum Biochemical Indices in Broiler Chickens

[0196] 1. Materials and Methods

[0197] The experiment selected 300 Ross 308 broilers, half male and half female, and randomly divided them into 5 treatment groups, with 6 replicates in each treatment. The treatment groups were: basic diet (control group), basic diet + 1×10 8 CFU / kg Clostridium butyricum (low-dose group), basal diet + 5×10 8 CFU / kg Clostridium butyricum (medium dose group), basal diet + 1×10 9 The basal diet composition is shown in Table 8. The experimental period was 42 days. Six chickens were randomly selected from each treatment group and slaughtered on days 21 and 42, respectively. Serum was collected and serum biochemical parameters of broiler chickens in each treatment group were measured. One-way analysis of variance was performed on the experimental data using SPSS 26.0. The F test was used for factor significance, and multiple comparisons were performed using Tukey and Dunnett's methods. Results are expressed as mean values, and P < 0.05 indicated a significant difference.

[0198] 2. Test results

[0199] The test results are shown in Table 13. As shown in Table 13, the addition of Clostridium butyricum to the diet had a significant effect on the serum biochemical parameters of broiler chickens at day 21 (P<0.05). The high-dose group supplemented with Clostridium butyricum can significantly reduce the serum GLU content of broilers (P<0.05), and the serum GLU content of each group supplemented with Clostridium butyricum is significantly lower than that of the antibiotic group (P<0.05); the serum TC and TG contents of each group supplemented with Clostridium butyricum are significantly lower than those of the antibiotic group (P<0.05); the serum TP content of the medium-dose group supplemented with Clostridium butyricum is significantly lower than that of the antibiotic group (P<0.05); the serum ALB content of the medium-dose group supplemented with Clostridium butyricum is significantly higher than that of the control group and the antibiotic group (P<0.05), and the serum ALB content of the low-dose and medium-dose groups supplemented with Clostridium butyricum is significantly higher than that of the antibiotic group (P<0.05); the serum UREA content of the medium-dose and high-dose groups supplemented with Clostridium butyricum is significantly lower than that of the control group and the antibiotic group (P<0.05); the serum CREA content of the low-dose group supplemented with Clostridium butyricum is significantly lower than that of the control group (P<0.05), and the serum CREA content of the high-dose group supplemented with Clostridium butyricum is significantly lower than that of the antibiotic group (P<0.05).

[0200] The addition of Clostridium butyricum to the diet had a significant effect on the serum biochemical parameters of broiler chickens at 42 days of age (P<0.05). The serum GLU content of broiler chickens in the high-dose Clostridium butyricum supplementation group was significantly lower than that in the antibiotic group (P<0.05); the serum TC content of broiler chickens in the medium-dose Clostridium butyricum supplementation group was significantly lower than that in the antibiotic group (P<0.05); the serum TG content of broiler chickens in the low-dose Clostridium butyricum supplementation group was significantly higher than that in the control group (P<0.05); the serum TP and ALB content of broiler chickens in the low-dose Clostridium butyricum supplementation group was significantly higher than that in the control group (P<0.05); the serum TP and ALB content of broiler chickens in all Clostridium butyricum supplementation groups was significantly higher than that in the antibiotic group (P<0.05); the serum UREA content of broiler chickens in the medium-dose and high-dose Clostridium butyricum supplementation groups was significantly lower than that in the control group (P<0.05); the serum CREA content of broiler chickens in the low-dose Clostridium butyricum supplementation group was significantly higher than that in the control group (P<0.05).

[0201] The above research results show that Clostridium butyricum can regulate the serum biochemical index of broiler chickens, promote the conversion of non-protein nitrogen, and benefit the growth and development of animals; it is more conducive to regulating lipid metabolism and protein synthesis in animals than antibiotics.

[0202] Table 13 Effects of Clostridium butyricum on serum biochemical indices of broiler chickens

[0203]

[0204]

[0205] Note: a,b,c,d The values ​​in the same row with different lowercase superscripts indicate significant differences (P<0.05);

[0206] GLU: glucose, TC: total cholesterol, TG: total triglycerides, TP: total protein, ALB: albumin, UREA: urea, CREA: inosine.

[0207] Example 8 Application of Clostridium butyricum in Improving the Antioxidant Capacity of Broiler Chickens

[0208] 1. Materials and Methods

[0209] The experiment selected 300 Ross 308 broilers, half male and half female, and randomly divided them into 5 treatment groups, with 6 replicates in each treatment. The treatment groups were: basic diet (control group), basic diet + 1×10 8 CFU / kg Clostridium butyricum (low-dose group), basal diet + 5×10 8 CFU / kg Clostridium butyricum (medium dose group), basal diet + 1×10 9CFU / kg of Clostridium butyricum (high-dose group) and an antibiotic group (120 mg / kg chlortetracycline). The basal diet composition is shown in Table 8. The experimental period was 42 days. Six chickens were randomly selected from each treatment group and slaughtered on days 21 and 42, respectively. Serum and liver were collected for analysis of antioxidant indicators. SPSS 26.0 was used to analyze the experimental data using a one-way analysis of variance. The F test was used for factor significance, and Tukey and Dunnett's methods were used for multiple comparisons. Results are expressed as mean values. P < 0.05 indicated a significant difference.

[0210] 2. Test results

[0211] 2.1 Effect of Clostridium butyricum on the antioxidant capacity of broiler serum

[0212] The experimental results are shown in Table 14. As shown in Table 14, the addition of Clostridium butyricum had a significant effect on the serum antioxidant capacity of broiler chickens at 21 days (P<0.05). The serum T-AOC of broiler chickens in the medium-dose and high-dose groups supplemented with Clostridium butyricum was significantly higher than that in the control group (P<0.05), and the serum T-AOC of all groups supplemented with Clostridium butyricum was significantly higher than that in the antibiotic group (P<0.05); the serum T-SOD content of all groups supplemented with Clostridium butyricum was significantly higher than that in the antibiotic group (P<0.05); the serum CAT content of broiler chickens in the medium-dose and high-dose groups supplemented with Clostridium butyricum was significantly higher than that in the control group and the antibiotic group (P<0.05); the serum GSH-Px content of broiler chickens in the medium-dose and high-dose groups supplemented with Clostridium butyricum was significantly higher than that in the antibiotic group (P<0.05); and the serum MDA content of all groups supplemented with Clostridium butyricum was significantly lower than that in the control group and the antibiotic group (P<0.05).

[0213] The supplementation of Clostridium butyricum significantly increased the serum antioxidant capacity of broiler chickens at 42 days of age (P<0.05). The serum T-AOC of broiler chickens in the medium-dose Clostridium butyricum supplementation group was significantly higher than that in the control group (P<0.05), and the serum T-AOC levels of the low-dose and medium-dose Clostridium butyricum supplementation groups were significantly higher than those in the antibiotic group (P<0.05). The serum T-SOD levels of broiler chickens in the C. butyricum supplementation group were significantly higher than those in the antibiotic group (P<0.05). The serum CAT levels of broiler chickens in the low-dose Clostridium butyricum supplementation group were significantly higher than those in the control group (P<0.05), and the serum CAT levels of broiler chickens in the C. butyricum supplementation groups were significantly higher than those in the antibiotic group (P<0.05). The serum GSH-Px levels of broiler chickens in the medium-dose and high-dose Clostridium butyricum supplementation groups were significantly higher than those in the control group (P<0.05), and the serum GSH-Px levels of broiler chickens in the C. butyricum supplementation groups were significantly higher than those in the antibiotic group (P<0.05). The serum MDA levels of broiler chickens in the C. butyricum supplementation groups were significantly lower than those in the control and antibiotic groups (P<0.05).

[0214] The above results indicate that Clostridium butyricum can improve the enzymatic and non-enzymatic antioxidant capacity of broiler serum and promote the balance of serum oxidative stress.

[0215] Table 14 Effect of Clostridium butyricum on the antioxidant capacity of broiler serum

[0216]

[0217] Note: a,b,c,d The values ​​in the same row with different lowercase superscripts indicate significant differences (P<0.05);

[0218] T-AOC: total antioxidant capacity, T-SOD: total superoxide dismutase, CAT: catalase, GSH-Px: glutathione peroxidase, MDA: malondialdehyde.

[0219] 2.2 Effect of Clostridium butyricum on the antioxidant capacity of broiler liver

[0220] The test results are shown in Table 15. As shown in Table 15, the addition of Clostridium butyricum had a significant effect on the antioxidant capacity of the liver of broiler chickens at 21 days (P<0.05). The T-AOC of broiler livers in the medium-dose and high-dose groups supplemented with Clostridium butyricum was significantly higher than that in the control group (P<0.05), and the T-AOC of livers in the high-dose group supplemented with Clostridium butyricum was significantly higher than that in the antibiotic group (P<0.05); the T-SOD content of livers in broiler livers in the low-dose and high-dose groups supplemented with Clostridium butyricum was significantly higher than that in the control group (P<0.05), and the T-SOD content of livers in the high-dose group supplemented with Clostridium butyricum was significantly higher than that in the antibiotic group (P<0.05); the CAT content of livers in broiler livers in the medium-dose and high-dose groups supplemented with Clostridium butyricum was significantly higher than that in the other groups (P<0.05); the GSH-Px content of livers in broiler livers in the low-dose and high-dose groups supplemented with Clostridium butyricum was significantly higher than that in the control group and the antibiotic group (P<0.05); the MDA content of livers in all groups supplemented with Clostridium butyricum was significantly lower than that in the control group (P<0.05).

[0221] The supplementation of Clostridium butyricum significantly increased the antioxidant capacity of broiler livers at 42 days of age (P<0.05). The T-AOC of the livers of broilers in the high-dose Clostridium butyricum supplementation group was significantly higher than that in the control group (P<0.05), and the T-AOC of the livers of the medium-dose and high-dose Clostridium butyricum supplementation groups was significantly higher than that in the antibiotic group (P<0.05). The T-SOD content of the livers of broilers in the high-dose Clostridium butyricum supplementation group was significantly higher than that in the control and antibiotic groups (P<0.05). The CAT content of the livers of broilers in all the Clostridium butyricum supplementation groups was significantly higher than that in the control and antibiotic groups (P<0.05). The GSH-Px content of the livers of broilers in the high-dose Clostridium butyricum supplementation group was significantly higher than that in the control and antibiotic groups (P<0.05). The MDA content of the livers of broilers in the medium-dose and high-dose Clostridium butyricum supplementation groups was significantly lower than that in the control group (P<0.05), and the MDA content of the livers of broilers in the high-dose Clostridium butyricum supplementation group was significantly lower than that in the antibiotic group (P<0.05).

[0222] The above results indicate that Clostridium butyricum can improve the enzymatic and non-enzymatic antioxidant capacity of broiler liver and promote the balance of liver oxidative stress.

[0223] Table 15 Effects of Clostridium butyricum on antioxidant capacity of broiler liver

[0224]

[0225] Note: a,b,c The values ​​in the same row with different lowercase superscripts indicate significant differences (P<0.05);

[0226] T-AOC: total antioxidant capacity, T-SOD: total superoxide dismutase, CAT: catalase, GSH-Px: glutathione peroxidase, MDA: malondialdehyde.

[0227] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Clostridium butyricum NCB, characterized in that The deposit number is CGMCC No.23377.

2. A bacterial agent containing the Clostridium butyricum according to claim 1.

3. A Clostridium butyricum preparation, characterized in that The Clostridium butyricum preparation is a liquid or dry powder, and the preparation method is as follows: (1) The activated Clostridium butyricum according to claim 1 is inoculated into a seed culture medium, cultured at 37° C. under anaerobic conditions for 24 to 32 hours, and then inoculated into a fermentation medium at an inoculum rate of 2% to 10% v / v, and cultured anaerobically for 48 to 72 hours to obtain a Clostridium butyricum fermentation broth, i.e., a Clostridium butyricum liquid; The seed culture medium comprises: peptone 0.8-1.2%, beef extract 0.8-1.2%, yeast powder 0.3-0.6%, glucose 0.6-1.0%, (NH4)2SO4 0.1%, NaCl 0.5%, K2HPO4·H2O 0.4%, MnSO4·H2O 0.02%, MgSO4·7H2O 0.05%, CaCO3 0.2%, pH 7.0±0.1; The fermentation medium comprises: 0.4-0.7% soy peptone, 0.3-0.7% tryptone, 0.1-0.4% yeast powder, 0.3-1.0% glucose, 0.8-1.2% corn flour, 0.1% (NH4)2SO4, 0.5% NaCl, 0.4% K2HPO4·H2O, 0.02% MnSO4·H2O, 0.05% MgSO4·7H2O, 0.2% CaCO3, 0.05-0.1% v / v defoamer, and a pH value of 7.0±0.

1. (2) mixing the Clostridium butyricum fermentation liquid and the carrier in a weight ratio of (0.6-1.0):1, then drying at low temperature and pulverizing to obtain Clostridium butyricum dry powder; The carrier is selected from one or more of bran, wheat bran, corn cob powder, glucose and corn gluten powder.

4. Use of the Clostridium butyricum according to claim 1, the bacterial agent according to claim 2, or the Clostridium butyricum preparation according to claim 3 in the preparation of a biological product for preventing ulcerative colitis in animals, and for resisting oxidative stress induced by dextran sodium sulfate and improving intestinal health.

5. Use of the Clostridium butyricum according to claim 1, the bacterial agent according to claim 2, or the Clostridium butyricum preparation according to claim 3 in improving the growth performance of broiler chickens.

6. Use of the Clostridium butyricum according to claim 1, the bacterial agent according to claim 2, or the Clostridium butyricum preparation according to claim 3 in improving the intestinal morphology of broiler chickens, wherein the use is for non-disease treatment purposes.

7. Use of the Clostridium butyricum according to claim 1, the bacterial agent according to claim 2, or the Clostridium butyricum preparation according to claim 3 in improving the immune function of broiler chickens, wherein the use is for non-disease treatment purposes.

8. Use of the Clostridium butyricum according to claim 1, the bacterial agent according to claim 2, or the Clostridium butyricum preparation according to claim 3 in regulating serum biochemical indices in broiler chickens, wherein the use is for non-disease treatment purposes.

9. Use of the Clostridium butyricum according to claim 1, the bacterial agent according to claim 2, or the Clostridium butyricum preparation according to claim 3 for improving the antioxidant capacity of broiler chickens, wherein the use is for non-disease treatment purposes.

10. Use of the Clostridium butyricum according to claim 1, the bacterial agent according to claim 2, or the Clostridium butyricum preparation according to claim 3 in improving the quality of chicken.

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