Monopterus albus source clostridium butyricum YF1 as well as culture method and application thereof

Abstract

The invention discloses ricefield eel source clostridium butyricum YF1 as well as a culture method and application thereof, and belongs to the technical field of microorganism isolated culture and aquatic animal feed. The clostridium butyricum YF1 is preserved in the China Center for Type Culture Collection, and the preservation number of the clostridium butyricum YF1 is CCTCC NO: M20242414. In addition, the invention further provides a culture method of the clostridium butyricum YF1, and the culture method comprises the step of culturing under the anaerobic condition of 35-37 DEG C. In addition, the invention also provides an application of the clostridium butyricum YF1 as a feed additive. The clostridium butyricum YF1 strain has the characteristics of high yield of acetic acid and butyric acid in a short time, tolerance to temperature, cholate and artificial gastrointestinal fluid and sensitivity to most antibiotics. By adding the clostridium butyricum YF1 strain into the feed, the digestive enzyme activity of the intestinal tract of the ricefield eel can be remarkably improved, the intestinal tract is kept healthy and stable, and ricefield eel breeding is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of microbial separation and culture and aquatic animal feed, and in particular to a strain of Clostridium butyricum YF1 from a rice field eel, and a culture method and application thereof. Background Art

[0002] The eel has tender meat and delicious taste, and its nutritional value is very high. Eel is rich in DHA and lecithin. In recent years, due to the gradual expansion of breeding scale and the increase in breeding density, the breeding environment of eels has deteriorated and diseases have occurred frequently, which seriously hinders the sustainable development of healthy eel breeding. The most commonly used method to control fish diseases is the use of antibiotics. However, excessive use of antibiotics will produce drug resistance and drug residues. The reduction of antibiotic use and the application of antibiotic substitutes have received widespread attention from the society. Among them, probiotics, Chinese herbal medicines, and immune enhancers are potential substitutes with good application prospects. Clostridium butyricum belongs to the Bacillaceae family and the genus Clostridium. It is an obligate anaerobic Gram-positive bacillus. It is widely present in healthy animals (gastrointestinal tract or feces) or soil and fermented foods. The main metabolite of Clostridium butyricum is butyric acid, which can promote the regeneration and repair of intestinal epithelial tissue cells.

[0003] Studies in recent years have shown that Clostridium butyricum, as a feed additive, can improve the growth performance of fish, increase feed utilization, enhance digestive enzyme activity, inhibit pathogen invasion, mediate immune response, improve disease resistance, regulate intestinal microorganisms and change the intestinal structure of fish. However, most strains come from terrestrial animals such as poultry and livestock, rather than aquatic animals. It is reported that probiotic strains from different sources show different characteristics. Yellow eel is one of the famous and excellent economic freshwater farmed fish in my country, and Clostridium butyricum has not yet been isolated from yellow eel. Therefore, isolating and culturing Clostridium butyricum from yellow eel, improving the intestinal health of yellow eel, and then helping yellow eel farming is a technical problem that the prior art needs to solve. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned technical deficiencies, provide a strain of Clostridium butyricum YF1 from rice field eel and its culture method and application, and solve the technical problem in the prior art of how to propose a new Clostridium butyricum to improve the intestinal health of rice field eel and thus contribute to rice field eel farming.

[0005] The invention provides a strain of Clostridium butyricum YF1 derived from yellow eel, which is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO:M20242414. The 16s rDNA sequence of the Clostridium butyricum YF1 derived from yellow eel is shown in SEQ ID NO:1.

[0006] In addition, the present invention also proposes a method for culturing the above-mentioned eel-derived Clostridium butyricum YF1, which comprises culturing under anaerobic conditions at 35-37°C.

[0007] In any embodiment, the culture medium used includes: 10-12g of tryptone, 10-12g of beef extract, 3-4g of yeast extract powder, 5g of glucose, 1-1.5g of soluble starch, 5-6g of sodium chloride, 3-4g of anhydrous sodium acetate, 0.5-0.6g of L-cysteine ​​salt, 20-22g of solid culture medium agar, and 1000-1100mL of distilled water.

[0008] In any embodiment, the culture medium used includes: 30-32 g yeast extract, 10-12 g glucose, 2-2.5 g sodium chloride, 0.25-0.28 g magnesium sulfate heptahydrate, 2-2.5 g dimethyl phosphate, 0.5-0.7 g L-cysteine ​​hydrochloride, and water to make up to 1000 mL.

[0009] In any embodiment, the culture medium used includes: 15.0-15.2 g / L of polyvalent peptone, 3.0-3.2 g / L of yeast extract, 3.0-3.2 g / L of soluble starch, 0.1-0.2 g / L of magnesium sulfate, 1.0-1.2 g / L of sodium thioglycolate, and 11.0-11.2 g / L of disodium hydrogen phosphate.

[0010] In addition, the present invention also proposes the use of the above-mentioned eel-derived Clostridium butyricum YF1 as a feed additive.

[0011] In any embodiment, the feed additive is a fish feed additive.

[0012] In any embodiment, the fish is rice field eel.

[0013] In any embodiment, the amount of eel-derived Clostridium butyricum YF1 added to the feed is 10 7 -10 9 CFU / g.

[0014] In addition, the present invention also provides a feed additive, comprising the above-mentioned Monopterus albus-derived Clostridium butyricum YF1.

[0015] In addition, the present invention also proposes the use of the above-mentioned Monopterus albus-derived Clostridium butyricum YF1 in producing acetic acid and / or butyric acid.

[0016] Compared with the prior art, the beneficial effects of the present invention include: the eel-derived Clostridium butyricum YF1 proposed in the present invention has a strong butyrate production ability and tolerance to the intestinal environment. When mixed into eel feed and fed, it can significantly improve the activity of amylase and lipase in the eel's intestine, maintain intestinal health and stability, contribute to eel farming, and have broad prospects in eel farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the colony morphology of the eel-derived Clostridium butyricum YF1 in the culture medium in Example 1 of the present invention.

[0018] Figure 2 This is the microscopic morphology of the colony of Clostridium butyricum YF1 from the yellow eel in Example 1 of the present invention.

[0019] Figure 3 This is the scanning electron microscope morphology of Clostridium butyricum YF1 from rice field eel in Example 1 of the present invention.

[0020] Figure 4 This is the evolutionary tree constructed from the 16SrDNA of strain YF1 in Example 1 of the present invention.

[0021] Figure 5 is the absorbance (OD) of the eel-derived Clostridium butyricum YF1 measured in Example 1 of the present invention 600 ) and pH results.

[0022] Figure 6 This is the result of determining the short-chain fatty acid production capacity of Clostridium butyricum YF1 from the yellow eel in Example 1 of the present invention.

[0023] Figure 7 This is the test result of temperature tolerance of Clostridium butyricum YF1 from rice field eel in Example 1 of the present invention.

[0024] Figure 8 This is the test result of the tolerance of the eel-derived Clostridium butyricum YF1 to bile salts in Example 1 of the present invention.

[0025] Fig. 9 This is the test result of the tolerance of the eel-derived Clostridium butyricum YF1 to artificial gastric juice in Example 1 of the present invention.

[0026] Fig.10 This is the test result of the tolerance of Clostridium butyricum YF1 from rice field eel to artificial intestinal fluid in Example 1 of the present invention.

[0027] Fig.11 This is the test result of the intestinal amylase activity of the rice field eel in Example 1 of the present invention.

[0028] Fig.12 This is the result of detecting the lipase activity in the intestine of the rice field eel in Example 1 of the present invention. DETAILED DESCRIPTION

[0029] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 60 to 120 and 80 to 110 is listed for a particular parameter, it is understood that a range of 60 to 110 and 80 to 120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In the present application, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0030] If there is no special explanation, the "include" and "comprising" mentioned in this application are open-ended or closed-ended. For example, the "include" and "comprising" may mean that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0031] If not specifically stated, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0032] This specific embodiment provides a strain of Clostridium butyricum YF1 from rice field eel, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M20242414.

[0033] The present specific embodiment also proposes a method for culturing the above-mentioned eel-derived Clostridium butyricum YF1, comprising culturing under anaerobic conditions at 35-37°C; the culture medium used comprises: 10-12g of tryptone, 10-12g of beef extract, 3-4g of yeast extract powder, 5g of glucose, 1-1.5g of soluble starch, 5-6g of sodium chloride, 3-4g of anhydrous sodium acetate, 0.5-0.6g of L-cysteine ​​acid salt, 20-22g of solid culture medium agar, and 1000-1100mL of distilled water; in some embodiments, the culture medium used comprises: 30-32g of yeast extract powder, 10-12g of glucose, 2-2.5g of sodium chloride, 0.25-0.28g of magnesium sulfate heptahydrate, 2-2.5g of dimethyl phosphate, 0.5-0.7g of L-cysteine ​​hydrochloride, and water is diluted to 1000mL; in some embodiments, the culture medium used comprises: 15.0 polyvalent peptone -15.2g / L, yeast extract 3.0-3.2g / L, soluble starch 3.0-3.2g / L, magnesium sulfate 0.1-0.2g / L, sodium thioglycolate 1.0-1.2g / L, disodium hydrogen phosphate 11.0-11.2g / L.

[0034] This specific embodiment also proposes the use of eel-derived Clostridium butyricum YF1 as a feed additive, wherein the feed additive is preferably a fish feed additive, the fish is preferably an eel, and the amount of eel-derived Clostridium butyricum YF1 added to the feed is preferably 10 7 -10 9 CFU / g.

[0035] This specific embodiment also proposes a feed additive, including the above-mentioned Clostridium butyricum YF1 from rice field eel.

[0036] This specific embodiment also proposes the use of the above-mentioned eel-derived Clostridium butyricum YF1 in the production of acetic acid and / or butyric acid.

[0037] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0038] In the present invention, reference is made to “some embodiments”, “this embodiment”, examples, etc., which describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0039] If similar descriptions of "first / second" appear in the application documents, the following instructions are added. In the following description, the terms "first\second\third" involved are merely used to distinguish similar objects and do not represent a specific ordering of the objects. It can be understood that "first\second\third" can be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0040] In this embodiment, the term "and / or" is merely a description of the association relationship between associated objects, indicating that three relationships may exist. For example, object A and / or object B may represent three situations: object A exists alone, object A and object B exist at the same time, and object B exists alone.

[0041] Hereinafter, the embodiments of the present application will be described. The embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If no specific techniques or conditions are indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0042] Example 1

[0043] This example proposes a strain of Clostridium butyricum YF1 from rice field eel, which is classified and named as Clostridium butyricum, and has been deposited in the China Center for Type Culture Collection on November 1, 2024. The deposit number is CCTCC NO: M20242414, and the deposit address is: China Center for Type Culture Collection of Wuhan University, Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0044] 1. Isolation and purification of Clostridium butyricum:

[0045] Twenty wild eels were collected from the wild in Wuhan, Hubei Province, China. Before dissection, the wild eels were first anesthetized with 0.05% 3-aminobenzoic acid ethyl methanesulfonate-222 (MS-222) solution. The intestines were collected aseptically, and the hindgut contents were gently scraped and diluted 10 times with 0.1M sodium phosphate buffer (PBS, pH 6.8). The dilution was placed in a water bath and heated at 70°C for 10 minutes. The sample was then inoculated into enhanced Clostridium medium (RCM) at a ratio of 5% and cultured anaerobically at 37°C (02≤1%) for 48 hours. Subsequently, the culture was serially diluted to 10 -4 , 10 -5 and 10 -6100 μL of the dilution was spread on trypsin-sulfite-neomycin (TSN) agar medium and cultured anaerobically (02 ≤ 1%) at 37°C for 24 h. A single colony was picked and streaked three times on TSN solid medium for purification.

[0046] A single colony was inoculated into a 2 mL centrifuge tube, and cultured at 37° C. for 24 h under anaerobism (02≤1%) to obtain a bacterial solution, which was stored at −80° C. Finally, a strain was screened out and named YF1.

[0047] The enhanced Clostridium agar medium (RCM) is composed of: 10 g tryptone, 10 g beef extract, 3 g yeast extract, 5 g glucose, 1 g soluble starch, 5 g sodium chloride, 3 g anhydrous sodium acetate, 0.5 g L-cysteine, 20 g solid culture medium agar, and distilled water to 1000 mL, pH 6.8±0.1, and sterilized by high-pressure steam at 121°C for 20 min.

[0048] The composition of the above-mentioned trypsin sulfite neomycin (TSN) agar medium is: 15g of tryptone, 10g of yeast extract powder, 1g of sodium sulfite, 0.5g of ferric citrate, 0.02g of polymyxin B, 0.05g of neomycin sulfate, 13.5g of agar, and distilled water to 1000mL, pH7.2±0.2, and sterilized by high-pressure steam at 121°C for 20min.

[0049] 2. Identification of Clostridium butyricum from eel:

[0050] (1) Observation of the morphological characteristics of the strain

[0051] The strain isolated and purified in step 1 was inoculated on RCM solid medium and cultured anaerobically at 37°C for 24 h. The colonies were observed to be round, with irregular edges, slightly convex, milky white, and sticky ( Figure 1 ), Gram-stain positive ( Figure 2 ), under a scanning electron microscope, the bacterial body has a blunt end, slightly enlarged in the middle, and is rod-shaped or slightly curved, appearing alone or in pairs ( Figure 3 ).

[0052] (2) Physiological and biochemical assays

[0053] The determination of physiological and biochemical characteristics was carried out according to the Manual of Identification of Common Bacteria and Bergey's Manual of Systematic Bacteriology. The results are shown in Table 1.

[0054] Table 1 Physiological and biochemical test results

[0055] project YF1 project YF1 Acid production + Glucose Phosphate Peptone Water (MR) + Gas production + Glucose Phosphate Peptone Water (VP) + lactose + Simon's Citrate - maltose + Hydrogen sulfide - Mannitol + Urea - sucrose + Semisolid agar + Peptone water - Starch hydrolysis +

[0056] Note: "+" represents positive, and "-" represents negative.

[0057] (3) Molecular biological identification of strains and construction of phylogenetic tree

[0058] ① Bacterial DNA extraction

[0059] Inoculate a single colony in a 2 mL centrifuge tube and culture anaerobically at 37°C for 24 hours. After centrifugation at 12,000 r / min for 5 minutes, collect the bacterial precipitate. Use a bacterial genomic DNA rapid extraction kit to extract genomic DNA according to the operating instructions.

[0060] ②PCR amplification of 16S rDNA

[0061] The genomic DNA obtained in step ① was used as an amplification template, and 16S rDNA universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGCTACCTTGTTACGACTT-3') were used for polymerase chain reaction (PCR) amplification. The PCR amplification system was 25 μL, including 1 μL DNA template, 0.4 μL upstream and downstream primers, 12.5 μL 2×Taq premix and 10.7 μL ddH20. The PCR reaction conditions were as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55°C annealing for 45 s, 72°C extension for 90 s, for a total of 35 cycles; 72°C extension for 7 min. After the molecular weight of the PCR product was verified by 1% agarose gel electrophoresis, it was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing.

[0062] ③Construction of phylogenetic tree based on 16S rDNA sequence

[0063] The 16S rDNA sequence obtained in step ② was uploaded to the NCBI website for BLASTN comparison, and the sequence with the highest homology was selected to construct a phylogenetic tree using the neighbor method in MEGA 7.0 software ( Figure 4 ).

[0064] The 16S rDNA sequence of strain YF1 is shown in SEQ ID NO: 1, and is as follows:

[0065]

[0066] According to the morphological characteristics, physiological and biochemical characteristics of the strain and the results of phylogenetic tree analysis based on the 16S rDNA sequence, the strain was identified as Clostridium butyricum and named Clostridium butyricum YF1 strain. The YF1 strain was sent to the China Center for Type Culture Collection on November 1, 2024, and its preservation number is CCTCC NO: M20242414.

[0067] Embodiment 2:

[0068] Determination of biological characteristics of Clostridium butyricum YF1 strain

[0069] 1. Growth characteristics and short-chain fatty acid production capacity of Clostridium butyricum YF1 from rice field eel

[0070] The eel-derived Clostridium butyricum YF1 obtained in Example 1 was inoculated into a Clostridium butyricum growth medium and cultured anaerobically at 37°C and 220 r / min. The medium was renewed every 4 h. The absorbance (OD 600 ) and pH value, the results are as follows Figure 5 At the same time, the culture medium was centrifuged at 4000g for 10 min, and the supernatant was collected and sent to Wuhan Mattwell Biotechnology Co., Ltd. for the determination of the content of short-chain fatty acids. Each group was measured three times.

[0071] The results of the short-chain fatty acid production capacity test are as follows Figure 6 As shown in the results, the eel-derived Clostridium butyricum YF1 can produce 10 short-chain fatty acids (decanoic acid, 2-methylbutyric acid, isocaproic acid, butyric acid, valeric acid, isovaleric acid, caproic acid, acetic acid, isobutyric acid and propionic acid) during the growth cycle, especially high production of acetic acid and butyric acid. Within 24 hours of culture, the content of acetic acid and butyric acid continued to rise and reached a peak at the 24th hour, at which time the production of acetic acid and butyric acid was 984.92±81.59mg / L and 515.42±55.07mg / L, respectively, and then the content showed a slow downward trend. The results show that the eel-derived Clostridium butyricum YF1 has the performance of short-chain fatty acid high production in a short time, especially high production of acetic acid and butyric acid, and has great probiotic potential.

[0072] The above-mentioned Clostridium butyricum growth liquid culture medium is composed of: yeast extract powder 30g, glucose 10g, sodium chloride 2g, magnesium sulfate heptahydrate 0.25g, dimethyl phosphate 2g, L-cysteine ​​hydrochloride 0.5g, distilled water to 1000mL, pH 7.0±0.1, sterilized by 121°C high-pressure steam for 20min, and deoxygenated with mixed gas (H2 5%, CO2 15%, N2 80%) for 30min.

[0073] Tolerance test of Clostridium butyricum YF1 from rice field eel

[0074] (1) Temperature tolerance

[0075] The eel-derived Clostridium butyricum YF1 obtained in Example 1 was inoculated into RCM liquid culture medium and cultured anaerobically at 37°C and 220r / min for 72 to 96h. The cultured spores were centrifuged at 4,000g for 5min to obtain a precipitate, which was then washed with PBS and resuspended. 0.5mL of spore liquid was placed in a water bath at different temperatures (50, 60, 70, 80, 90 and 100°C) and heated for 10min respectively. After treatment, the bacterial liquid was spread on iron sulfite agar medium, cultured anaerobically for 24h, and then the number of colonies on the plate was counted. Three replicates were set for each treatment. The results of the temperature tolerance test of Clostridium butyricum YF1 from eel are shown in Figure 7 When the culture temperature was 70℃, the survival rate of YF1 reached 84.7%, indicating that the eel-derived Clostridium butyricum YF1 had good high temperature resistance.

[0076] The above RCM liquid culture medium is composed of: 10g tryptone, 10g beef extract, 3g yeast extract powder, 5g glucose, 1g soluble starch, 5g sodium chloride, 3g anhydrous sodium acetate, 0.5g L-cysteine ​​salt, and distilled water to 1000mL, pH 6.8±0.1, and sterilized by high-pressure steam at 121°C for 20min.

[0077] The above-mentioned iron sulfite agar medium is composed of: 15g tryptone, 5g soy peptone, 5g yeast extract, 1g sodium metabisulfite, 1g ammonium ferric citrate, 20g agar, and distilled water to 1000mL, pH 7.5-7.7, and sterilized by high-pressure steam at 121°C for 20min.

[0078] (2) Tolerance to bile salts

[0079] The eel-derived Clostridium butyricum YF1 obtained in Example 1 was inoculated into RCM liquid culture medium and cultured anaerobically at 37°C and 220r / min for 72 to 96h. The cultured spores were centrifuged at 4,000g for 5min to obtain a precipitate, which was then washed with PBS and resuspended. According to an inoculation amount of 10%, 0.2mL of the spore suspension was inoculated into bile salt solutions with concentrations of 0.1%, 0.3% and 0.5%, respectively. After anaerobic culture at 37°C for 3h, the bacterial solution was spread on iron sulfite agar medium to evaluate the number of live bacteria. Three replicates were set for each treatment. The results of the tolerance test of Clostridium butyricum YF1 from eel to bile salts are shown in Figure 8 Within the concentration range of 0.5% for 3 hours, the survival rate of YF1 reached more than 90%.

[0080] (3) Tolerance to artificial gastrointestinal fluid

[0081] The eel-derived Clostridium butyricum YF1 obtained in Example 1 was inoculated into RCM liquid culture medium and cultured anaerobically at 37°C and 220r / min for 72 to 96h. The cultured spores were centrifuged at 4,000g for 5min to obtain a precipitate, which was then washed and resuspended with PBS. 0.2mL of the spore suspension was inoculated into artificial gastric juice and artificial intestinal juice at an inoculation rate of 10%. After being treated at 37°C for 0, 1, 2, 3 and 4h, the samples were collected and coated on iron sulfite agar medium to evaluate the number of viable bacteria. Three replicates were set for each treatment.

[0082] Artificial gastric juice and intestinal juice are prepared as follows. Artificial gastric juice: Mix 16.4 mL of dilute hydrochloric acid with about 800 mL of water, then add 10 g of pepsin. Stir the mixture thoroughly, then add water to make up to 1000 mL (pH = 1.5 ± 0.1). Artificial intestinal juice: Dissolve 6.8 g of potassium dihydrogen phosphate in 500 mL of water. Use 0.4% sodium hydroxide solution to adjust the pH to 6.8. Take another 10 g of trypsin and dissolve it in water, mix the two liquids thoroughly, and then add water to make the total volume reach 1000 mL (pH = 6.8 ± 0.1).

[0083] The results of the test on the tolerance of Clostridium butyricum YF1 from rice field eel to artificial gastric juice are shown in Fig. 9 Compared with the control group, the survival rate of YF1 spores was significantly reduced to 72%, 68%, 69% and 60% after 1, 2, 3 and 4 hours of artificial gastric juice treatment, respectively. Fig.10 Compared with the control group, the survival rate of YF1 spores did not change significantly after 1, 2, 3 and 4 hours of artificial intestinal fluid treatment. The above experimental results show that the butyric acid Clostridium YF1 from the eel has a strong tolerance to artificial gastric fluid, and its tolerance to artificial intestinal fluid is stronger than that to artificial gastric fluid.

[0084] Based on the experimental results of (1-3), it can be seen that the eel-derived Clostridium butyricum YF1 showed good tolerance to high temperature, bile salts and artificial gastrointestinal fluid, indicating that YF1 has strong stress resistance.

[0085] Antimicrobial susceptibility testing

[0086] The eel-derived Clostridium butyricum YF1 obtained in Example 1 was inoculated into RCM liquid culture medium and cultured anaerobically at 37°C and 220 r / min for 18 to 24 h. 5 CFU / mL) was inoculated on iron sulfite agar medium, and different antibiotic susceptibility tablets were placed in the center of the medium. After anaerobic culture at 37°C for 48 hours, the diameter of the inhibition zone was measured. Three replicates were set for each treatment. According to the "Disc Diffusion Method for Determination of Bacterial Resistance in Animals and Their Products" (SN / T 1944-2016) and the "Manual for Determination of Bacterial Drug Susceptibility Tests", the results of the drug sensitivity test were divided into: resistant, moderately sensitive, and sensitive according to the diameter of the inhibition zone. The results of drug sensitivity test are shown in Table 2. Strain YF1 was highly sensitive to 21 antibiotics, including ampicillin, carbenicillin, cefazolin, cefoperazone, cefradine, ceftriaxone, cefuroxime, cephalexin, chloramphenicol, ciprofloxacin, clindamycin, doxycycline, erythromycin, furazolidone, minocycline, norfloxacin, ofloxacin, penicillin, piperacillin, tetracycline and vancomycin; moderately sensitive to 4 antibiotics, including amikacin, kanamycin, midecamycin and oxacillin; and strongly resistant to 5 antibiotics, including ceftazidime, cotrimoxazole, gentamicin, neomycin and polymyxin B. The results showed that YF1 was sensitive to most antibiotics and had low drug resistance.

[0087] Table 2. Results of drug sensitivity test

[0088]

[0089] Example 3

[0090] Experiment on feeding eel seedlings with Clostridium butyricum YF1 from monopterus albus

[0091] 1. Formula of basic feed

[0092] The basic feed formula for yellow eel used in this experiment is, calculated by mass percentage, including: 40% fish meal, 12% chicken meal, 12.15% soybean meal, 8% squid meal, 21% pregelatinized starch, 1.5% soybean oil, 0.35% choline chloride, 1% compound multivitamin, 1.5% compound multimineral, 1.5% monocalcium phosphate, and 1% microcrystalline cellulose. Among them, 1kg of composite multi-mineral components includes: Ca(H2PO4)2 1800mg, KH2PO4 1350mg, NaCl 500mg, MgSO4·7H2O 750mg, NaH2PO4·2H2O 650mg, KI 1.5mg, COSO4·6H2O 2.5mg, CuSO4·5H2O 15mg, ZnSO4·7H2O 350mg, FeSO4·7H2O1250mg, MnSO4·4H2O 80mg, and Na2SeO3 6.00mg. 1kg of complex multivitamin components includes: VB1 50mg, VB2 200mg, VB6 50mg, VB12 20mg, folic acid 15mg, pantothenic acid 400mg, inositol 1500mg, D-biotin (2%) 5mg, niacin 750mg, VA2.5mg, VE (50%) 160mg, VD3 2mg, VK20 mg.

[0093] The eel-derived Clostridium butyricum YF1 obtained in Example 1 was inoculated into RCM liquid culture medium and cultured anaerobically at 37°C and 220 r / min for 24 h. Then, it was inoculated into spore-forming broth culture medium at a 2% inoculum amount and cultured anaerobically at 37°C and 220 r / min for at least 72 h. The spores were checked under a microscope to ensure that all spores were formed. The precipitates were obtained by centrifugation at 4000 g for 10 min, and then resuspended in distilled water to 10 5 , 10 6 , 10 7 , 10 8 , 10 9 CFU / mL five concentration gradients. The spore suspension was stored in a 4°C refrigerator and renewed once a week. The basic feed was prepared according to the above formula, and the bacterial suspension was evenly sprayed on the basic feed. The control group was sprayed with the same volume of distilled water. During the spraying process, the mixture was stirred continuously and then fed. The feed fed daily was prepared and used immediately.

[0094] The formula of spore-forming broth medium (pH=7.8±0.1) is: 15.0 g / L of polyvalent peptone, 3.0 g / L of yeast extract, 3.0 g / L of soluble starch, 0.1 g / L of magnesium sulfate, 1.0 g / L of sodium thioglycolate, and 11.0 g / L of disodium hydrogen phosphate.

[0095] 2. Feeding eels

[0096] The feeding experiment of eels was conducted at Fenggong Agriculture Co., Ltd. in Xiantao City, Hubei Province for 3 weeks. Healthy and uniform eel seedlings (weight 4.49±0.96g, body length 18.42±1.46cm) were selected and placed in 18 net frames (0.6*0.5*0.35m) on average. They were divided into six groups, with three replicates in each group and 70 eels in each replicate. The eels in the control group (CK) were fed with the basic bait sprayed with distilled water, and the eels in the treatment groups (T1, T2, T3, T4 and T5) were fed with 10 5 , 10 6 , 10 7 , 10 8 , 10 9 CFU / mL eel-derived Clostridium butyricum YF1 spore suspension. During the feeding period, eels were fed once at 10:00 am every day, and the sewage was sucked out once 1 hour after feeding and at 15:00 pm. The water temperature for eel rearing was 29.8±1.8℃.

[0097] 3. Intestinal digestive enzyme activity detection

[0098] Six eels were randomly selected from each group in the feeding experiment. They were first anesthetized with iodine, and then the intestinal tissue was dissected and ground evenly. The contents of amylase and lipase in the eel intestine were detected using a kit (Nanjing Jiancheng Bioengineering Co., Ltd.). The results of the intestinal amylase and lipase activity detection of the eel are shown in Table 1. Fig.11 and Fig.12 Compared with the control group, after feeding for 3 weeks, the addition concentration of 10 8 The amylase activity of the group treated with CFU / mL butyric Clostridium YF1 from rice field eel (T4) was significantly higher than that of the control group (P<0.05), and the lipase activity of the five treatment groups with added Clostridium butyricum was significantly higher than that of the control group (P<0.05). The results showed that butyric Clostridium YF1 from rice field eel can significantly enhance the intestinal digestive enzyme activity of rice field eel, thereby improving the intestinal health of the body.

[0099] In summary, the eel-derived Clostridium butyricum YF1 isolated and screened from the intestinal tract of the eel of the present invention has a strong acid production capacity, especially high production of acetic acid and butyric acid, strong tolerance to high temperature, bile salts and artificial gastrointestinal fluid, and sensitivity to most antibiotics. When an appropriate concentration of eel-derived Clostridium butyricum YF1 is mixed and fed, the intestinal digestive enzyme activity of the eel can be significantly improved, thereby protecting the intestinal health, and has a good application prospect in the breeding and production of the eel.

[0100] The specific implementation of the present invention described above does not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A strain of Clostridium butyricum YF1 derived from yellow eel, characterized in that: It is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO:M20242414.

2. The eel-derived Clostridium butyricum YF1 according to claim 1, characterized in that The 16s rDNA sequence of the eel-derived Clostridium butyricum YF1 is shown in SEQ ID NO:

1.

3. A method for culturing the eel-derived Clostridium butyricum YF1 according to any one of claims 1 to 2, characterized in that: This includes culturing under anaerobic conditions at 35-37°C.

4. The culture method according to claim 3, characterized in that The culture medium used includes: yeast extract powder 30-32g, glucose 10-12g, sodium chloride 2-2.5g, magnesium sulfate heptahydrate 0.25-0.28g, dimethyl phosphate 2-2.5g, L-cysteine ​​hydrochloride 0.5-0.7g, and water to 1000mL; and / or, the culture medium used includes: tryptone 10-12g, beef extract 10-12g, yeast extract powder 3-4g, glucose 5g, soluble starch 1-1.5g, chloride 5-6g sodium chloride, 3-4g anhydrous sodium acetate, 0.5-0.6g L-cysteine ​​salt, 20-22g solid culture medium agar, 1000-1100mL water; and / or, 15.0-15.2g / L polyvalent peptone, 3.0-3.2g / L yeast extract, 3.0-3.2g / L soluble starch, 0.1-0.2g / L magnesium sulfate, 1.0-1.2g / L sodium thioglycolate, and 11.0-11.2g / L disodium hydrogen phosphate.

5. Use of the eel-derived Clostridium butyricum YF1 as claimed in any one of claims 1 to 2 as a feed additive.

6. The use according to claim 5, characterized in that: The feed additive is a fish feed additive.

7. The use according to claim 6, characterized in that: The fish is yellow eel.

8. The use according to claim 5, characterized in that: The amount of Clostridium butyricum YF1 from eel added to the feed was 10 7 -10 9 CFU / g.

9. A feed additive, characterized in that: It includes the eel-derived Clostridium butyricum YF1 as described in any one of claims 1-2.

10. Use of the eel-derived Clostridium butyricum YF1 according to any one of claims 1 to 2 in producing acetic acid and / or butyric acid.

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