Clostridium butyricum as well as culture method and application thereof

Abstract

The invention discloses clostridium butyricum as well as a culture method and application thereof, the classification number of the clostridium butyricum CB-a is named as Clostridium butyricum CB-a, the preservation number of the clostridium butyricum CB-a is CCTCC NO: M20242645, and the preservation date of the clostridium butyricum CB-a is December 4, 2024. The culture medium of the clostridium butyricum CB-a is simple in formula, mild in culture condition, easy to control and beneficial to saving of industrial large-scale production cost; when the clostridium butyricum CB-a is used as a pig feed additive, the diarrhea rate of piglets can be remarkably reduced while the feed conversion ratio is reduced, and the clostridium butyricum CB-a has relatively high application and popularization values.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to Clostridium butyricum and a culture method and application thereof. Background Art

[0002] Clostridium butyricum, also known as Clostridium butyricum, butyric acid bacteria, butyric acid bacteria, Clostridium butyricum, Clostridium butyricum, Miyairi bacteria, belongs to the genus Clostridium of Bacillaceae, is a Gram-positive anaerobic short rod, which can become a Gram-negative bacillus in the later stage of cultivation. It is widely present in the intestines and soil of humans and animals and has strong resistance to adverse environments. At present, Clostridium butyricum is mainly derived from animal intestines, feces, soil and wine cellars. As a probiotic, it is widely used in medical treatment, food additives, feed additives and other aspects.

[0003] Clostridium butyricum can maintain the biological intestinal barrier by promoting intestinal morphology and development, regulating the expression of occludens, and promoting biological growth by improving intestinal enzyme activity and metabolic capacity and regulating intestinal flora homeostasis. The metabolite of Clostridium butyricum is mainly butyric acid, which can regulate intestinal pH. At the same time, it can increase beneficial flora and reduce harmful flora to correct intestinal flora disorder, reduce enterotoxins, and thus reduce the inflammatory response of intestinal cells. Enterotoxigenic Escherichia coli can cause diarrhea and damage intestinal cells in animals, and in severe cases can lead to death. Clostridium butyricum can reduce the expression of adhesion genes and enterotoxin genes of enterotoxigenic Escherichia coli, mediate the mTOR signaling pathway to inhibit the inflammatory response of pig intestinal epithelial cells induced by enterotoxigenic Escherichia coli, and increase the expression of tight junction proteins, thereby reducing the damage of enterotoxigenic Escherichia coli to pig intestinal epithelial cells. Heat-killed Clostridium butyricum upregulated the expression of FcRn through the TLR2 / 4-MyD88 / TRIF-NF-κB signaling pathway, playing a role in reducing enteritis. Clostridium butyricum also has the function of synthesizing vitamins (especially biotin) and amylase, and has strong resistance to macrolide and polypeptide antibiotics, and has a health-care effect in the animal intestine.

[0004] Feeding poultry with Clostridium butyricum as a feed additive can, on the one hand, increase the content of short-chain fatty acids by enhancing the diversity of intestinal flora, and at the same time promote the development of the small intestine, enhance the antioxidant capacity of the intestinal mucosa, improve the intestinal morphology, and increase the absorption rate of nutrients by poultry, thereby improving their growth performance. On the other hand, Clostridium butyricum can enhance the intestinal barrier and immune function of poultry, thereby maintaining their health. In addition, Clostridium butyricum can also increase the average daily egg weight of poultry and reduce the egg breakage rate, thereby increasing its economic benefits.

[0005] Adding an appropriate amount of Clostridium butyricum to feed can accelerate the changes in the composition and concentration of fecal microbiota and the determination of potential probiotics, regulate animal metabolism, and improve the growth performance of intestinal mucosa. Since the performance of different strains of Clostridium butyricum varies greatly, it is of great significance to screen and obtain a strain of Clostridium butyricum with excellent comprehensive performance. Summary of the invention

[0006] The purpose of the present invention is to provide a Clostridium butyricum CB-a and a culture method and application thereof. The culture method of the present invention has a fast growth rate and simple nutritional requirements for bacterial growth. Experiments have verified that the strain can be used as a pig feed additive to reduce the feed-to-meat ratio and significantly reduce the diarrhea rate of piglets. It has high application and promotion value and lays a foundation for large-scale promotion and application.

[0007] To achieve the above object, the present invention is implemented by the following technical solutions:

[0008] The butyricum clostridium of the present invention is named as Clostridium butyricum CB-a, with a preservation number of CCTCCNO: M20242645 and a preservation date of November 27, 2024.

[0009] The screening method of Clostridium butyricum of the present invention is as follows:

[0010] (1) Culture medium configuration

[0011] Clostridium proliferation medium (RCM medium): yeast extract powder 3g, beef extract 10g, tryptone 10g, glucose 5g, soluble starch 1g, sodium chloride 5g, sodium acetate trihydrate 3g, cysteine ​​hydrochloride 0.5g, 0.5% methylene blue 0.2mL, distilled water 1000mL, sterilize at 115℃ for 20min.

[0012] Selection medium (TSN medium): 15 g tryptone, 10 g yeast extract powder, 1 g sodium sulfite, 0.5 g ferric citrate, 0.05 g polymyxin, 0.02 g novobiocin, 1000 mL distilled water, sterilized at 115°C for 20 min.

[0013] (1) Screening of Clostridium butyricum

[0014] Take 1.0g chicken manure sample and dissolve it in 100mL sterile water, culture it at 37℃ for 1h at a speed of 180r / min, kill non-spore-forming bacteria in a water bath at 80℃ for 10min, and then inoculate it into RCM medium for anaerobic culture at 37℃ for 48h. Take 200μL of RCM medium with a large amount of gas produced and incubate it in 80℃ water bath for 10min to kill non-spore-forming bacteria, and then inoculate it into TSN medium for anaerobic culture at 37℃ for 48h. Take TSN medium with bubbles produced and dilute it 10 times to 10 -4, 10 -5 , 10 -6 , and then inoculated into Clostridium count medium, and cultured anaerobically at 37℃ for 24h. Pick the black single colony in Clostridium count medium and culture it in RCM medium for expansion, culture it anaerobically at 37℃ for 24h, and select the strains that produce a lot of gas for strain preservation and Gram staining.

[0015] The experiment isolated 10 target strains by enrichment culture and selection culture, respectively, see the 10 strain numbers in Table 1 below, among which strain c and strain S are facultative anaerobes, which do not meet the culture characteristics of Clostridium butyricum, and the specific culture characteristics are shown in Table 1. The Gram staining results showed that strain b was a Gram-negative bacterium, while the remaining strains were all Gram-positive bacteria. The strain of Clostridium butyricum CB-a (a) of the present invention was rod-shaped, the bacterial body was spindle-shaped, single or oppositely arranged, and the spores were mostly subterminal or separated from the vegetative body.

[0016] Table 1

[0017]

[0018] Note: “+” means positive test result, “-” means negative test result.

[0019] The selected strains were subjected to molecular biological identification. According to Gram staining, physiological and biochemical characteristics and 16SrDNA sequence comparison analysis, the seven strains were determined to be Clostridium butyricum, and the strains were named CB-1, CB-2, CB-3, CB-4, CB-5, CB-a, and CB-H, respectively. The sequencing results of Clostridium butyricum CB-a of the present invention are shown in the attached sequence table.

[0020] The present invention also studies the optimum culture temperature of the above-mentioned strains, see the attached Figure 1As can be seen from the figure, there is no significant difference in the growth of CB-1, CB-2 and CB-4 when cultured at 34℃ and 37℃, and there is a significant difference in the growth of strains cultured at 37℃ and 40℃, indicating that the optimal culture temperature of these three strains is between 34 and 40℃; there is no significant difference in the growth of CB-3 strains when cultured at 34℃ and 37℃, there is a significant difference in the growth of strains cultured at 37℃ and 40℃, and there is no significant difference in the growth of strains cultured at 34℃ and 40℃, indicating that the strain The optimum culture temperature of the strain is 37℃; there is no significant difference in the culture of CB-5 strain at 34℃, 37℃, 40℃ and 43℃, and the optimum culture temperature of the strain is 37℃; there are significant differences in the culture of CB-a strain at 37℃, 34℃ and 40℃, indicating that the optimum culture temperature of the strain is 37℃; there is no significant difference in the culture of CB-H strain at 34℃, 37℃ and 40℃, and there is a significant difference in the culture of 40℃ and 43℃, indicating that the optimum culture temperature of the strain is between 34 and 43℃. In summary, the suitable culture temperature of several strains is between 34 and 40℃, and the optimum culture temperature is 37℃.

[0021] The present invention also provides a method for culturing Clostridium butyricum CB-a. The method comprises the following raw materials, calculated by weight percentage: 3% glucose, 2.5% soy peptone, 1.5% yeast extract powder, 2% peptone, 0.5% calcium carbonate, 0.05% potassium dihydrogen phosphate, 0.5% sodium chloride, 0.1% magnesium sulfate heptahydrate, and 0.02% manganese sulfate. The initial pH is 6, 3% Clostridium butyricum CB-a is inoculated by volume percentage, and the culture is performed at 34°C for 24 hours.

[0022] The number of viable bacteria in the culture medium of the present invention reaches 4.56×10 9 CFU / mL.

[0023] The present invention also provides an application of Clostridium butyricum CB-a.

[0024] The Clostridium butyricum CB-a of the present invention is screened from chicken manure. Experiments have verified that the strain can be used as a pig feed additive to reduce the feed-to-meat ratio and significantly reduce the diarrhea rate of piglets, and has high application and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the most suitable culture temperature diagram of CB-1, CB-2, CB-3, CB-4, CB-5, CB-a, and CB-H, where A represents strain CB-1; B represents strain CB-2; C represents strain CB-3; D represents strain CB-4; E represents strain CB-5; F represents strain CB-a; and G represents strain CB-H (p<0.05);

[0026] Figure 2 It is the content diagram of the main metabolites of Clostridium butyricum;

[0027] Figure 3 This is the antibacterial experiment of Example 5 of the present invention, wherein Ⅰ is Weizmannella coagulans; Ⅱ is Bacillus subtilis; Ⅲ is Escherichia coli; Ⅳ is Staphylococcus aureus; ACB-1; B CB-2; C CB-3; D CB-4; E CB-5; F CB-a; G CB-H;

[0028] Figure 4 It is the alcohol tolerance of Example 6 of the present invention, A strain CB-1; B strain CB-2; C strain CB-3; D strain CB-4; E strain CB-5; F strain CB-a; G strain CB-H (p<0.05);

[0029] Figure 5 A is the effect of carbon source on the biomass of Clostridium butyricum CB-a; B is the effect of glucose addition on the biomass of Clostridium butyricum CB-a;

[0030] Figure 6 is the effect of different nitrogen sources on the biomass of Clostridium butyricum;

[0031] Figure 7 is the effect of different nitrogen source additions on the biomass of Clostridium butyricum;

[0032] Figure 8 The effect of different culture conditions on the biomass of Clostridium butyricum. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0034] Example 1

[0035] A butyricum Clostridium in this example has a classification number named Clostridium butyricum CB-a, a preservation number of CCTCC NO: M20242645, and a preservation date of December 4, 2024.

[0036] Example 2 Determination of butyric acid content

[0037] The 7 strains of Clostridium butyricum obtained by screening, CB-1, CB-2, CB-3, CB-4, CB-5, CB-a, CB-H, and Examples 2-6 were respectively studied for the content of metabolite butyrate, artificial stomach and intestinal fluid, bile salts, alcohol resistance, and high temperature resistance.

[0038] The determination of the butyric acid content of the metabolite of Clostridium butyricum in this embodiment includes the following steps:

[0039] (1) Initial fermentation medium: 10 g yeast extract, 20 g glucose, 25 g peptone, 5 g calcium carbonate, 0.5 g potassium dihydrogen phosphate, 5 g sodium chloride, 1 g magnesium sulfate heptahydrate, 0.2 g manganese sulfate, and 1000 mL distilled water, sterilize at 115°C for 20 min;

[0040] (2) Clostridium butyricum CB-1, CB-2, CB-3, CB-4, CB-5, CB-a, and CB-H strains were inoculated at a 2% (v / v) initiation rate into an initial fermentation medium, cultured at 37° C. for 24 h, and the fermentation broth was centrifuged at 4° C. at 12,000 rpm for 10 min. The obtained supernatant was mixed with formic acid at a ratio of 100:1 for acidification, and filtered through a 0.22 μm membrane to obtain the strain fermentation supernatant.

[0041] (3) The butyric acid content was determined using a gas chromatograph equipped with an automatic injector and a flame ionization detector, and a DB-WAX 250 fused silica capillary column (30 m×0.32 mm, 0.25 μm). The program settings were: initial temperature 70°C for 1 min, heating to 135°C at 3°C / min, injection port temperature 250°C, front FID injection port temperature 290°C, split ratio 10:1, injection volume 5 μL. The peak time and peak area of ​​the mixed standard under the above gas chromatography conditions were used to quantitatively analyze the fermentation supernatant of the strain.

[0042] The results of gas chromatography are shown in Table 1 below. The main metabolites of several strains are butyric acid, followed by acetic acid. Among them, no butanol is produced in the metabolites of several strains of Clostridium butyricum. No propionic acid and isobutyric acid were detected in the fermentation broth of CB-1, CB-a and CB-H strains. A small amount of propionic acid and isobutyric acid were detected in the fermentation broth of CB-2, CB-3, CB-4 and CB-5 strains. A small amount of ethanol was detected in the metabolites of several strains.

[0043] The content of Clostridium butyricum metabolites Figure 2 As shown, in the fermentation broth, the acetic acid content of the CB-2 strain was significantly different from that of the other strains, and there was no significant difference between the CB-1, CB-3, CB-4, CB-5, CB-a, and CB-H strains; the butyric acid content of the CB-3 strain was significantly different from that of the CB-2 and CB-5 strains, and there was no significant difference between the CB-1, CB-4, CB-a, and CB-H strains, and there was no significant difference between the CB-1, CB-2, CB-4, CB-5, CB-a, and CB-H strains. Among these Clostridium butyricum strains, the CB-2 strain and the CB-5 strain had higher butyric acid content, and the CB-2 strain had the highest acetic acid content.

[0044] Table 1 Determination of the content of Clostridium butyricum metabolites (mg / L)

[0045]

[0046]

[0047] Example 3 Analysis of tolerance to artificial gastric juice and artificial intestinal juice

[0048] 1 mL of live bacteria of CB-1, CB-2, CB-3, CB-4, CB-5, CB-a, and CB-H were placed in 9 mL of artificial gastric fluid, mixed and treated at 37°C for 2 h, 3 h, and 5 h respectively. The treated solution was diluted 10 times with sterile saline and then diluted to 10 -4 ~10 -6 Take 200 μL of each of the three gradients, add to the RCM solid medium that has been dissolved in a test tube and cooled to about 50°C, mix well and incubate at 37°C for 24 hours, observe and count, and take untreated Clostridium butyricum for 10 -4 ~10 -6 Three gradients were used as controls to calculate their survival rates.

[0049] The results are shown in Table 2. When treated with artificial gastric juice for 2 hours, the CB-a strain had the best tolerance, followed by CB-1, CB-2, CB-3, CB-4, and CB-H. The CB-5 strain had the worst tolerance. As the time of artificial gastric juice treatment increased, the survival rate of the strains decreased. The survival rate of the CB-a strain decreased significantly when treated for 3 hours. When treated with artificial gastric juice for 5 hours, except for the CB-a strain, the survival rates of the other strains were less than 5%. In summary, the strain with the best tolerance to artificial gastric juice was the CB-a strain, and the CB-5 strain had the worst tolerance.

[0050] Table 2 Artificial gastric juice tolerance (survival rate %)

[0051]

[0052] Take 1 mL of live bacteria from CB-1, CB-2, CB-3, CB-4, CB-5, CB-a, and CB-H, add 9 mL of 0.2%, 0.3%, 0.4%, and 0.5% bile salt solution, mix well, and treat at 37°C for 30 min, 1.5 h, and 2.5 h, respectively. After treatment, dilute the solution 10-fold with sterile saline and -4 ~10 -6 Take 200 μL of each of the three gradients, add to the RCM solid medium that has been dissolved in a test tube and cooled to about 50°C, mix well and incubate at 37°C for 24 hours, observe and count, and take Clostridium butyricum that has not been treated with bile salts for 10 hours. -4 ~10 -6 Three gradients were used as controls.

[0053] The results of artificial intestinal fluid tolerance are shown in Table 3. The survival rates of several strains decreased with the increase of artificial intestinal fluid treatment time. Among them, the CB-4 strain had the worst artificial intestinal fluid tolerance, with a survival rate of only 17.39% after 0.5h of artificial intestinal fluid treatment. The survival rates of CB-2 and CB-3 strains decreased significantly after 1h of artificial intestinal fluid treatment, and the survival rates of CB-1, CB-5, CB-a, and CB-H strains decreased significantly after 1.5h of artificial intestinal fluid treatment. Overall, the strains with better artificial intestinal fluid tolerance are CB-5, CB-1, CB-a, and CB-H.

[0054] Table 3 Artificial intestinal fluid tolerance (survival rate %)

[0055]

[0056] Example 4 Bile Salt Tolerance Analysis

[0057] Take 1 mL of live bacteria from CB-1, CB-2, CB-3, CB-4, CB-5, CB-a, and CB-H, add 9 mL of 0.2%, 0.3%, 0.4%, and 0.5% bile salt solution, mix well, and treat at 37°C for 30 min, 1.5 h, and 2.5 h, respectively. After treatment, dilute the solution 10-fold with sterile saline and -4 ~10 -6 Take 200 μL of each of the three gradients, add to the RCM solid medium that has been dissolved in a test tube and cooled to about 50°C, mix well and incubate at 37°C for 24 hours, observe and count, and take Clostridium butyricum that has not been treated with bile salts for 10 hours. -4 ~10 -6 Three gradients were used as controls.

[0058] The results are shown in Table 4. The survival rates of several strains decreased with the increase of time. When the bile salt concentration was 0.5% for 2.5 hours, the survival rates of CB-1, CB-3, CB-4 and CB-5 were all less than 30%, among which the survival rates of CB-3 and CB-4 were less than 20%; when the bile salt concentration was 0.2% for 2.5 hours, the survival rates of CB-1 and CB-H were less than 40%. In general, the bile salt tolerance of strains CB-2 and CB-H was stronger, followed by strain CB-a.

[0059] Table 4 Bile salt tolerance

[0060]

[0061] Example 5 Antibacterial Analysis

[0062] Cultivation of indicator bacteria: Escherichia coli, Staphylococcus aureus, and Bacillus subtilis were cultured in LB medium, and Bacillus coagulans was cultured in MRS medium for 24 hours.

[0063] Indicator bacteria coating plate: Pour 15-20 mL of MRS culture medium into each culture dish. After solidification, take 200 μL of indicator bacteria cultured for 24 hours and dilute 10 -2 Post-coating.

[0064] Oxford cup antibacterial operation: Place the Oxford cup in the culture dish, use sterile distilled water as the negative control, add 200μL of Clostridium butyricum solution into the Oxford cup. The culture dish is placed in a constant temperature box at 37℃ for incubation. Except for the indicator bacteria Bacillus coagulans, which is cultured for 48h, other indicator bacteria are cultured for 24h.

[0065] The criteria for determining the antibacterial effect are: “Insensitive”: DIZ=7.8mm; “Low sensitive”: 7.8mm<DIZ≤10.0nm; “Medium sensitive”: 10.0nm<DIZ≤12.0mm; “Highly sensitive”: DIZ greater than 12.0nm.

[0066] The results are shown in Table 5 and Figure 3 As shown in the figure, several strains were moderately sensitive to Weizmannella coagulans and Bacillus subtilis, and there was no significant difference; several strains were highly sensitive to Escherichia coli, among which CB-1, CB-2, CB-5, CB-a, and CB-H had no significant difference in inhibition, and CB-3 had the worst inhibition on Escherichia coli; CB-a and CB-2 were highly sensitive to Staphylococcus aureus, and CB-1, CB-3, CB-4, and CB-5 were moderately sensitive to Staphylococcus aureus. Overall, CB-a had the best antibacterial activity.

[0067] Table 5 Antibacterial experiment

[0068]

[0069] Note: p<0.05, "Insensitive": DIZ=7.8mm; "Low sensitive": 7.8mm<DIZ≤10.0mm; "Medium sensitive": 10.0mm<DIZ≤12.0mm; "Highly sensitive": DIZ greater than 12.0mm.

[0070] Example 6 Alcohol tolerance analysis

[0071] The ethanol concentration in the liquid fermentation medium was adjusted to 3%, 5%, 7%, 9%, 11%, 13% and 15% respectively with anhydrous ethanol. The CB-1, CB-2, CB-3, CB-4, CB-5, CB-a and CB-H butyric Clostridium butyricum strains activated with RCM liquid medium were inoculated into the medium at a 2% inoculation rate. The pH was adjusted to 7.0 and the cultured cells were cultured in a constant temperature incubator at 37°C for 24 hours. The absorbance at a wavelength of 600 nm was measured.

[0072] Alcohol tolerance results such as Figure 4 As shown in Table 6, different strains have great differences in tolerance to alcohol. When the alcohol concentration is 5%, the survival rate of the CB-a strain is still 92.07%. When the alcohol concentration is higher than 9%, the survival rates of the seven strains are significantly reduced. In summary, the data show that the CB-a strain has a stronger alcohol tolerance.

[0073] Table 6 Alcohol tolerance (%)

[0074]

[0075] Example 7 High temperature tolerance analysis

[0076] 2 mL of CB-1, CB-2, CB-3, CB-4, CB-5, CB-a, and CB-H Clostridium butyricum strains activated by RCM liquid medium were taken into 5 mL centrifuge tubes, respectively, and treated in 80 °C water bath for 30 min, with 37 °C as the control. After treatment, the solution was diluted 10 times with sterile saline and then diluted in 10 -4 ~10 -6 (control group) and 10 -3 ~10 -5 (Treatment group) Take 0.2 mL of each of the three gradients, add into test tube and dissolve in RCM solid medium cooled to about 50°C, mix well and culture at 37°C for 24 h, observe and count, and calculate the survival rate.

[0077] The high temperature tolerance of the seven strains was determined by the viable bacteria counting method. The results are shown in Table 7. After being treated in a water bath at 80°C for 30 min, the strain with the best tolerance was CB-3, followed by CB-5. The tolerance of CB-4, CB-a, and CB-H strains was similar, while CB-2 and CB-1 had the worst tolerance.

[0078] Table 7 High temperature tolerance

[0079]

[0080] Example 8

[0081] In order to further explore the application potential of Clostridium butyricum CB-a strain, this example also optimizes the culture medium carbon source, nitrogen source and addition amount, as well as the culture conditions such as culture temperature and initial pH for Clostridium butyricum CB-a strain.

[0082] (1) Carbon source optimization

[0083] Sucrose, soluble starch, glucose, glycerol, lactose, maltose, fructose, xylan, and chitosan were selected as carbon sources in the initial fermentation medium (see Example 2), and the initial carbon source addition amount was 2%. Other components were kept constant, and the pH was adjusted to 7. The culture medium was placed in a 250 mL conical flask, covered with 1 cm of liquid paraffin, and inoculated with 2% (v / v) culture solution of Clostridium butyricum CB-a, with 3 replicates per group. Cultured at 37°C for 24 h, and the OD was measured. 600 , determine the optimal carbon source.

[0084] The experimental results are as follows Figure 5 As shown, the biomass of Clostridium butyricum was the largest when glucose was used as the single carbon source in the culture medium, and there was a significant difference with other carbon sources (p<0.05); there was no significant difference when fructose and starch were used as the single carbon source, there was a significant difference between sucrose and chitosan, there was no significant difference when maltose, lactose, glycerol and xylitol were used as the single carbon source, and there was no significant difference with the carbon source and chitosan. In summary, glucose was selected as the single carbon source for optimization of addition amount.

[0085] (2) Optimization of carbon source addition

[0086] After determining the best carbon source, its addition amount was set to 1%, 2%, 3%, 4%, and 5%, respectively, and other components remained unchanged, and the inoculation amount and culture conditions were the same as above.

[0087] During the growth of Clostridium butyricum, adding too little carbon source will result in limited growth of Clostridium butyricum due to insufficient carbon source, while adding too much carbon source will cause the osmotic pressure to be too high, affecting the growth of the strain. Figure 5 As shown in B, the optimal addition amount of glucose is 3%.

[0088] (3) Nitrogen source optimization

[0089] Tryptone, yeast extract, beef extract, peptone, soy peptone, urea, ammonium sulfate, ammonium nitrate, and sodium nitrate were selected as nitrogen sources, and the initial nitrogen source addition amount was 2%. The carbon source was the optimal carbon source and addition amount measured above. Other components remained unchanged, and the inoculation amount and culture conditions were the same as above.

[0090] When glucose was used as the sole carbon source and the addition amount was 3%, nitrogen source optimization was carried out, among which ammonium nitrate, sodium nitrate, ammonium sulfate, and urea were used as inorganic nitrogen sources, and beef extract, yeast extract powder, tryptone, soy peptone, and peptone were used as organic nitrogen sources to cultivate Clostridium butyricum. The results are as follows Figure 6 As shown in the figure, when soybean peptone was used as a single nitrogen source, the growth of Clostridium butyricum was the largest, followed by yeast extract powder and peptone. Therefore, soybean peptone, yeast extract powder and peptone were selected as composite nitrogen sources to optimize the addition amount.

[0091] (4) Optimization of nitrogen source addition

[0092] According to the experimental results of nitrogen source optimization, three organic nitrogen sources (soybean peptone, peptone, and yeast extract powder) with the most significant effect on the growth of CB-a strain were selected, and the addition amounts were set to 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, and 4.0%, respectively. The other components remained unchanged, and the inoculation amount and culture conditions were the same as above.

[0093] According to the experimental results, a three-factor three-level orthogonal experiment was designed to configure different culture media, while other components remained unchanged, and the inoculation amount and culture conditions were the same as above.

[0094] The results are as follows Figure 7 As shown, there was no significant difference (p<0.05) when the addition amount of soy peptone was 2%, 2.5%, 3%, 3.5%, and 4%, but there was a significant difference with 1% and 1.5%, and the optimal addition amount of soy peptone was 2%; when the addition amount of yeast extract powder was 2.5%, there was a significant difference with the addition amount of 2% and 3%, and the optimal addition amount was 2.5%. When the addition amount of peptone was 3%, there was a significant difference with 2.5%, but there was no significant difference with 3.5%, and the optimal addition amount was 3%. Based on this result, an orthogonal experiment was designed (see Table 8) to determine the best nitrogen source combination.

[0095] From Table 8, we can see that the order of extreme difference is R B >R A >R C , as shown in Table 9, the significance of variance test is B>A>C. It can be seen that yeast extract powder has a greater effect on strain growth than soy peptone, and soy peptone has a greater effect on strain growth than peptone. Since the variance P of each factor is greater than 0.05, it shows that the three factors at this level have no significant effect on the results (p<0.05). Therefore, the theoretical optimal combination of orthogonal experiment is determined to be A3B2C2, that is, the best organic nitrogen source combination is 2.5% soy peptone, 1.5% yeast extract powder, and 2% peptone.

[0096] The results of viable bacteria count showed that after the culture medium was optimized, the number of viable bacteria reached 3.53×10 9 CFU / mL.

[0097] Table 8 Results of orthogonal test for nitrogen source optimization

[0098]

[0099] Table 9 Analysis of variance for orthogonal experiment of nitrogen source optimization

[0100]

[0101]

[0102] (5) Optimization of temperature, pH, and inoculum size

[0103] According to the above test results, single factor optimization of culture temperature, initial pH and inoculation amount was performed, where culture temperature was set to 34°C, 37°C, 40°C, 43°C, and 46°C; initial pH was set to 4, 5, 6, 7, and 8; inoculation amount was set to 1%, 2%, 3%, 4%, and 5%. According to the test results, a three-factor three-level orthogonal experiment was designed, see Table 10.

[0104] Table 10 Orthogonal experimental design of culture conditions

[0105]

[0106] like Figure 8 As shown in the figure, when the initial pH is 4, the strain growth is significantly different from the other initial pHs, and the biomass is the largest when the initial pH is 6; when the inoculation amount is 1% to 5%, there is no significant difference in biomass, and the biomass is the largest when the inoculation amount is 3%; when the culture temperature is 34℃, there is a significant difference with 37℃, and there is no significant difference when the culture temperature is 40℃, 43℃ and 46℃. Based on this result, an orthogonal experiment was designed, see Table 11, to determine the optimal culture conditions.

[0107] From Table 11, we can see that the order of extreme difference is R C >R A >R B , as shown in Table 12, the significance of the variance test is C>A>B. It can be seen that the influence of culture temperature on strain growth is greater than that of initial pH, and the influence of initial pH on strain growth is greater than that of inoculation amount. The variance P of culture temperature is less than 0.05, which proves that culture temperature has a significant effect on the result (p<0.05). The variance P of initial pH and inoculation amount are both greater than 0.05, indicating that the initial pH and inoculation amount have no significant effect on the result at this level (p<0.05). It is thus determined that the theoretical optimal combination of the orthogonal test is A1B2C1, that is, the optimal culture conditions are initial pH of 6, inoculation amount of 3%, and culture temperature of 34°C.

[0108] Table 11 Orthogonal test results of culture conditions

[0109]

[0110] Table 12 Analysis of variance of orthogonal test of culture conditions

[0111]

[0112] (6) Optimized culture of Clostridium butyricum

[0113] Cultivate Clostridium butyricum for 24 hours using the optimized culture medium or culture conditions, take the culture solution and dilute it 10 times in a gradient manner, inoculate it into Clostridium counting medium, and count the live bacteria.

[0114] The results of live bacteria count under the optimized conditions showed that the number of live bacteria reached 4.56×10 9 CFU / mL. Therefore, the optimal culture of Clostridium butyricum CB-a of the present invention is obtained by experiment. According to the weight percentage, it includes the following raw materials: 3% glucose, 2.5% soy peptone, 1.5% yeast extract powder, 2% peptone, 0.5% calcium carbonate, 0.05% potassium dihydrogen phosphate, 0.5% sodium chloride, 0.1% magnesium sulfate heptahydrate, 0.02% manganese sulfate, the initial pH is 6, 3% of Clostridium butyricum CB-a is inoculated by volume percentage, and cultured at 34°C for 24 hours. The number of viable bacteria in the culture medium described in this embodiment reaches 4.56×10 9 CFU / mL.

[0115] Example 9

[0116] In the present embodiment, Clostridium butyricum CB-a is used as a pig feed additive. The liquid fermentation liquid of Clostridium butyricum CB-a is dried and prepared into a powder as a pig feed additive, wherein the number of live bacteria is uniformly adjusted to 1×10 8 CFU / g.

[0117] Animals and experimental design. The experimental site was located in Huangshi City, Hubei Province. A total of 120 weaned piglets (Du × Chang × Da; weaning age 25 ± 2d; average weight 6.81 ± 0.53kg) were selected for the experiment. The piglets were divided into 12 pens according to weight, sex, etc. to ensure uniformity. After 3 days of pre-feeding, they were divided into 4 treatments according to a completely randomized block design: control group (CK; fed as usual, without any probiotics added to the diet), strain CB-a group, strain CB-H group and strain CB-1 group. In the three strain groups, 0.5×10 8 CFU of Clostridium butyricum. Probiotic group, each treatment was tested with 3 replicates. The experiment was divided into 2 phases (Phase I: 1-14 days; Phase II: 15-18 days).

[0118] Feeding management and sample collection. Wash and disinfect the pig house thoroughly before the piglets are transferred to the pen. Clean the pig house once a day during feeding and disinfect it regularly (3 times a week). During the experiment, observe at any time to ensure that the pigs have sufficient feeding and drinking water and can eat freely. Record the feed intake every day in pens. Weigh the piglets at the beginning and end of each stage, and calculate the daily weight gain, daily feed intake and feed-to-meat ratio. Observe the growth of the piglets in a timely manner every day, score the feces, and record the diarrhea. The growth performance is shown in Tables 13-16 below.

[0119] Table 13 Growth of weaned piglets in stage I (1-14 days)

[0120]

[0121] Table 14 Growth of weaned piglets in Phase II (15-18 days)

[0122]

[0123] Table 15 Growth of weaned piglets during the entire period (1-28 days)

[0124]

[0125] Table 16 Diarrhea rate of weaned piglets at different stages

[0126]

[0127] As can be seen from Tables 13-16 above, there was no significant difference in daily feed consumption in each stage (P>0.05), and there were significant differences in daily weight gain (P<0.001), while the feed-to-meat ratio was significantly different in stage II and the entire stage (P<0.05). In stage I, the daily weight gain of strain CB-a was significantly higher than that of the control group, CB-H group and strain CB-1 group. In stage II, the daily weight gain of strain CB-a group was significantly higher than that of the control group, CB-H group and strain CB-1 group. Combining the two stages, adding strain CB-a to the diet has the best effect. In addition, adding strain CB-a to the diet can significantly reduce the diarrhea rate of piglets, see Table 16.

[0128] It can be seen that the Clostridium butyricum CB-a of the present invention, as a pig feed additive, can reduce the feed-to-meat ratio and significantly reduce the diarrhea rate of piglets, and has good application and promotion value.

Claims

1. A Clostridium butyricum, characterized in that: Its classification number is named Clostridium butyricum CB-a, the deposit number is CCTCC NO: M20242645, and the deposit date is December 4, 2024.

2. The method for culturing Clostridium butyricum according to claim 1, wherein: Calculated by weight percentage, the following raw materials are included: 3% glucose, 2.5% soy peptone, 1.5% yeast extract powder, 2% peptone, 0.5% calcium carbonate, 0.05% potassium dihydrogen phosphate, 0.5% sodium chloride, 0.1% magnesium sulfate heptahydrate, 0.02% manganese sulfate, the initial pH is 6, 3% Clostridium butyricum CB-a is inoculated by volume percentage, and the culture is carried out at 34°C for 24 hours.

3. The method for culturing Clostridium butyricum according to claim 2, characterized in that: The number of viable bacteria of Clostridium butyricum CB-a in the culture medium reached 4.56×10 9 CFU / mL.

4. Use of Clostridium butyricum as claimed in claim 1.

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