Clostridium butyricum, fermentation inoculant, and preparation method and application of clostridium butyricum and fermentation inoculant

Abstract

The invention relates to clostridium butyricum, a fermentation inoculant and a preparation method and application of the clostridium butyricum, the clostridium butyricum is named as clostridium butyricum CB03, the clostridium butyricum is preserved in the China Center for Type Culture Collection, the preservation date is April 17, 2025, and the preservation number is CCTCC NO: M 2025813. According to the preparation method disclosed by the invention, the probiotics with efficient bacteriostasis and broad spectrum are obtained by optimizing strain fermentation conditions; the process for improving the stability of the clostridium butyricum is obtained by researching the storage stability of the strain; by applying the microecological preparation to aquaculture animals, the high-quality microecological preparation capable of promoting growth, improving immunity and improving intestinal flora is obtained. The clostridium butyricum microecological preparation provided by the invention is simple in treatment mode operation and low in cost, and can be well applied to aquaculture.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a Clostridium butyricum, a fermentation bacterium agent, and a preparation method and application thereof. Background Art

[0002] With the high-density and intensive development of aquaculture, the problem of aquaculture diseases has become increasingly serious and has become one of the main factors restricting the healthy development of the aquaculture industry. Currently, the prevention and control of aquaculture animal diseases mainly use antibiotics and chemical drugs. Excessive use often leads to drug residues and environmental pollution, and at the same time causes the enhancement of pathogen drug resistance, thus threatening food safety and human health. Probiotics and prebiotics and other microecological agents have become the current hotspots and have gradually become the main force in reducing and replacing antibiotics.

[0003] Currently, Bacillus, Lactobacillus, and Saccharomyces cerevisiae have become the main economic markets for microecological agents. However, in a complex aquatic environment, the survival ability of these microorganisms is poor and their competitiveness is weak. For example, water quality parameters, competition from other microorganisms, and the immune status of the host itself may all affect the effects of these beneficial microorganisms. Even if these microorganisms can survive and reproduce in water, it is difficult to maintain their activity for a long time in the aquaculture system and continuously provide protection for fish, and they need to be frequently added to maintain a sufficient concentration to exert their effects. Summary of the Invention

[0004] Based on the above description, the present invention provides a Clostridium butyricum, a fermentation bacterium agent, and a preparation method and application thereof to solve the problem of how to improve the antibacterial effect and stability of the bacterium agent.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The present invention provides a Clostridium butyricum named Clostridium butyricum CB03, which has been deposited in the China Center for Type Culture Collection. The deposit date is April 17, 2025, and the deposit number is CCTCC NO: M 2025813. The deposit address is Wuhan University, China.

[0007] The present invention also proposes an application of Clostridium butyricum in preparing a feed additive, and the feed additive is used to antagonize pathogenic bacteria of aquatic animals.

[0008] The present invention also proposes a fermentation bacterium agent, and the fermentation bacterium agent is a fermentation product of Clostridium butyricum, and the fermentation product of Clostridium butyricum is fermented from the aforementioned Clostridium butyricum.

[0009] Furthermore, the fermentation bacterium agent further includes a Clostridium butyricum protectant, and the Clostridium butyricum protectant includes xanthan gum, skim milk, and lecithin.

[0010] Furthermore, in the fermentation inoculum, the mass ratio of xanthan gum is 0.1% - 0.2%, the mass ratio of skim milk is 0.1% - 0.5%, and the mass ratio of lecithin is 0.1% - 0.5%.

[0011] The present invention also provides a preparation method of the fermentation inoculum as described above, comprising the following steps:

[0012] S1. Seed liquid fermentation: Inoculate Clostridium butyricum CB03 onto a specific culture medium, pick monoclonal colonies into a liquid culture medium for anaerobic fermentation culture to obtain a fermentation seed liquid;

[0013] S2. Small fermenter fermentation: Inoculate the fermentation seed liquid into a fermenter filled with a primary fermentation medium at a volume ratio of 1% - 3% for the first expansion culture, and perform anaerobic fermentation to obtain an expanded culture solution;

[0014] S3. Large fermenter fermentation: Inoculate the expanded culture solution into a fermenter filled with a secondary fermentation medium at a volume ratio of 3% - 10% for the second expansion culture, and perform anaerobic fermentation to obtain a fermentation inoculum.

[0015] Furthermore, in step S1, the fermentation temperature is 30°C - 37°C, the fermentation time is 24h - 48h, ferment on a shaker, and the shaker speed is 200rpm - 220rpm; and / or,

[0016] In step S2, the fermentation temperature is 30°C - 37°C, the fermentation time is 24h - 48h, ferment on a shaker, and the shaker speed is 100rpm - 150rpm; and / or,

[0017] In step S3, the fermentation temperature is 30°C - 37°C, the fermentation time is 16h - 24h, the pressure of the fermenter is 0.2MPa, ferment on a shaker, and the shaker speed is 100rpm - 150rpm.

[0018] Furthermore, in step S1, by mass, every 100 parts of the liquid culture medium comprises the following components: 1.5 - 2.0 parts of glucose, 0.8 - 1.0 parts of peptone, 0.3 - 0.5 parts of yeast powder, 0.1 - 0.2 parts of dipotassium hydrogen phosphate, 0.3 - 0.5 parts of sodium acetate, 0.1 - 0.2 parts of diammonium hydrogen citrate, 0.005 - 0.01 parts of Tween 80, 0.1 - 0.3 parts of magnesium sulfate, 0.005 - 0.05 parts of manganese sulfate, and the balance is purified water, and the pH value of the liquid culture medium is 6.0 - 7.2; and / or,

[0019] In step S2, by mass, every 100 parts of the primary fermentation medium comprises the following components: 1.0 - 2.0 parts of peptone, 0.1 - 0.2 parts of diammonium hydrogen citrate, 3.0 - 4.0 parts of molasses, 0.05 - 0.1 parts of Tween 80, 0.1 - 0.5 parts of sodium acetate, 0.1 - 0.2 parts of dipotassium hydrogen phosphate, 0.01 - 0.05 parts of magnesium sulfate, 0.01 - 0.03 parts of manganese sulfate, with the balance being purified water, and the pH value of the primary fermentation medium is 6.0 - 7.2; and / or,

[0020] In step S3, by mass, every 100 parts of the secondary fermentation medium comprises the following components: 1.0 - 2.0 parts of peptone, 0.1 - 0.2 parts of diammonium hydrogen citrate, 3.5 - 4.0 parts of sucrose, 0.08 - 0.2 parts of Tween 80, 0.1 - 0.5 parts of sodium acetate, 0.2 - 0.3 parts of dipotassium hydrogen phosphate, 0.01 - 0.05 parts of magnesium sulfate, 0.01 - 0.03 parts of manganese sulfate, with the balance being purified water, and the pH value of the primary fermentation medium is 3.5 - 4.5.

[0021] The present invention also provides an application of a fermentation bacterium agent in the preparation of an antibacterial agent, wherein the fermentation bacterium agent comprises the aforementioned fermentation bacterium agent or a fermentation bacterium agent prepared according to the preparation method of the aforementioned fermentation bacterium agent.

[0022] Compared with the prior art, the technical solution of the present application has the following beneficial technical effects:

[0023] (1) The Clostridium butyricum CB03 of the present invention has a significant antibacterial effect against various fish pathogenic bacteria such as Aeromonas hydrophila, Streptococcus agalactiae, Edwardsiella tarda, Vibrio spp., etc.; it has the potential to be developed into a probiotic preparation and can be used to inhibit pathogenic bacteria and reduce the contamination of the above-mentioned bacteria in feed;

[0024] (2) Through process improvement, the Clostridium butyricum CB03 of the present invention has improved the stability of Clostridium butyricum CB03 and ensured the stability of the product;

[0025] (3) When the Clostridium butyricum CB03 of the present invention is used as a feed additive to feed fish, it can improve the growth performance of fish, improve the intestinal flora, and enhance the resistance of fish to pathogenic bacteria;

[0026] (4) The method for preparing the Clostridium butyricum CB03 of the present invention into a probiotic preparation is simple and has a low production cost. At the same time, it can effectively reduce the risks such as drug residues and pathogenic bacteria resistance caused by the abuse of antibiotics, and has good application prospects. Description of the Drawings

[0027] Figure 1 It is a diagram showing the antibacterial result of Clostridium butyricum CB03 in Example 1 of the present invention;

[0028] Figure 2 It is the morphological feature diagram of Clostridium butyricum CB03 in Example 2 of the present invention;

[0029] Figure 3 It is the statistical chart of the high temperature resistance test results of Clostridium butyricum CB03 in Example 3 of the present invention;

[0030] Figure 4 It is the physical diagram of the high temperature resistance test results of Clostridium butyricum CB03 in Example 3 of the present invention;

[0031] Figure 5 It is the statistical chart of the acid and alkali resistance test results of Clostridium butyricum CB03 in Example 3 of the present invention;

[0032] Figure 6 It is the physical diagram of the acid and alkali resistance test results of Clostridium butyricum CB03 in Example 3 of the present invention;

[0033] Figure 7 It is the statistical chart of the enzyme resistance test results of Clostridium butyricum CB03 in Example 3 of the present invention;

[0034] Figure 8 It is the physical diagram of the enzyme resistance test results of Clostridium butyricum CB03 in Example 3 of the present invention;

[0035] Figure 9 It is the bacteriostatic effect diagram of Clostridium butyricum CB03 under different carbon sources in Example 4 of the present invention;

[0036] Figure 10 It is the bacteriostatic effect diagram of Clostridium butyricum CB03 under different nitrogen sources in Example 4 of the present invention;

[0037] Figure 11 It is the bacteriostatic effect diagram of Clostridium butyricum CB03 under different carbon-nitrogen ratios in Example 4 of the present invention;

[0038] Figure 12 It is the bacteriostatic effect diagram of Clostridium butyricum CB03 at different temperatures in Example 4 of the present invention;

[0039] Figure 13 It is the bacteriostatic effect diagram of Clostridium butyricum CB03 at different initial pH values in Example 4 of the present invention;

[0040] Figure 14 It is the bacteriostatic effect diagram of Clostridium butyricum CB03 at different times in Example 4 of the present invention;

[0041] Figure 15 It is the screening diagram of the protectant of Clostridium butyricum CB03 in Example 5 of the present invention;

[0042] Figure 16 It is the diagram of the mixed protectant of Clostridium butyricum CB03 in Example 5 of the present invention;

[0043] Figure 17 This is the diagram showing the effect of Clostridium butyricum CB03 on the intestinal immune genes in Example 6 of the present invention;

[0044] Figure 18 This is the diagram showing the effect of Clostridium butyricum CB03 on the liver immune genes in Example 7 of the present invention;

[0045] Figure 19 This is the diagram showing the effect of Clostridium butyricum CB03 on the intestinal flora of grass carp in Example 7 of the present invention. Detailed implementation manners

[0046] To facilitate the understanding of this application, the following will describe this application more comprehensively with reference to the relevant attached drawings. Embodiments of this application are shown in the attached drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of this application more thorough and comprehensive.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of this application in this specification are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0048] Currently, Bacillus, Lactobacillus, and Saccharomyces cerevisiae have become the main economic markets for probiotics. However, in a complex aquatic environment, the survival ability of these microorganisms is poor and their competitiveness is weak. For example, water quality parameters, competition from other microorganisms, and the immune status of the host itself may all affect the effects of these beneficial microorganisms. Even if these microorganisms can survive and reproduce in water, it is difficult to maintain their activity for a long time in a farming system and continuously provide protection for fish, and they need to be frequently added to maintain a sufficient concentration to exert their effects.

[0049] Clostridium butyricum is a type of probiotic widely present in nature, with various probiotic effects such as improving the intestinal microecology, increasing feed conversion rate, promoting animal growth, and enhancing host immunity. Clostridium butyricum is an anaerobic bacterium, which accounts for a relatively high proportion in the digestive tract and colonizes in the animal intestine for a long time. The development of its probiotic preparation is particularly important. During production and storage, it is prone to a decrease in activity, resulting in a decline in the effect of the live bacteria preparation. In order to better promote and apply Clostridium butyricum in the market, this study isolated Clostridium butyricum from animal-source intestines and screened strains with antibacterial effects; through fermentation optimization, the antibacterial effect and storage stability of the strains were improved; through mixing and feeding in aquaculture, the effect of Clostridium butyricum on aquatic animals was verified.

[0050] In view of this, the present invention provides a Clostridium butyricum, named Clostridium butyricum CB03, which has been deposited in the China Center for Type Culture Collection. The deposit date is April 17, 2025, and the deposit number is CCTCC NO: M 2025813.

[0051] In the technical solution of the present invention, Clostridium butyricum CB03 is obtained by screening, and it has a significant antibacterial effect against various fish pathogenic bacteria such as Aeromonas hydrophila, Streptococcus agalactiae, Edwardsiella tarda, and Vibrio, and can be used to inhibit pathogenic bacteria and reduce the contamination of the above-mentioned bacteria in feed.

[0052] The present invention also proposes an application of Clostridium butyricum in the preparation of a feed additive, and the feed additive is used to antagonize aquatic animal pathogenic bacteria.

[0053] In the technical solution of the present invention, by feeding fish with Clostridium butyricum CB03 as a feed additive, it can improve the growth performance of fish, improve the intestinal flora, and enhance the resistance of fish to pathogenic bacteria.

[0054] The present invention also proposes a fermentation bacterium agent, which is a fermentation product of Clostridium butyricum, and the fermentation product of Clostridium butyricum is fermented from the aforementioned Clostridium butyricum.

[0055] Furthermore, the fermentation bacterium agent further includes a Clostridium butyricum protectant, and the Clostridium butyricum protectant includes xanthan gum, skim milk, and lecithin.

[0056] In the technical solution of the present invention, by adding the Clostridium butyricum protectant to the fermentation bacterium agent, the biological activity of Clostridium butyricum in the fermentation bacterium agent can be improved, thereby improving the antibacterial effect of the fermentation bacterium agent.

[0057] Furthermore, in the fermentation bacterium agent, the mass ratio of xanthan gum is 0.1% - 0.2%, the mass ratio of skim milk is 0.1% - 0.5%, and the mass ratio of lecithin is 0.1% - 0.5%.

[0058] The present invention also proposes a preparation method of the aforementioned fermentation bacterium agent, including the following steps:

[0059] S1. Seed liquid fermentation: Inoculate Clostridium butyricum CB03 on a specific medium, pick monoclonal colonies into a liquid medium for anaerobic fermentation culture to obtain a fermentation seed liquid;

[0060] S2. Small fermenter fermentation: Inoculate the fermentation seed liquid into a fermenter filled with a primary fermentation medium at a volume ratio of 1% - 3% for the first stage of scale-up culture, and perform anaerobic fermentation to obtain an enlarged culture solution;

[0061] S3. Fermentation in a large fermenter: inoculate the enlarged culture solution into a fermenter containing a secondary fermentation medium at a volume ratio of 3% - 10%, and conduct a second enlarged culture and anaerobic fermentation to obtain a fermentation inoculum.

[0062] Further, in step S1, the fermentation temperature is 30°C - 37°C, the fermentation time is 24h - 48h, and the fermentation is carried out on a shaker with a shaker speed of 200rpm - 220rpm; and / or,

[0063] In step S2, the fermentation temperature is 30°C - 37°C, the fermentation time is 24h - 48h, and the fermentation is carried out on a shaker with a shaker speed of 100rpm - 150rpm; and / or,

[0064] In step S3, the fermentation temperature is 30°C - 37°C, the fermentation time is 16h - 24h, the pressure in the fermenter is 0.2MPa, and the fermentation is carried out on a shaker with a shaker speed of 100rpm - 150rpm.

[0065] In the technical solution of the present invention, by adjusting the fermentation conditions, the biological activity of Clostridium butyricum CB03 in the fermentation inoculum can be further improved; it should be noted that the fermentation conditions of steps S1, S2, and S3 can be defined separately or simultaneously. When defined simultaneously, the biological activity and antibacterial stability of Clostridium butyricum CB03 in the fermentation inoculum can be further improved.

[0066] Further, in step S1, by mass, every 100 parts of the liquid medium comprises the following components: 1.5 - 2.0 parts of glucose, 0.8 - 1.0 parts of peptone, 0.3 - 0.5 parts of yeast powder, 0.1 - 0.2 parts of dipotassium hydrogen phosphate, 0.3 - 0.5 parts of sodium acetate, 0.1 - 0.2 parts of diammonium hydrogen citrate, 0.005 - 0.01 parts of Tween 80, 0.1 - 0.3 parts of magnesium sulfate, 0.005 - 0.05 parts of manganese sulfate, and the balance is purified water, and the pH value of the liquid medium is 6.0 - 7.2; and / or,

[0067] In step S2, by mass, every 100 parts of the primary fermentation medium comprises the following components: 1.0 - 2.0 parts of peptone, 0.1 - 0.2 parts of diammonium hydrogen citrate, 3.0 - 4.0 parts of molasses, 0.05 - 0.1 parts of Tween 80, 0.1 - 0.5 parts of sodium acetate, 0.1 - 0.2 parts of dipotassium hydrogen phosphate, 0.01 - 0.05 parts of magnesium sulfate, 0.01 - 0.03 parts of manganese sulfate, and the balance is purified water, and the pH value of the primary fermentation medium is 6.0 - 7.2; and / or,

[0068] In step S3, by mass parts, every 100 parts of the secondary fermentation medium comprises the following components: 1.0 - 2.0 parts of peptone, 0.1 - 0.2 parts of diammonium hydrogen citrate, 3.5 - 4.0 parts of sucrose, 0.08 - 0.2 parts of Tween 80, 0.1 - 0.5 parts of sodium acetate, 0.2 - 0.3 parts of dipotassium hydrogen phosphate, 0.01 - 0.05 parts of magnesium sulfate, 0.01 - 0.03 parts of manganese sulfate, and the balance is purified water. The pH value of the primary fermentation medium is 3.5 - 4.5.

[0069] In the technical solution of the present invention, different culture media are used for culturing, which can meet the nutrients required for different growth and development stages of Clostridium butyricum, provide a suitable growth environment, further improve the antibacterial effect of the fermentation agent, and simultaneously inhibit the growth of miscellaneous bacteria to a certain extent.

[0070] It should be noted that the components of the liquid medium, the primary fermentation medium and the secondary fermentation medium can limit any one of them, or can be limited simultaneously; when limited simultaneously, the stability of the fermentation agent in inhibiting pathogenic bacteria can be further improved.

[0071] The present invention also provides an application of a fermentation agent in the preparation of an antibacterial agent, and the fermentation agent includes the fermentation agent as described above or the fermentation agent prepared according to the preparation method of the fermentation agent as described above.

[0072] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0073] The materials, reagents, etc. used in the following examples can be obtained from commercial channels without special instructions.

[0074] Example 1 Isolation and Screening of Clostridium butyricum CB03

[0075] The intestinal contents of healthy grass carp from Wuhan City, Hubei Province were gradient diluted with sterile normal saline and then spread on the culture medium, and cultured at 37°C for 48 - 72 h. The growth of the strains was observed and single colonies were picked for purification.

[0076] Using pathogenic bacteria such as Streptococcus agalactiae, Aeromonas hydrophila, Edwardsiella tarda, and Vibrio harveyi as indicator bacteria, the antibacterial activity of the fermentation broth against the above pathogenic bacteria was detected by the Oxford cup method. It was found that the supernatant of Clostridium butyricum CB03 had good antibacterial effects on all 6 kinds of original bacteria, and the antibacterial effect on Aeromonas hydrophila was the strongest, with an average antibacterial circle diameter reaching 19.91 ± 0.15 mm (as Figure 1 shown).

[0077] Example 2 Identification of Clostridium butyricum CB03

[0078] (1) Morphological characteristics and Gram staining

[0079] The single colony morphology of CB03 is as Figure 2 shown in Figure A in the middle. The colony has a neat edge, is small, and white; the Gram staining result of CB03 is as Figure 2 shown in Figure B in the middle. It is a Gram-positive bacterium, presenting a short rod shape.

[0080] (2) Physiological and biochemical identification

[0081] The genus of the isolated strain was preliminarily identified according to the commonly used bacterial systematic identification manual and Bergey's systematic identification manual. A total of 15 physiological and biochemical identifications were carried out on the isolated strain. The results showed that the isolated strain CB03 could utilize sucrose, xylose, fructose, raffinose, arabinose, and maltose (Table 1).

[0082] Table 1 Physiological and biochemical results of CB03

[0083]

[0084]

[0085] Note: - indicates negative, + indicates positive.

[0086] Example 3 Tolerance of antibacterial substances of Clostridium butyricum CB03

[0087] (1) High temperature resistance detection of CB03

[0088] The supernatant of CB03 was treated at 40°C, 60°C, 80°C, and 100°C for 30 min, and the untreated supernatant was used as a negative control. The Oxford cup method was used to determine the inhibitory effect on pathogenic bacteria. The results showed that the supernatant of CB03 had a good inhibitory effect on Aeromonas hydrophila after being treated at 100°C for 30 min ( Figure 3 and Figure 4 ).

[0089] (2) Acid and alkali resistance detection of CB03

[0090] The supernatant of CB03 was placed in media with pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. 1 mL of the filtered solution was added to 9 mL of different pH media and incubated overnight at 4°C. The untreated supernatant was used as a negative control. The Oxford cup method was used to detect the inhibitory effect of the supernatant of CB03 on pathogenic bacteria. The results showed that the supernatant of CB03 had an inhibitory effect on Aeromonas hydrophila under strong acid and strong alkali conditions ( Figure 5 and Figure 6 ).

[0091] (3) Enzyme resistance detection of CB03

[0092] The CB03 supernatant was separately added with proteinase K, trypsin, and alkaline protease (under the optimal pH conditions of the three enzymes, reacting at 37 °C for 2 h), and the final mass concentration of the enzyme was 1 mg / ml. The untreated supernatant was used as a negative control. The Oxford cup method was used to detect the inhibitory effect of the CB03 supernatant on pathogenic bacteria. The results showed that CB03 had a good inhibitory effect on Aeromonas hydrophila under the treatment of proteinase K, trypsin, and alkaline protease ( Figure 7 and Figure 8 ).

[0093] Example 4 Optimization of the antibacterial effect of Clostridium butyricum CB03

[0094] 1) Antibacterial effect of CB03 under different carbon sources

[0095] The inhibitory effect of CB03 on Aeromonas hydrophila under different carbon sources was detected. The results showed that CB03 had the greatest antibacterial effect on Aeromonas hydrophila under the condition of sucrose ( Figure 9 ).

[0096] 2) Antibacterial effect of CB03 under different nitrogen sources

[0097] The inhibitory effect of CB03 on Aeromonas hydrophila under different nitrogen sources was detected. The results showed that there was no significant difference in the antibacterial effect of CB03 on Aeromonas hydrophila under different nitrogen source conditions ( Figure 10 ).

[0098] 3) Antibacterial effect of CB03 under different carbon-nitrogen ratios

[0099] The inhibitory effect of CB03 on Aeromonas hydrophila under different carbon-nitrogen ratios was detected. The results showed that CB03 had the greatest antibacterial effect on Aeromonas hydrophila under the condition of a carbon-nitrogen ratio of 1.25 / 1 ( Figure 11 ).

[0100] 4) Antibacterial effect of CB03 at different temperatures

[0101] The inhibitory effect of CB03 on Aeromonas hydrophila at different temperatures was detected. The results showed that there was no significant difference in the antibacterial effect of CB03 on Aeromonas hydrophila between 28 °C and 37 °C, indicating that CB03 has a wide range of temperature adaptability ( Figure 12 ).

[0102] 5) Antibacterial effect of CB03 at different initial pH values

[0103] The inhibitory effect of CB03 on Aeromonas hydrophila at different initial pH values was detected. The results showed that CB03 had a greater antibacterial effect on Aeromonas hydrophila under the condition of an initial pH of 4 ( Figure 13 ).

[0104] 6) Antibacterial effect of CB03 at different times

[0105] The inhibitory effect of CB03 on Aeromonas hydrophila at different times was detected. The results showed that the inhibitory effect of CB03 on Aeromonas hydrophila gradually increased with the fermentation time( Figure 14 ).

[0106] 7) Antibacterial effects before and after fermentation optimization of CB03

[0107] After multiple fermentation optimizations and price references, the final fermentation medium was: peptone 1.0 - 2.0%, diammonium hydrogen citrate 0.1 - 0.2%, sucrose 3.5 - 4.0%, Tween 80 0.08 - 0.2%, sodium acetate 0.1 - 0.5%, dipotassium hydrogen phosphate 0.2 - 0.3%, magnesium sulfate 0.01 - 0.05%, manganese sulfate 0.01 - 0.03%, and the rest was purified water, with a pH value of 3.5 - 4.5. The supernatants of CB03 before and after optimization were compared. The results showed that the optimized CB03 had improved effects on various pathogenic bacteria, and its improvement effect on Aeromonas hydrophila was the most obvious, with an improvement rate of 26.32% (Table 2).

[0108] Table 2 Optimization of the antibacterial effect of Clostridium butyricum CB03

[0109]

[0110] Example 5 Preparation of microecological agents and their stability process

[0111] 1) Fermentation process of Clostridium butyricum CB03

[0112] Seed liquid preparation: Streak the cryopreserved tube CB03 on the medium, pick a single colony and inoculate it into the first-stage seed medium, and culture it at 30 - 37°C and 200 - 220 r / min for 24 h. Small fermenter fermentation: Inoculate the secondary seed liquid at an inoculation amount of 1 - 2%, and culture it at 30 - 37°C and 150 - 200 r / min for 24 h. Large fermenter fermentation: Transfer the secondary seed liquid prepared in the previous step to the fermenter at a ratio of 3 - 4%, control the tank temperature at 30 - 37°C, and the stirring speed at 100 - 200 rpm / min, and culture for 24 h to obtain the fermentation broth. After multiple process optimizations, the viable count of Clostridium butyricum CB03 was 10 9 cfu / ml (Table 3).

[0113] Table 3 Fermentation level of Clostridium butyricum CB03

[0114]

[0115] 2) Preparation of microecological agents and their stability

[0116] Six kinds of protectants were screened, and the results showed that xanthan gum, skim milk, and lecithin had a protective effect on the viable count of CB03 ( Figure 15 ). After mixing the screened protectants evenly according to 0.1-0.2% xanthan gum, 0.1-0.5% skim milk, and 0.1-0.5% lecithin, water was added while stirring. After the xanthan gum was dissolved and swollen, it was placed in the CB03 fermenter and stirred evenly. Through the screening of various protectants, the results showed that the combined use of xanthan gum, skim milk, and lecithin could play a synergistic protective role. Therefore, this scheme could improve the stability of Clostridium butyricum CB03 ( Figure 16 ).

[0117] Example 6 Safety of Clostridium butyricum CB03 in aquaculture

[0118] Healthy grass carps were randomly divided into 3 groups, with 3 replicates in each group and 10 grass carps in each replicate. They were fed for two weeks to make them stable. During the experiment, the water temperature was controlled at 25-28 °C and there was sufficient oxygen. The experimental groups were respectively injected with the L3-9 strain at 1×10 8 colony-forming units per milliliter (cfu / ml) and Aeromonas hydrophila at 1×10 6 cfu / ml. The control group was injected with normal saline. They were continuously observed for 14 days, and the living status and death of the grass carps were observed and recorded every day. The results showed that the grass carps injected with the CB03 strain had a good growth status and no death occurred. While in the group injected with Aeromonas hydrophila, the survival rate of the grass carps was 40.33%, indicating that the CB03 strain would not affect the survival of grass carps and had high safety (Table 4).

[0119] Table 4 Animal safety experiment (survival rate%)

[0120]

[0121] Note: Different superscripts in the same row indicate significant differences, and the same below.

[0122] Example 7 Application of Clostridium butyricum CB03 in aquaculture

[0123] 1) Influence of CB03 on the growth performance of grass carps

[0124] Healthy grass carps were randomly selected and divided into 2 groups, with 3 replicates in each group and 10 grass carps in each replicate. They were acclimated and fed for one week to adapt to the experimental environment, and the water temperature was controlled at 25-28 °C. The feeding method was used in the experiment. 1×10 8The feed was sprayed with CB03 strain at cfu / ml and mixed evenly. The grass carp in the control group were fed with ordinary feed, and the feeding amount was 2% of the body weight of the grass carp. They were fed continuously for 42 days, once in the morning and once in the evening every day. The grass carp were weighed on the 1st day and the 42nd day at the beginning of feeding, and their initial weight and final weight were recorded. The weight gain rate (WG), feed coefficient (FE), specific growth rate (SGR) and survival rate (SR) were calculated using the following formulas. Weight gain rate (WG) = ((final weight - initial weight) / initial weight) × 100%; Feed coefficient (FE) = total feeding amount / (final weight - initial weight); Survival rate SR = (number of grass carp tails at the end of the experiment / number of grass carp tails at the start of the experiment) × 100%; Specific growth rate (SGR) = ((ln final weight - ln initial weight) / number of feeding days) × 100%. The results showed that the final weight, weight gain rate and specific growth rate of the grass carp fed with CB03 group were significantly higher than those of the control group, and the feed coefficient was significantly lower than that of the control group, indicating that feeding CB03 strain could significantly increase the weight of grass carp (Table 5).

[0125] Table 5 Effects of Clostridium butyricum CB03 on the growth performance of grass carp

[0126]

[0127] 2) Effects of CB03 on the immunity of grass carp

[0128] After CB03 was added to the feed and fed for 42 days, the intestinal tracts of grass carp in each group were taken respectively to detect the expression of immune genes in the tissues. The results showed that adding CB03 to the feed could significantly increase the relative expression levels of IL-6, TLR-8, TLR-4 and TGF-β1 in the intestinal tissues of grass carp, and significantly reduce the relative expression level of IL-10 ( Figure 17 ). At the same time, adding CB03 to the feed could significantly increase the relative expression levels of IL-6, TLR-8, IL-10, TLR-4 and TGF-TGF-β1 in the liver tissues of grass carp ( Figure 18 ).

[0129] 3) Protection rate of CB03 on grass carp

[0130] After adding CB03 strain to the feed and feeding grass carp for 42 days, each group was injected with Aeromonas hydrophila into grass carp, and the death situation of grass carp was continuously observed and recorded within 14 days (Table 6). The results showed that the survival rate of grass carp in the control group was 33.3% after 14 days of challenge, and the survival rate of the CB03 feeding group was 62.50% after 14 days. From the results, it can be seen that the survival rate of grass carp fed with CB03 strain was significantly higher than that of the control group. Therefore, feeding Clostridium butyricum CB03 can improve the survival rate of grass carp infected with Aeromonas hydrophila and enhance the disease resistance of grass carp.

[0131] Table 6 Protection rate experiment of CB03 strain on grass carp (survival rate%)

[0132]

[0133] 4) Effects of CB03 on the intestinal flora of grass carp

[0134] After adding CB03 strain to the feed and feeding grass carp for 42 days, under sterile conditions, the intestinal microorganisms of each group of grass carp were taken separately and quickly placed in liquid nitrogen, and then intestinal microorganism detection was carried out. The detection results showed that the CB03 group could increase the abundance of Lactobacillaceae, while reducing the abundances of Aeromonadaceae and Enterobacteriaceae( Figure 19 ). It shows that adding Clostridium butyricum CB03 to the feed can increase the abundance of Lactobacillaceae and improve the abundance of the host intestinal flora.

[0135] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

[0136] In summary, the technical solution of this application has the following beneficial technical effects:

[0137] (1) The Clostridium butyricum CB03 of the present invention has a significant antibacterial effect against various fish pathogenic bacteria such as Aeromonas hydrophila, Streptococcus agalactiae, Edwardsiella tarda, Vibrio, etc.; it has the potential to be developed into a probiotic preparation and can be used to inhibit pathogenic bacteria and reduce the contamination of the above bacteria in the feed.

[0138] (2) The Clostridium butyricum CB03 of the present invention has improved process, which improves the stability of Clostridium butyricum CB03 and ensures the stability of the product.

[0139] (3) The Clostridium butyricum CB03 of the present invention, as a feed additive for feeding fish, can improve the growth performance of fish, improve the intestinal flora, and enhance the resistance of fish to pathogenic bacteria.

[0140] (4) The method for preparing the Clostridium butyricum CB03 of the present invention into a probiotic preparation is simple, with low production cost. At the same time, it can effectively reduce the risks such as drug residues and pathogenic bacteria drug resistance caused by the abuse of antibiotics, and has good application prospects.

Claims

1. A Clostridium butyricum, characterized in that, The Clostridium butyricum is named Clostridium butyricum CB03, and the Clostridium butyricum has been deposited in the China Center for Type Culture Collection. The deposit date is April 17, 2025, and the deposit number is CCTCC NO: M 2025813.

2. Application of Clostridium butyricum in preparing feed additive, characterized in that, The feed additive is used for antagonizing pathogenic bacteria of aquatic animals.

3. A fermentation inoculant, characterized in that, The fermentation inoculant is a fermentation product of Clostridium butyricum, and the fermentation product of Clostridium butyricum is fermented from Clostridium butyricum as described in claim 1.

4. The fermenting inoculant according to claim 3, wherein The fermentation inoculant further includes a Clostridium butyricum protectant, and the Clostridium butyricum protectant includes xanthan gum, skim milk, and lecithin.

5. The fermenting inoculant according to claim 4, wherein In the fermentation inoculant, the mass ratio of the xanthan gum is 0.1% - 0.2%, the mass ratio of the skim milk is 0.1% - 0.5%, and the mass ratio of the lecithin is 0.1% - 0.5%.

6. A preparation method of the fermentation inoculum according to any one of claims 3 to 5, characterized in that, It includes the following steps: S1. Seed liquid fermentation: Inoculate Clostridium butyricum CB03 on a specific culture medium, pick monoclonal colonies into a liquid culture medium for anaerobic fermentation culture to obtain a fermentation seed liquid; S2. Small fermenter fermentation: Inoculate the fermentation seed liquid into a fermenter containing a primary fermentation medium at a volume ratio of 1% - 3% for the first expansion culture and anaerobic fermentation to obtain an expanded culture solution; S3. Large fermenter fermentation: Inoculate the expanded culture solution into a fermenter containing a secondary fermentation medium at a volume ratio of 3% - 10% for the second expansion culture and anaerobic fermentation to obtain a fermentation inoculant.

7. The preparation method of the fermentation inoculum according to claim 6, wherein In step S1, the fermentation temperature is 30°C - 37°C, the fermentation time is 24h - 48h, and it is fermented on a shaker with a shaker speed of 200rpm - 220rpm; and / or, In step S2, the fermentation temperature is 30°C - 37°C, the fermentation time is 24h - 48h, and it is fermented on a shaker with a shaker speed of 100rpm - 150rpm; and / or, In step S3, the fermentation temperature is 30°C - 37°C, the fermentation time is 16h - 24h, the pressure of the fermenter is 0.2MPa, and it is fermented on a shaker with a shaker speed of 100rpm - 150rpm.

8. The preparation method of the fermentation inoculum according to claim 6, wherein In step S1, by mass fraction, every 100 parts of the liquid culture medium includes the following components: 1.5 - 2.0 parts of glucose, 0.8 - 1.0 parts of peptone, 0.3 - 0.5 parts of yeast powder, 0.1 - 0.2 parts of dipotassium hydrogen phosphate, 0.3 - 0.5 parts of sodium acetate, 0.1 - 0.2 parts of diammonium hydrogen citrate, 0.005 - 0.01 parts of Tween 80, 0.1 - 0.3 parts of magnesium sulfate, 0.005 - 0.05 parts of manganese sulfate, and the balance is purified water. The pH value of the liquid culture medium is 6.0 - 7.2; and / or, In step S2, per 100 parts by mass of the primary fermentation medium, it comprises the following components: 1.0 - 2.0 parts of peptone, 0.1 - 0.2 parts of diammonium hydrogen citrate, 3.0 - 4.0 parts of molasses, 0.05 - 0.1 parts of Tween 80, 0.1 - 0.5 parts of sodium acetate, 0.1 - 0.2 parts of dipotassium hydrogen phosphate, 0.01 - 0.05 parts of magnesium sulfate, 0.01 - 0.03 parts of manganese sulfate, with the balance being purified water, and the pH value of the primary fermentation medium is 6.0 - 7.2; and / or, In step S3, per 100 parts by mass of the secondary fermentation medium, it comprises the following components: 1.0 - 2.0 parts of peptone, 0.1 - 0.2 parts of diammonium hydrogen citrate, 3.5 - 4.0 parts of sucrose, 0.08 - 0.2 parts of Tween 80, 0.1 - 0.5 parts of sodium acetate, 0.2 - 0.3 parts of dipotassium hydrogen phosphate, 0.01 - 0.05 parts of magnesium sulfate, 0.01 - 0.03 parts of manganese sulfate, with the balance being purified water, and the pH value of the primary fermentation medium is 3.5 - 4.

5.

9. Use of a fermentation bacterium agent in the preparation of a drug for treating fish pathogenic bacteria infection, characterized in that, The fermentation inoculum includes the fermentation inoculum according to any one of claims 3 to 5 or the fermentation inoculum prepared by the preparation method of the fermentation inoculum according to any one of claims 6 to 8.

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