A Clostridium butyricum strain and its applications

Abstract

The present invention relates to a strain of Clostridium butyricum and its application, belonging to the field of microbiology. The Clostridium butyricum JBH-BD1 of the present invention is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M2022151. The Clostridium butyricum of the present invention is natural, safe, acid-resistant, alkali-resistant, heat-resistant, and has strong abilities to produce protease, amylase and organic acids. At the same time, it has antibacterial effects on Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Pasteurella multocida of porcine origin and Actinobacillus pleuropneumoniae, and can convert inorganic ferrous iron into organic ferrous iron; when used in the aquaculture industry, it can not only regulate the balance of intestinal flora, promote the growth of livestock and poultry, but also effectively improve the utilization rate of iron element, reduce its addition amount, prevent iron deficiency anemia in livestock and poultry, improve the absorption and utilization efficiency of iron by livestock and poultry, and increase the aquaculture benefits.

Description

Technical Field

[0001] The present invention relates to a Clostridium butyricum strain and its application, belonging to the field of microbial technology. Background Art

[0002] Trace elements play an indispensable role in the life activities of livestock and poultry, such as growth, development, and reproduction. Currently, in the breeding industry, a certain amount of trace elements is mainly added to the diet to meet the physiological needs of livestock and poultry. However, there are risks such as large toxic and side effects and certain environmental pollution when feeding inorganic trace elements in the diet. Most importantly, the biological utilization efficiency of inorganic trace elements is low. A large number of studies have shown that organic ligands participate in the entire absorption and metabolism process of trace elements. By feeding organic trace elements, the biological utilization efficiency of metal elements can be significantly improved, the usage amount can be reduced, and the effect can be achieved with half the effort. Currently, the development of organic trace elements mainly includes two methods: chemical synthesis and biological transformation. Since microorganisms can utilize inorganic trace elements and convert them into organic forms, screening and preparing beneficial microbial preparations with strong tolerance, enrichment, and synthesis abilities for trace elements for the production and enrichment of organic trace elements have become one of the hotspots in the feed additive industry at home and abroad.

[0003] Microecological preparations are biological preparations and live bacterial preparations of microbial cells and their metabolites obtained by probiotic fermentation and post-processing. Therefore, they have the functions of maintaining the balance of intestinal microecology, improving feed conversion efficiency, improving the growth performance of livestock and poultry, enhancing immune function, antioxidant and anti-cancer effects, and improving the environment and reducing the generation of harmful substances, and are increasingly sought after by the breeding industry.

[0004] Clostridium butyricum belongs to the genus Clostridium and is also known as Clostridium butyricum and Clostridium tyrobutyricum. It was first discovered and named as Miyairi bacteria by Professor Miyagi in Japan. Clostridium butyricum is a unique intestinal probiotic among Gram-positive bacteria. The bacterial cells are straight rods or slightly curved, surrounded by flagella, produce spores, and the endospores are oval. During fermentation and cultivation, the bacteria can produce butyric acid, acetic acid, lactic acid and propionic acid, accompanied by a small amount of hydrogen sulfide and hydrogen. In recent years, Clostridium butyricum has been widely used as a probiotic in the clinical treatment of gastrointestinal diseases, such as intestinal flora imbalance, drug-related enteritis, acute and chronic diarrhea, irritable bowel syndrome and constipation. Especially in the context of the feed antibiotic ban in 2020, Clostridium butyricum has increasingly developed into a feed microecological preparation due to its good repair and enhancement functions of the intestinal barrier function of livestock and poultry. Compared with other probiotic preparations, this bacterium has good characteristics such as fast growth rate, strong stress resistance, continuous colonization in the intestine, production of organic acids, butyricin and other substances. This makes Clostridium butyricum have good application potential in the livestock and poultry breeding industry. The organic acids secreted by Clostridium butyricum can lower the pH of the intestinal lumen, inhibit the reproduction of pathogenic microorganisms, reduce intestinal toxins, and reduce the incidence of diarrhea. At the same time, the active substance butyricin produced by Clostridium butyricum has the ability to kill and antagonize the colonization of pathogenic microorganisms in the intestine. It can be seen that the existing technology mainly focuses on the research of Clostridium butyricum in aspects such as strain breeding, inhibition of harmful pathogenic microorganisms, regulation of the intestinal environment of livestock and poultry, regulation of the body's immunity and antioxidant ability, and no Clostridium butyricum with the ability to enrich trace element iron has been found. Summary of the Invention

[0005] The object of the present invention is to provide a strain of Clostridium butyricum with the ability to enrich iron.

[0006] The present invention also provides the application of the above-mentioned Clostridium butyricum.

[0007] In order to achieve the above object, the technical solution adopted by the Clostridium butyricum of the present invention is as follows:

[0008] Clostridium butyricum JBH-BD1, which is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M2022151.

[0009] The Clostridium butyricum JBH-BD1 of the present invention is screened from the intestinal fecal samples of healthy piglets, which is natural, safe, acid-resistant, alkali-resistant, heat-resistant, and has strong abilities to produce protease, amylase and organic acids. At the same time, it has antibacterial effects on Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Pasteurella multocida of porcine origin and Actinobacillus pleuropneumoniae, and can convert inorganic ferrous iron into organic ferrous iron. When the Clostridium butyricum of the present invention is used in the breeding industry, it can not only regulate the balance of intestinal flora, promote the growth of livestock and poultry, but also effectively improve the utilization rate of iron element, reduce its addition amount, prevent iron deficiency anemia in livestock and poultry, improve the absorption and utilization efficiency of iron by livestock and poultry, and increase the breeding benefits.

[0010] The technical solution adopted for the application of the Clostridium butyricum of the present invention is as follows:

[0011] The application of the Clostridium butyricum JBH-BD1 in inhibiting Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Pasteurella multocida of porcine origin or Actinobacillus pleuropneumoniae. The Clostridium butyricum of the present invention has significant inhibitory effects on Escherichia coli of porcine origin, Salmonella, Staphylococcus aureus, Clostridium perfringens, Pasteurella multocida of porcine origin and Actinobacillus pleuropneumoniae, and the order of inhibitory ability is: Clostridium perfringens type C > Clostridium perfringens type A > Actinobacillus pleuropneumoniae > Salmonella typhimurium > Pasteurella multocida of porcine origin > Escherichia coli > Staphylococcus aureus.

[0012] The application of the Clostridium butyricum JBH-BD1 in iron enrichment. The Clostridium butyricum of the present invention can convert inorganic ferrous iron into organic ferrous iron in an in vitro co-culture environment, thereby improving the absorption and utilization of iron by livestock and poultry.

[0013] The application of the Clostridium butyricum JBH-BD1 in iron enrichment includes the following steps: anaerobically culturing the Clostridium butyricum JBH-BD1 in a medium containing ferrous ions.

[0014] Further, the culture medium containing ferrous ions is obtained by adding ferrous salts to the reinforced Clostridium medium (RCM medium) and mixing evenly; the concentration of the ferrous ions is 200-600 mg / L. The ferrous salt is one or any combination of ferrous sulfate and ferrous chloride. The RCM medium is an RCM liquid medium. For example, the composition of the RCM liquid medium is: peptone 10.0 g, beef powder 10.0 g, yeast powder 3.0 g, glucose 5.0 g, soluble starch 1.0 g, sodium acetate 3.0 g, sodium chloride 5.0 g, L-cysteine hydrochloride 0.5 g, distilled water 1000 mL, pH 6.8±0.2.

[0015] Further, the anaerobic culture time is 24-36 h, for example, 24-30 h.

[0016] Using the live bacteria preparation of the above-mentioned Clostridium butyricum JBH-BD1, compared with the traditionally used antibiotic products, it is safe, non-toxic, does not produce drug resistance, and can be used to regulate the intestinal microflora balance of young livestock and poultry. Especially compared with directly feeding inorganic iron to livestock and poultry, feeding this live bacteria preparation can effectively improve the utilization rate of iron element, reduce its addition amount, prevent iron deficiency anemia in livestock and poultry, and improve the absorption and utilization efficiency of iron by livestock and poultry.

[0017] Further, the live bacteria preparation is a probiotic preparation for pig feed additives. Description of the Drawings

[0018] Figure 1 It is the colony morphology of Clostridium butyricum JBH-BD1 of the present invention on the RCM agar plate;

[0019] Figure 2 It is the microscopic examination picture of the colony of Clostridium butyricum JBH-BD1 of the present invention;

[0020] Figure 3 It is the metabolic kinetic growth curve and pH change curve graph of Clostridium butyricum JBH-BD1 in Example 2;

[0021] Figure 4 It is the 16S rRNA gene phylogenetic tree of Clostridium butyricum JBH-BD1 of the present invention;

[0022] Figure 5 It is the change curve of the organic acid content in the fermentation broth of Clostridium butyricum JBH-BD1 with time in Example 3;

[0023] Figure 6 It is the protease activity graph of Clostridium butyricum JBH-BD1 in Example 3;

[0024] Figure 7It is the graph of the amylase production ability of Clostridium butyricum JBH-BD1 in Example 3;

[0025] Figure 8 It is the curve graph of the changes in the viable count and survival rate of Clostridium butyricum JBH-BD1 over time during the high-temperature resistance test in Example 3;

[0026] Figure 9 It is the curve graph of the changes in the survival rate of Clostridium butyricum JBH-BD1 at different pH values over time during the acid resistance test in Example 3;

[0027] Figure 10 It is the graph of the changes in the survival rate and viable count of Clostridium butyricum JBH-BD1 at different porcine bile salt concentrations during the bile salt resistance test in Example 3;

[0028] Figure 11 It is the graph of the antibacterial result of Clostridium butyricum JBH-BD1 in Example 3;

[0029] Figure 12 It is the graph of the influence of the culture time on the iron enrichment and cell growth of Clostridium butyricum JBH-BD1 in Example 3;

[0030] Figure 13 It is the graph of the influence of the culture time on the iron enrichment rate and organic conversion rate of Clostridium butyricum JBH-BD1 in Example 3. Specific Embodiments

[0031] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0032] The step methods in the following embodiments are all conventional methods unless otherwise specified; the raw materials involved are all ordinary commercially available products and can be obtained through market purchase unless otherwise specified.

[0033] The RCM agar plate (i.e., the RCM agar medium plate) is prepared by adding agar at 18 g / L to the RCM basal medium and autoclaving to make plates. The RCM liquid medium is prepared by omitting agar from the RCM basal medium. The reinforced clostridial agar medium is obtained by adding agar at 18 g / L to the RCM basal medium and then autoclaving.

[0034] The composition of the RCM basal medium: peptone 10.0 g, beef powder 10.0 g, yeast powder 3.0 g, glucose 5.0 g, soluble starch 1.0 g, sodium acetate 3.0 g, sodium chloride 5.0 g, L-cysteine hydrochloride 0.5 g, agar 0.5 g, distilled water 1000 mL, pH 6.8 ± 0.2.

[0035] Example 1 Obtaining of Clostridium butyricum JBH-BD1

[0036] The Clostridium butyricum JBH-BD1 of the present invention was screened by a method comprising the following steps:

[0037] 1) Sample treatment: Fresh feces of healthy piglets from a certain farm were aseptically collected using a fecal sampler, rinsed repeatedly 3 times with 0.85% physiological saline, mixed well, centrifuged, and then diluted successively by 10-fold serial dilution. Diluents of different dilution gradients were selected for standby.

[0038] 2) Isolation and culture: Ferrous sulfate was added to the reinforced Clostridium agar medium and mixed well to obtain a ferrous-containing reinforced Clostridium agar medium with an iron content of 600 mg / L. It was autoclaved at 121 °C for 20 min, cooled to about 50 °C, and poured into petri dishes. After the medium in the petri dishes cooled, diluents of different dilution gradients in 1) were spread on the RCM agar medium plates and placed in an anaerobic incubator for culturing at 37 °C for 16 h. The purpose of adding ferrous sulfate to the reinforced Clostridium agar medium was to exclude Clostridium butyricum and other miscellaneous bacteria that are intolerant to iron, facilitating the screening of Clostridium butyricum that is tolerant to iron.

[0039] 3) Purification: Different morphological single colonies were selected under a stereomicroscope and transferred to RCM agar medium plates for purification.

[0040] 4) Preservation: The purified single colonies were picked and inoculated into RCM liquid medium for anaerobic overnight culture at 37 °C. 500 μL of the culture solution and 500 μL of 50% sterilized glycerol were mixed at a ratio of 1:1 (V / V) and stored in a -80 °C ultra-low temperature refrigerator;

[0041] 5) Activation: The strains separated and preserved in step 4) were inoculated into RCM liquid medium, activated at 37 °C for 16 h, and then inoculated into RCM liquid medium at an inoculation amount of 2% and cultured at 37 °C for 16 h to obtain a Clostridium butyricum culture solution;

[0042] 6) Separation: The culture solution in step 5) was centrifuged to collect the supernatant, and the supernatant was filtered through a 0.22 μm microporous membrane and stored refrigerated at 2 - 8 °C; The centrifugation speed was 7000 r / min, and the centrifugation time was 15 min;

[0043] 7) Screening: The pathogen indicator bacteria after culturing for 24 h were added to LB medium, mixed well, and poured into plates. After cooling and solidifying, 9 mm holes were punched, 3 holes per plate. 200 μL of the filtrate prepared in step 6) was added to each hole and cultured at 37 °C for 16 h. The diameter of the inhibition zone was measured with a vernier caliper, and strains with excellent antibacterial properties were screened according to the size of the inhibition zone diameter, namely Clostridium butyricum JBH-BD1. The indicator bacteria used were Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Porcine Pasteurella multocida, and Actinobacillus pleuropneumoniae. The concentration of each bacterium added to LB medium was 1.0×10 8 CFU / mL.

[0044] The screened Clostridium butyricum JBH-BD1 was spread on an enhanced Clostridium agar medium plate. After cultivation, the enhanced Clostridium agar culture plate was observed as follows Figure 1 shown. From Figure 1 it can be seen that the strain formed white irregular-sized colonies on the plate, slightly protruding, with a diameter of about 1-3 mm. The microscopic examination results are as shown in Figure 2 shown. From Figure 2 it can be seen that it is Gram-positive, has spores, and the spores are oval-shaped.

[0045] Example 2 Identification of Clostridium butyricum JBH-BD1

[0046] 1) Physiological and biochemical tests:

[0047] The physiological and biochemical tests were carried out on the screened strain Clostridium butyricum JBH-BD1 using micro biochemical tubes. Referring to the "Manual for Systematic Identification of Common Bacteria" and the "Bergey's Manual of Determinative Bacteriology", the physiological and biochemical characteristics of the strain were analyzed: The 24-hour culture solution of the test bacterium was inoculated into 24 micro fermentation tubes such as salicin, melezitose, melibiose, lactose, etc., with the opening facing down, and cultured in an anaerobic incubator at 37°C for 24 hours. The results were observed and recorded, and the results are shown in Table 1.

[0048] Table 1 Results of physiological and biochemical tests of JBH-BD1 strain

[0049] Item Result Item Result Salicin + Sucrose + Melibiose + Trehalose + Melezitose - Mannose + Lactose + Xylose + Galactose + Litmus reduction test + Maltose + Hydrolyzed starch test - Glucose + Indole test - Mannitol + Nitrate reduction test + Sorbitol - Hydrolyzed gelatin test - Raffinose + Hydrolyzed casein test - Fructose + Acetoin test - Cellobiose + Catalase test -

[0050] As can be seen from Table 1, the Clostridium butyricum JBH-BD1 strain can ferment and utilize salicin, melibiose, lactose, galactose, glucose, mannitol, raffinose, fructose, sucrose, trehalose, mannose, xylose, cellobiose; it can reduce litmus and nitrate.

[0051] 2) Metabolic kinetics of JBH-BD1 strain:

[0052] A single colony of Clostridium butyricum JBH-BD1 was picked and inoculated into RCM liquid medium, anaerobically cultured at 37°C at 220 r / min for 16 h. An appropriate amount of the culture solution was transferred to 100 mL of sterile RCM liquid medium at a ratio of 1%, and anaerobically cultured at 37°C at 220 r / min. The absorbance value and pH value of the bacterial culture solution were measured every 2 h at 600 nm.

[0053] The Clostridium butyricum JBH-BD1 strain grew well in the RCM liquid medium, and its growth curve and pH change curve are shown in Figure 3 . From Figure 3It can be seen that JBH-BD1 can enter the logarithmic growth phase in RCM medium within 4 h. During this period, the bacterial count increases rapidly. Since organic acids are produced during growth, the pH value of the medium also drops sharply. The logarithmic growth phase of strain JBH-BD1 lasts for about 8 h and then enters the stationary growth phase. After that, the bacterial count no longer increases, and the OD 600 value of the culture solution remains basically unchanged at about 1.7, and its pH value is stable between 4.5 and 4.7.

[0054] 3) Molecular biological identification

[0055] Using the genome of Clostridium butyricum JBH-BD1 as a template, a 1505-bp gene sequence was amplified by PCR with universal primers for bacterial 16S rRNA. The nucleotide sequence is shown in SEQ ID NO.1. After homology alignment, the homology between strain JBH-BD1 and Clostridium butyricum reached 100%, belonging to Clostridium butyricum. The constructed phylogenetic tree is shown in Figure 4 , and strain JBH-BD1 and Clostridium butyricum strain BSCBM01 from Sichuan, China (GenBank accession number: KY435713.1) clustered into one group with a confidence level of 99%, indicating that they have the closest genetic relationship.

[0056] Clostridium butyricum JBH-BD1 was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC, address: Wuhan University, Wuhan, China, postcode 430072) on February 25, 2022, with the deposit number CCTCC NO: M2022151.

[0057] Example 3 Performance test of Clostridium butyricum JBH-BD1

[0058] 1) Acid production ability

[0059] a) Test sample treatment: The test product was diluted 10 times with ultrapure water and filtered through a 0.22-μm microporous filter membrane for standby.

[0060] b) Preparation of reference substances: Accurately pipette 1 ml of butyric acid, acetic acid, and lactic acid solutions into a 100-ml volumetric flask respectively, and make up to the mark with ultrapure water. After mixing evenly, it was used for standby. At this time, the contents of butyric acid, acetic acid, and lactic acid in the reference substances were 19.16, 21.00, and 21.16 g / L respectively. The above three organic acid reference substances were diluted to appropriate concentrations with ultrapure water. A standard curve was established with the concentration and the corresponding peak area.

[0061] c) Chromatographic conditions: The chromatographic column was an ion exchange chromatographic column KC811; the mobile phase was 0.1% phosphoric acid solution; the injection volume was 5 μl; the maximum pressure was 5 MPa; the flow rate was 1 ml / min; the detection wavelength was 210 nm.

[0062] Inoculate a single colony of Clostridium butyricum JBH-BD1 into RCM liquid medium, anaerobically culture it at 37°C with a rotation speed of 220 r / min for 16 h. Then, transfer an appropriate amount of the culture broth to 100 mL of sterile RCM liquid medium at a ratio of 1%, and anaerobically culture it at 37°C with a rotation speed of 220 r / min. Start timing from the moment of inoculation into the RCM liquid medium. Every 4 h, take 1 mL from the JBH-BD1 fermentation broth as a sample to be measured, and respectively test the contents of lactic acid, acetic acid, and butyric acid in the fermentation broth. The measurement results of the contents of organic acids such as lactic acid, acetic acid, and butyric acid in the JBH-BD1 fermentation broth within 36 h are shown in Figure 5 . It can be seen from Figure 5 that after 20 h, the yields of butyric acid, acetic acid, and lactic acid no longer increase. At 36 h of fermentation, the contents of butyric acid, acetic acid, and lactic acid in the fermentation broth are 10 g / L, 7.8 g / L, and 5 g / L respectively.

[0063] 2) Enzyme production ability

[0064] Pick a single colony of Clostridium butyricum JBH-BD1 and inoculate it into RCM liquid medium. Anaerobically culture it at 37°C with a rotation speed of 220 r / min for 16 h. Then, transfer an appropriate amount of the culture broth to 100 mL of sterile RCM liquid medium at a ratio of 1%, and anaerobically culture it at 37°C with a rotation speed of 220 r / min for 16 h. Take 2 mL of the fermentation broth and centrifuge it at 5000 r / min for 15 min for standby. Establish a tyrosine standard curve according to the Folin-Ciocalteu method, measure the protease activity in the JBH-BD1 fermentation broth at 10 h of anaerobic fermentation, and use the blank RCM liquid medium as a control. The results are shown in Figure 6 . The results show that the protease activity of JBH-BD1 measured by the Folin-Ciocalteu method is 33.54 U / mL, which is much higher than the protease activity of the control group (specifically 1.45 U / mL).

[0065] Dilute the Clostridium butyricum JBH-BD1 bacterial solution 10-fold to an appropriate concentration. Take 100 μL of the bacterial solution and spread it on the starch medium. After anaerobic culture for 16 h, stain the plate with colorimetric iodine solution, as shown in Figure 7 . Observe the diameter (D) of the transparent circle around the colony and the diameter (C) of the colony, and calculate the starch production ability (i.e., the D / C ratio). The results are shown in Table 2.

[0066] Table 2 Starch amylase production ability of Clostridium butyricum JBH-BD1

[0067] Strain D (mm) C (mm) D / C JBH - BD1 2.50 1.00 2.50

[0068] It can be seen from Table 2 that the starch amylase production ability (D / C) of Clostridium butyricum JBH-BD1 is 2.5. Thus, it can be seen that strain JBH-BD1 has the ability to produce protease and amylase.

[0069] 3) Stress resistance detection

[0070] High temperature resistance test: Take the culture solution of Clostridium butyricum JBH-BD1 in a 250 mL conical flask, place it in a water bath at 85 °C and heat for 10 min. Samples are taken before the water bath heating, at 5 min and 10 min of heating, the viable cell count is measured and the survival rate is calculated. The results are shown in Figure 8 . The culture solution used in the high temperature resistance test was obtained by picking a single colony of Clostridium butyricum JBH-BD1 and inoculating it into RCM liquid medium, and anaerobically culturing at 37 °C and 220 r / min for 16 h.

[0071] Acid resistance test: Inoculate Clostridium butyricum JBH-BD1 at an inoculation amount of 1% into test tubes containing sterile phosphate buffer at pH 1.0, pH 2.0, and pH 3.0 respectively, place them in an anaerobic incubator at 37 °C, and perform viable cell counting at 0 h, 1 h, 2 h, and 3 h respectively to calculate the survival rate. The results are shown in Figure 9 .

[0072] Bile salt resistance test: Inoculate Clostridium butyricum JBH-BD1 at an inoculation amount of 1% into RCM liquid medium containing 0.10%, 0.20%, 0.30%, 0.50%, and 1.00% porcine bile salt by mass fraction respectively. After culturing for 36 h, calculate the survival rate. The results are shown in Figure 10 .

[0073] The results show that after Clostridium butyricum JBH-BD1 is treated at 85 °C for 5 min and 10 min, its survival rate is above 95%, as shown in Figure 8 . In a strongly acidic environment with a pH of 1.0, the survival rate of Clostridium butyricum JBH-BD1 is still above 95%, as shown in Figure 9 . When acting for 36 h at a bile salt concentration below 0.3%, the survival rate of Clostridium butyricum JBH-BD1 is above 95%. At a bile salt concentration of 0.3%, the survival rate of Clostridium butyricum JBH-BD1 is above 90%. At relatively large bile salt concentrations of 0.5% and 1.0%, the survival rate of Clostridium butyricum JBH-BD1 is still above 85%, as shown in Figure 10 .

[0074] 4) Antibacterial effect test

[0075] Dip a medical cotton swab sterilized by high temperature and high pressure in 7 pathogenic bacterial strains, namely Escherichia coli of porcine origin, Salmonella typhimurium, Staphylococcus aureus, Clostridium perfringens type A, Clostridium perfringens type C, Pasteurella multocida of porcine origin, and Actinobacillus pleuropneumoniae, and evenly coat them on the RCM agar medium plate. Then, use a 1 mL pipette tip (outer diameter: 9 mm) to punch holes on the RCM agar medium plate, and seal the bottom with a flame for standby. Pipette 200 μL of the JBH-BD1 fermentation broth into the corresponding holes. Place the petri dish with the added sample in a refrigerator at 4 °C and let it stand for 2 h, then transfer it to an incubator at 37 °C. Among them, the Clostridium perfringens plate is anaerobically cultured. After 12 h, measure and record the diameter of the inhibition zone on the plate to judge the antibacterial effect of the sample to be tested. The method for obtaining the JBH-BD1 fermentation broth is as follows: Pick a single colony of Clostridium butyricum JBH-BD1 and inoculate it into RCM liquid medium, anaerobically culture it at 37 °C at 220 r / min for 16 h. Pipette an appropriate amount of the culture broth and transfer it to 100 mL of sterile RCM liquid medium at a ratio of 1%, and anaerobically culture it at 37 °C at 220 r / min for 16 h to obtain the JBH-BD1 fermentation broth.

[0076] The antibacterial effects of Clostridium butyricum JBH-BD1 strain against 7 different porcine pathogenic bacteria are shown in Figure 11 . The results show that the JBH-BD1 strain has significant inhibitory effects on the above 7 different pathogenic bacteria, and the order of inhibitory ability is: Clostridium perfringens type C > Clostridium perfringens type A > Actinobacillus pleuropneumoniae > Salmonella typhimurium > Pasteurella multocida of porcine origin > Escherichia coli > Staphylococcus aureus.

[0077] 5) Detection of iron enrichment rate

[0078] Pick a single colony of Clostridium butyricum JBH-BD1 and inoculate it into RCM liquid medium, anaerobically culture it at 37 °C at 220 r / min for 16 h. Pipette an appropriate amount of the culture broth and transfer it to 100 mL of sterile RCM liquid medium containing ferrous ions (calculated as iron, ferrous ion concentration is 600 mg / L) (formed by adding ferrous sulfate to the RCM liquid medium) at a ratio of 1%, and anaerobically culture it at 37 °C at 220 r / min for 72 h. Take the fermentation broth at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h respectively to detect the iron enrichment rate of the bacteria.

[0079] The operation for measuring the iron enrichment rate of the bacterial cells is as follows: Take 2 ml of the fermentation broth and centrifuge it at 5000 r / min for 15 min for standby. Discard the supernatant, wash it repeatedly 3 times with 0.85% normal saline, dry the obtained bacterial cell precipitate in an oven at 70 °C until a constant mass is reached and weigh it, accurate to 0.0001 g. Take 0.5000 g of the dry bacterial cells, add 5 mL of concentrated nitric acid, and simultaneously dropwise add 3 - 5 drops of hydrogen peroxide with a volume fraction of 30%, and perform digestion treatment at 80 °C. Dissolve the digestion residue with a 1.4 mol / L nitric acid solution, and then make up the volume to 5 mL with deionized water. Measure the iron content in the digestion solution with an atomic absorption spectrophotometer. The calculation formula is as follows:

[0080] Total amount of iron enriched in the bacterial cells (mg) = Iron content in the added digestion solution (mg / L) × Volume of the added digestion solution (L);

[0081] Amount of iron enriched (mg / g) = Total amount of iron enriched in the bacterial cells (mg) / Mass of the dry bacterial cells (g);

[0082] Iron enrichment rate = [Total amount of iron enriched in the bacterial cells (mg) / Total amount of iron in the culture broth (mg)] × 100%.

[0083] The detection results of the iron enrichment rate and the bacterial cell content of Clostridium butyricum JBH - BD1 are shown in Figure 12 . The results show that with the extension of the culture time, the bacterial cell yield and the iron enrichment rate of Clostridium butyricum JBH - BD1 show an upward trend. At 36 h of culture, its bacterial cell yield and iron enrichment rate can reach 3.20 g / L and 54.20% respectively, and the upward amplitude is weaker thereafter. And the iron enrichment rate is the highest at 60 h, about 58.3%. Considering comprehensively, the optimal culture time of Clostridium butyricum JBH - BD1 is 36 h.

[0084] 6) Detection of iron enrichment form

[0085] Take the dry bacterial cells obtained after treating the fermentation broth taken at different time points in the iron enrichment detection in 5), quickly grind them in a mortar with an appropriate amount of liquid nitrogen, add 10 mL of deionized water, mix well, and flow through D101 macroporous adsorption resin. Wash the resin with 1% dilute nitric acid and collect the washing solution, and measure its iron content with an atomic absorption spectrophotometer, which is the content of inorganic iron elements. Then the amount of organic iron = total amount of iron in the bacterial cells - amount of inorganic iron, and the units are both mg. Iron organic conversion rate = (amount of organic iron / total amount of iron in the bacterial cells) × 100%.

[0086] The detection results of the iron enrichment rate and the organic conversion rate of Clostridium butyricum JBH - BD1 are shown in Figure 13 . The results show that with the extension of the culture time, the iron organic conversion rate of Clostridium butyricum JBH - BD1 shows an upward trend. At 36 h of culture, the iron organic conversion rate reaches the highest, which is 11.45%, and then shows a slightly decreasing trend with the extension of time. Therefore, the iron organic conversion rate of Clostridium butyricum JBH - BD1 is the highest at 36 h of culture.

[0087] In summary, the Clostridium butyricum JBH-BD1 provided by the present invention can convert inorganic ferrous iron into organic ferrous iron. Compared with traditional antibiotic products, it is safe, non-toxic, and does not produce drug resistance. The Clostridium butyricum JBH-BD1 of the present invention can be widely applied to the aquaculture industry, improve the absorption and utilization efficiency of iron by livestock and poultry, while regulating the balance of intestinal flora, promoting the growth of livestock and poultry, and increasing the aquaculture benefits.

Claims

1. Clostridium butyricum ( Clostridium butyricum ), JBH-BD1, characterized in that: The preservation number is CCTCC NO: M2022151.

2. The application of Clostridium butyricum ( Clostridium butyricum ) JBH-BD1 in the preparation of a preparation for inhibiting Escherichia coli, Salmonella, Staphylococcus aureus, Clostridium perfringens, Pasteurella multocida of porcine origin or Actinobacillus pleuropneumoniae.

3. The application of Clostridium butyricum ( Clostridium butyricum ) JBH-BD1 in iron enrichment.

4. The application according to claim 3, characterized in that: It includes the following steps: Anaerobically culture the Clostridium butyricum ( Clostridium butyricum ) JBH-BD1 in a culture medium containing ferrous ions.

5. The application according to claim 4, characterized in that: The culture medium containing ferrous ions is obtained by adding ferrous salts to the reinforced Clostridium medium and mixing evenly; calculated as iron, the concentration of the ferrous ions is 200 - 600 mg / L.

6. The application according to claim 5, wherein: The ferrous salt is one or any combination of ferrous sulfate and ferrous chloride.

7. The application according to claim 4 or 5 or 6, characterized in that: The time for anaerobic culture is 24 - 36 h.

Images