CN118028161A - Clostridium pelteobagrus-source clostridium butyricum B3 capable of producing short-chain fatty acid in high yield and application thereof - Google Patents
Clostridium pelteobagrus-source clostridium butyricum B3 capable of producing short-chain fatty acid in high yield and application thereof Download PDFInfo
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- CN118028161A CN118028161A CN202410172466.3A CN202410172466A CN118028161A CN 118028161 A CN118028161 A CN 118028161A CN 202410172466 A CN202410172466 A CN 202410172466A CN 118028161 A CN118028161 A CN 118028161A
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- Prior art keywords
- clostridium butyricum
- pelteobagrus fulvidraco
- chain fatty
- short
- acid
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- 241000193171 Clostridium butyricum Species 0.000 title claims abstract description 101
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- 241000193403 Clostridium Species 0.000 title description 3
- 241000376029 Tachysurus fulvidraco Species 0.000 claims abstract description 43
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- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims description 25
- 239000001963 growth medium Substances 0.000 claims description 23
- 235000021391 short chain fatty acids Nutrition 0.000 claims description 15
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims description 14
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 claims description 14
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/52—Propionic acid; Butyric acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/54—Acetic acid
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/145—Clostridium
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- Polymers & Plastics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Food Science & Technology (AREA)
- Animal Husbandry (AREA)
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- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
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Abstract
The invention discloses a high-yield short-chain fatty acid clostridium butyricum B3 of pelteobagrus fulvidraco and application thereof, wherein the clostridium butyricum B3 of pelteobagrus fulvidraco isolated by the invention is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20231711. The clostridium butyricum B3 has the characteristics of short-time high-yield short-chain fatty acid, high environmental tolerance and sensitivity to antibiotics, and can obviously improve the growth rate of pelteobagrus fulvidraco, improve the liver digestive enzyme activity and the liver nonspecific immunity and antioxidation capability, change the composition and diversity of intestinal microorganisms, and concentrate dominant strains in o-spora and bacillus (P < 0.01). The invention provides a clostridium butyricum which has the characteristics of short-time high-yield short-chain fatty acid, safety, no toxicity, simple separation operation and high absorption rate of freshwater fish, can be used as a feed additive, can be used for rapidly conditioning intestinal tracts of pelteobagrus fulvidraco, and can be applied to promoting rapid growth of pelteobagrus fulvidraco.
Description
Technical Field
The invention belongs to microorganism separation culture and aquatic animal feed, and particularly relates to clostridium butyricum B3 of pelteobagrus fulvidraco sources with high yield of short-chain fatty acid and application thereof.
Background
Pelteobagrus fulvidraco (Pelteobagrus fulvidraco) is a rare freshwater economic fish commonly called Huang Lading, yellow drum fish and the like, has tender meat quality, no intramuscular thorns, multiple fat, contains multiple amino acids required by multiple human bodies, and has high nutritive value. In recent years, due to the expansion of the cultivation scale and the improvement of the cultivation density, how to promote the germplasm resources, improve the cultivation conditions and increase the mu yield is a great difficulty for pelteobagrus fulvidraco cultivation. Probiotics such as lactobacillus, bacillus, bifidobacterium and the like are widely used in fresh water aquiculture at present, and clostridium butyricum has high difficulty in separation culture and is not widely applied to fresh water aquiculture at present.
Clostridium butyricum (Clostridium butyricum) is a probiotic that improves gut flora composition and maintains gut health in the host. It can inhibit invasion of pathogenic bacteria by improving microorganism composition of host or its surrounding environment, and can reach intestinal flora to balance state. The major metabolites Short Chain Fatty Acids (SCFAs), such as acetate, propionate and butyrate, are important energy sources for the intestinal flora and host epithelial cells and are also important mediators of communication between the intestinal flora and host metabolism. SCFAs can regulate host intestinal immunity and reduce intestinal inflammatory response through various regulation mechanisms. SCFAs can regulate host intestinal immunity and reduce intestinal inflammatory response through various regulation mechanisms.
Clostridium butyricum has been widely used as probiotics in aquaculture, however, most probiotics used in fresh water aquaculture are derived from terrestrial animals at present, few probiotics derived from aquatic animals lack host specificity, and the fish colonization rate is difficult to guarantee. And clostridium butyricum with different sources is difficult to ensure safety, and the expression efficiency of functional genes is low. The clostridium butyricum for aquatic products, which has been reported at present, has limitations, and the clostridium butyricum which is a fish source and is easy to separate and culture, high in short-chain fatty acid output efficiency and high in safety is comprehensively considered to be a main research and development direction.
Disclosure of Invention
The invention aims to: aiming at the problems in the prior art, the invention provides the clostridium butyricum B3 which is a yellow catfish source and can effectively improve the host absorption rate and promote the host growth, so as to solve the problems in the prior freshwater aquaculture technology.
It is a further object of the invention to provide the use of the strain.
The technical scheme is as follows: in order to achieve the aim, the clostridium butyricum B3 which is the high-yield short-chain fatty acid and is derived from pelteobagrus fulvidraco is identified as clostridium butyricum (Clostridium butyricum) and is preserved in China center for type culture collection, the preservation time is 2023 and 09 months 18, and the preservation number is CCTCCNO: M20231711.
The clostridium butyricum B3 is applied to short-time high-yield short-chain fatty acid.
Wherein the clostridium butyricum B3 can produce short-chain fatty acids with high yield within 24 hours, and the short-chain fatty acids comprise acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and caproic acid.
Preferably, the clostridium butyricum B3 is capable of producing short-chain fatty acids in high yield within 24 hours, wherein the short-chain fatty acids comprise acetic acid and butyric acid.
The clostridium butyricum B3 is applied to fish feed additives.
Wherein the fish is pelteobagrus fulvidraco.
The clostridium butyricum B3 is used as a feed additive for rapidly conditioning intestinal tracts of pelteobagrus fulvidraco and promoting rapid growth of pelteobagrus fulvidraco.
The clostridium butyricum B3 is used as a feed additive and is particularly applied to improving the growth performance of pelteobagrus fulvidraco, liver digestive enzyme activity and regulating liver nonspecific immunity and antioxidation capability.
The method for culturing clostridium butyricum B3 is characterized by being rapid and simple, wherein the separation operation process is strictly operated in an anaerobic environment (the oxygen concentration is less than 0.5 percent), and the solid culture medium and the liquid culture medium used in the culturing process are strictly deoxidized.
Wherein the culture medium is an RCM anaerobic solid culture medium and mainly comprises 8-12g of tryptone, 8-12g of beef extract, 2-4g of yeast extract powder, 4-6g of glucose, less than or equal to 1g of soluble starch, 4-6g of sodium chloride, 2-4g of anhydrous sodium acetate, less than or equal to 0.5g of L-cysteine acid salt, 15-25g of agar and 1000mL of ultrapure water, wherein the pH value of the agar is 6.9+/-0.5; the liquid medium does not contain the agar.
Preferably, the culture medium is an RCM anaerobic solid culture medium and mainly comprises 10g of tryptone, 10g of beef extract, 3g of yeast extract powder, 5g of glucose, 1g of soluble starch, 5g of sodium chloride, 3g of anhydrous sodium acetate, 0.5g of L-cysteine salt, 20g of agar, 1000mL of ultrapure water, and pH of 6.9+/-0.1, wherein the liquid culture medium does not contain the agar.
The clostridium butyricum B3 separated and screened by the method is obtained by separating the clostridium butyricum from a fish source for the first time, is used as an anaerobic bacterium capable of forming spores, mainly exists in the intestinal tract of a host, has extremely high temperature resistance and extremely acid resistance, and is sensitive to antibiotics. According to the invention, the clostridium butyricum can not only produce short-chain fatty acid in a short time and high yield, but also feed the clostridium butyricum with proper concentration to be beneficial to improving the growth performance of pelteobagrus fulvidraco and regulating the steady state of intestinal flora. The main metabolite short chain fatty acid of clostridium butyricum, such as acetic acid, butyric acid and the like, can maintain higher content, and has certain indication effect on the growth performance of pelteobagrus fulvidraco.
The isolated and screened clostridium butyricum B3 of the pelteobagrus fulvidraco can not only produce short-chain fatty acid in short time and have the characteristics of high environmental tolerance and sensitivity to antibiotics, but also can obviously improve the growth speed and liver digestive enzyme activity of pelteobagrus fulvidraco and enhance the liver nonspecific immunity and antioxidation capability, change the composition and diversity of intestinal microorganisms, and concentrate dominant strains in o-spora and bacillus (P < 0.01).
Further, short chain fatty acids produced by clostridium butyricum B3 of the present invention include, but are not limited to, acetic acid, butyric acid; the high-temperature tolerance rate is very strong (the survival rate at 80 ℃ is more than 85 percent) and the strong acid can still survive in the reverse environment from strong base; is sensitive to most antibiotics, and has strong inhibition effect, including ampicillin, chloramphenicol, erythromycin, lincomycin, penicillin, tetracycline and ciprofloxacin.
The invention can only separate clostridium butyricum B3 with strange condition, such as anaerobic gas generating bag, anaerobic operation table, anaerobic culture solution and anaerobic fermentation solution. Clostridium butyricum B3 is heat-resistant, heated at 80deg.C for 10min, and the tolerance is maintained at 85.12%. Clostridium butyricum B3 tolerance to pH: acid resistance is lowest at ph=3; after alkali resistance at pH >7, the decrease was significant and at ph=7, the optimum growth pH. Clostridium butyricum B3 has strong inhibition effect on most antibiotics, has no drug resistance and has higher safety. When clostridium butyricum B3 is used for feeding pelteobagrus fulvidraco, the feeding dosage is that: 3.0X10 7 CFU/g, remarkably improves growth performance, liver digestive enzyme activity and regulates liver nonspecific immunity and antioxidant capacity.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
The clostridium butyricum B3 separated and screened by the invention has the characteristics of short-time high yield of short-chain fatty acid, high environmental tolerance and sensitivity to antibiotics, particularly has the characteristic of extremely high temperature resistance, can obviously improve the growth speed and liver digestive enzyme activity of pelteobagrus fulvidraco and enhance the liver nonspecific immunity and oxidation resistance, changes the composition and diversity of intestinal microorganisms, and has the advantages that dominant strains are concentrated in o-spora and bacillus (P < 0.01).
The fish source clostridium butyricum B3 has the characteristics of short-time high-yield short-chain fatty acid, safety, no toxicity, simple separation operation and high absorption rate of freshwater fish, can be used as a feed additive, can be used for rapidly conditioning intestinal tracts of pelteobagrus fulvidraco, and can be applied to promoting rapid growth of pelteobagrus fulvidraco.
Drawings
FIG. 1 is a graph of Clostridium butyricum B3 colonies;
FIG. 2 is a microstructure of Clostridium butyricum B3;
FIG. 3 is a diagram showing the subcellular structure of Clostridium butyricum B3;
FIG. 4 is a C.butyricum B3 16S rDNA phylogenetic tree;
FIG. 5 is the effect of different pH stresses on Clostridium butyricum B3;
FIG. 6 is a TOP plot of dominant bacteria relative abundance at the intestinal content gate, mesh, genus level of pelteobagrus fulvidraco after feeding with clostridium butyricum B3 at different concentrations;
FIG. 7 is a diagram of LEfSe analysis of differential species between groups;
FIG. 8 is a graph of significant differences in the functional enrichment of the intestinal flora KEGG-Level3 predicted by the Tax4Fun analysis (P < 0.01);
FIG. 9 is a graph showing the change of liver nonspecific immunity enzyme activity after feeding Clostridium butyricum at different concentrations;
FIG. 10 is a graph showing changes in liver antioxidant enzyme activity after feeding Clostridium butyricum at different concentrations;
FIG. 11 is a graph showing changes in liver digestive enzyme activity after feeding Clostridium butyricum at different concentrations;
FIG. 12 shows the variation of the amount of short chain fatty acids in the fermentation broth with fermentation time.
Detailed Description
The invention is further described below with reference to the drawings and examples.
The raw materials and the reagents in the invention are commercially available unless otherwise specified.
The fish used in the embodiment of the invention is commercial conventional pelteobagrus fulvidraco.
Example 1
1. Isolation of clostridium butyricum
Under the aseptic condition, 1g of intestinal contents of commercial pelteobagrus fulvidraco are collected into an aseptic EP tube (placed at the temperature of minus 20 ℃ for standby), transferred into a 15mL centrifuge tube containing 9mL PBS (pH 7.4), immediately prepared into intestinal content diluent, heated for 10min at the temperature of 80 ℃ in a water bath kettle, taken out, oscillated and mixed uniformly, and the bacterial liquid after water bath is inoculated with RCM liquid culture medium for enrichment culture in an amount of 5% by volume, and cultured for 24h at the temperature of 37 ℃ and at the speed of 200 rpm. Carrying out gradient dilution on the enrichment culture solution, absorbing 20 mu L of each dilution solution to an RCM solid culture medium, uniformly coating the surface, after the gradient dilutions are completely absorbed, inverting, placing an RCM flat plate into a 15L self-sealing bag, adding a proper amount of anaerobic gas production bag before sealing, culturing for 48 hours at 37 ℃ in an anaerobic mode (O 2 is less than or equal to 1%), picking a bacterial strain with a rod-shaped microscopic morphology, carrying out subsequent 16S rDNA sequence strain identification and characteristic identification, and screening to obtain a high-quality bacterial strain named as B3.
The composition of the Reinforced Clostridium (RCM) culture medium is as follows: 10g of tryptone, 10g of beef extract, 3g of yeast extract powder, 5g of glucose, 1g of soluble starch, 5g of sodium chloride, 3g of anhydrous sodium acetate, 0.5g of L-cysteine acid salt, (20 g of solid culture medium agar), 1000mL of distilled water, pH 6.9+/-0.1, sterilizing by high-pressure steam at 121 ℃ for 20min, and deoxidizing the mixed gas (H 2 5％、CO2 15％、N2%) for 30min.
2. Identification of the isolated and screened Strain B3
(1) Colony characteristics: the colony is round, irregular in edge, slightly convex, milky white, viscous and sour and odorous, as shown in figure 1.
(2) Cell morphology characterization: rod-shaped, the top end is round and is on. The microscopic form and the scanning electron microscope form are shown in fig. 2 and 3.
(3) Identification of strain molecular biology and construction of phylogenetic tree:
1) Enrichment of bacterial cells
And (3) picking a single colony B3 after purification, inoculating the single colony B3 into an RCM liquid culture medium, shaking the bacteria to a logarithmic phase, and preserving the bacteria at 4 ℃.
2) PCR amplification and construction of phylogenetic tree
Using the bacterial solution obtained in the step 1) as an amplification template, and adopting bacterial 16S rDNA universal primers 27F:5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R: performing PCR amplification on 5'-TACGGYTACCTTGTTACGA CTT-3'; the PCR amplification system is as follows: 2X RAPID TAQ MASER Mix 10. Mu.L, bacterial liquid 1. Mu.L, 27F 0.8. Mu.L, 14992R 0.8. Mu.L, H 2 O7.4. Mu.L. The PCR reaction conditions were: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1min for 35 cycles; extending at 72℃for 10min. mu.L of the PCR product was electrophoresed in 1% agarose gel to verify the molecular weight of the product. The amplified 16S rNDA sequence was sent to the south Beijing sequencing department of Shanghai Biotechnology Co., ltd for sequencing. And (3) uploading the sequencing sequence to an NCBI nucleic acid comparison website for comparison, selecting a sequence with highest sequence homology, and constructing a phylogenetic tree by using MEGA 8.0 software. Phylogenetic tree based on 16S rDNA is shown in FIG. 4.
The 16S rDNA sequence of the strain B3 is identified by sequencing:
CGGCTTACCATGCAGTCGAGCGATGAAGCTCCTTCGGGAGTGGATTAGCG
GCGGACGGGTGAGTAACACGTGGGTAACCTGCCTCATAGAGGGGAATAG
CCTTTCGAAAGGAAGATTAATACCGCATAAGATTGTAGTACCGCATGGTA
CAGCAATTAAAGGAGTAATCCGCTATGAGATGGACCCGCGTCGCATTAGC
TAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGA
GAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGG
AGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCAA
CGCCGCGTGAGTGATGACGGTCTTCGGATTGTAAAGCTCTGTCTTTAGGG
ACGATAATGACGGTACCTAAGGAGGAAGCCACGGCTAACTACGTGCCAG
CAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTACTGGGCGT
AAAGGGAGCGTAGGTGGATATTTAAGTGGGATGTGAAATACCCGGGCTTA
ACCTGGGTGCTGCATTCCAAACTGGATATCTAGAGTGCAGGAGAGGAAAG
GAGAATTCCTAGTGTAGCGGTGAAATGCGTAGAGATTAGGAAGAATACCA
GTGGCGAAGGCGCCTTTCTGGACTGTAACTGACACTGAGGCTCGAAAGCG
TGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATG
AATACTAGGTGTAGGGGTTGTCATGACCTCTGTGCCGCCGCTAACGCATT
AAGTATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATT
GACGGGGGCCCGCACAAGCAGCGGAGCATGTGGTTTAATTCAGAACCAA
CGAGAAGCACTTTACATAGACGAGACATCTCGAGAATTTCTGTGTAAGAG
AGGAACCCATTTCGGTCGCAAGAAGACAGGTGGCGCATGGTTGTTCTCAG
CTCGTGTCGTGAGATGTTGGGATATGTCCCGCAACGAGCGCACCCTTTATT
GATAGTCGCTCCCATTTAGGTGCGCTCTATAGCGAGACTCCCCGGGTTCAC
CGGGAAGAAGGTGGGGATGATGTCATATCATCATGCCCCTTATGTATAGG
GATACACACGTGATACAATGGTCGGTACAATGAGACGCAACCTCGAGAG
AGAGAGCAAAACTATAAAACCGATCTCAGTTGGGATTGTAGGGTGAATCT
CGCATACAAGAAGGGGGAGTTTATTGTAATCGAGAATCAGAATGTCGCGG
AGAATACTTTCCCGCGCCTTGCACACCCCCCCTCTCACCCCATGAGAGTTCGCAATACCACAAATTTCGTGAGCTAACCGCAAGGAGGCAGCGACGT。
Based on physiological and biochemical properties and on treeing analysis based on 16s rDNA sequences, as well as genomic analysis, the strain was identified as Clostridium butyricum (Clostridium butyricum) and designated Clostridium butyricum (Clostridium butyricum) B3. The strain B3 is delivered to China with the preservation time of 2023 and 09 months and 18 days, and the preservation number is CCTCC NO: M20231711.
Example 2
Tolerance test of clostridium butyricum B3
1) PH tolerance of clostridium butyricum B3
Clostridium butyricum B3 obtained in example 1 was inoculated onto RCM liquid medium, cultured at 37℃under shaking at 200rpm, centrifuged at 4000rpm at 4℃for 5min after the logarithmic phase, the supernatant was discarded, 1g of Clostridium butyricum B3 cells were added to 15mL centrifuge tubes containing 9mL of sterile 1 XPBS solution having pH of 1.0, 3.0, 5.0, 7.0, 9.0, 11.0 and 13.0, and after culturing in a 37℃water bath for 2h, gradient dilution was performed, and the gradient dilution was placed on RCM solid medium, evenly spread, and cultured at 37℃under anaerobic conditions for 48h, followed by colony counting. Clostridium butyricum B3 pH tolerance was examined using the LOG10 value of colony count as an index.
As shown in FIG. 5, when the pH is 1.0 to 5.0, the Clostridium butyricum B3 cell concentration is decreased and then increased significantly; when the pH is 5.0-7.0, clostridium butyricum B3 is presented; at a pH of 7.0, clostridium butyricum B3 reached the highest biomass; the concentration of the bacterial cells gradually decreases between pH 9.0 and 13.0, and clostridium butyricum B3 can still grow at pH 13.0. Clostridium butyricum B3 has a certain degree of acid and alkali resistance, and the pH is the optimal growth pH of clostridium butyricum B3 when the pH is 7.0.
2) High temperature tolerance of clostridium butyricum B3
Clostridium butyricum B3 obtained in example 1 is inoculated into an anaerobic RCM liquid culture medium, after shaking at 37 ℃ and 200rpm for 24 hours, equal amount of bacteria liquid is respectively placed at 37 ℃ (contrast), 80 ℃ and 90 ℃ for 10min, 10min and 5min of water bath treatment, gradient dilution is carried out, the gradient dilution is placed into an anaerobic RCM solid culture medium, uniform coating is carried out, anaerobic (O 2 is less than or equal to 1%) culture is carried out for 48 hours, and colony counting is carried out. Clostridium butyricum B3 high temperature tolerance was examined using the colony number as an index.
The results are shown in Table 1, and the B3 tolerance rate of 80 ℃ treatment reaches 85.12% compared with the control group; the tolerance rate of B3 treated at 90 ℃ still keeps 60.82%, and the above effects can be achieved by multiple tests. Clostridium butyricum B3 has excellent high temperature resistance.
TABLE 1 Clostridium butyricum B3 tolerance under high temperature stress
Example 3
Antibiotic resistance detection of clostridium butyricum B3
Clostridium butyricum B3 obtained in example 1 is inoculated into RCM liquid culture medium, shaken at 37 ℃ for 24 hours at 200rpm, evenly coated on RCM culture medium flat plate, bacteria can grow, after bacteria liquid is completely absorbed, different antibiotic drug sensitive tablets are clamped to the center of the flat plate by using sterile forceps, anaerobic culture is carried out at 37 ℃ for 48 hours, and the diameter of a bacteria inhibition zone is observed. According to the general bacteria identification handbook, the drug sensitivity experimental results are divided into the following three stages: (0-5 mm) no effect, (6-15 mm) weak effect and (> 15 mm) strong inhibition effect.
As shown in table 2, clostridium butyricum B3 has strong inhibition effect on most antibiotics, has no drug resistance and has higher safety.
TABLE 2 Clostridium butyricum antibiotic resistance test results
Example 4
Feeding experiment
The experimental place for cultivation is the institute of water production at the university of Nanjing, and the experimental young fish is self-bred in the institute. The cells were temporarily maintained in a glass box (2.0X1.0X0.8m) for 2 weeks, during which time the Tongwei expanded compound feed (crude protein 40%, crude fat > 5%) was fed. The raw materials of the feed (the basic feed puffed compound feed) are crushed and pass through a 60-mesh sieve, then clostridium butyricum microbial inoculum is added, water is added and uniformly stirred, and a double-screw extruder is used for extruding the raw materials into strips and preparing the 1.0mm pellet feed for standby. The live bacterial values of Clostridium butyricum B3 in test feeds CB1, CB2 and CB3 were 3.0X10 5、3.0×107 and 3.0X10 9 CFU/g, respectively. The breeding experiment selects pelteobagrus fulvidraco juvenile fish with healthy constitution and consistent specification (initial weight is 6.75+/-0.13 g), randomly divides the pelteobagrus fulvidraco juvenile fish into 4 treatment groups, and feeds the following three feeds respectively: CB1, CB2, CB3 and basal feed group (puffed compound feed) (Control group). Each treatment group was repeated 3 times, one tank (0.8X0.5X0.5 m) each, 65 fish per tank. Feeding was done satiety twice daily at 9:00 a day and 18:00 a day later during the cultivation experiments, weighing and recording the feed weight. During the test, the water temperature is 20-30 ℃, the pH is about 7.6, and the dissolved oxygen is more than 6mg/L. After the cultivation test is finished for 30 days, the body weight and the body length of the tested fish are respectively measured, then intestinal tracts and liver tissues are collected, and intestinal tract contents are aseptically collected, so that the sequencing of the intestinal microorganism 16S rDNA is completed.
Growth data for the 30 day culture experiments are shown in table 3. When the initial weight is equal to the daily feed intake as compared with the Control group, the final weight is decreased and then increased with the increase of the clostridium butyricum B3 addition concentration, the specific growth rate (Specific Growth Rate, SGR) is decreased and then increased, and the feed conversion rate (Feed Conversion Ratio, FCR) is increased and then decreased. The three groups of growth data are synthesized, and the CB3 group pelteobagrus fulvidraco growth performance shows the most beneficial growth effect (P < 0.05) and is specifically expressed as follows: when the initial weight and daily feed intake basal numbers are the same, the weight gain rate (WEIGHT GAIN RATE, WGR) of the CB3 group is maximum, the SGR value is maximum and the FCR value is minimum compared with other groups.
TABLE 3 influence of Clostridium butyricum B3 on the growth Performance of pelteobagrus fulvidraco
Note that: different lowercase letters indicate significant differences (P < 0.05).
Intestinal microbiota analysis
The 15 intestinal tract content samples were taken and the 16S rRNA gene V4 variable region was sequenced to obtain 1953, 431reads, and OTU numbers from 92 to 251.
The α -diversity index calculation results are shown in Table 4, in which, except Shannon, simpson indexes are highest in CB1 group and there is a significant difference (P < 0.05), the observed features, chao, PD white tree indexes are all highest in Control group, while ace and good coverage indexes are not significantly different (P < 0.05).
TABLE 4 influence of Clostridium butyricum B3 on the alpha-diversity index of the intestinal flora
Note that: data are mean ± standard error (n=4); * Indicating significant differences from each other (P < 0.05).
As can be obtained by sequencing the 16S rRNA, dominant bacteria at the gate level in the intestinal contents of pelteobagrus fulvidraco are mainly Proteus (Proteus), fusobacterium (Fusobacteriota), bacteroides (Bacteroidota) and Thick-walled bacteria (Firmicutes), and are shown in FIG. 6. The relative abundance of Proteus and Thick-walled bacteria in the CB1, CB2, and CB3 groups was increased compared to the control group. Wherein, the Proteus gate is obviously increased, and the relative abundance CB1 (92.66%) > CB3 (34.19%) > CB2 (14.89%) > control group (14.41%). On the genus level, the dominant bacteria were O. Plesiomonas (Control: 11.10%; CB1:91.50%; CB2:12.52%; CB3: 27.09%), C. Cetobacterium (Control: 55.99%; CB1:2.66%; CB2:0.05%; CB3: 59.54%), A.aeromonas (Control: 0.07%; CB1:0.19%; CB2:4.85%; CB3: 1.95%), B.bacillus (Control: 0.17%; CB1:0.80%; CB2:0.03%; CB3: 0.58%), C. Romboutsia (Control: 3.56%; CB1:0.46%; CB2:3.48%; CB3:0.01%, C.1 Clostridium_sendsu_1 (Control: 1.53%; CB1:0.32%; CB2:4.67%; C.7%) and FIG. 7.
The species of difference between the experimental and control groups were analyzed by LEfSe, as shown in fig. 7. Control group significantly enriched with firmicutes, CB1 group significantly enriched with Actinomyces _ MASSILIENSIS (actinomycota), sutturella _ timonensis (actinomycota), oscillibacter (firmicutes), tub_ borchii _ symbiont _b_17bo (bacteroides), CB2 group significantly enriched with clostridium_sendu_stricto_1 (firmicutes), CB3 group significantly enriched with Eionea _nigra (actinomycota), poseidonibacter _ lekithochrous (archaea), macrococcus (firmicutes) [064] predicted intestinal flora function using Tax4Fun analysis to obtain KEGG 3 levels and ko relative abundance information, and analysis of variance found a significant difference (P < 0.01) pathway, relative abundance TOP4 pathway see fig. 8.
Liver-related enzyme activities are shown in FIGS. 9-11. Among the non-specific immunoenzymes (fig. 9), ACP activity was significantly increased in all three treatment groups compared to Control group, AKP activity was significantly higher in CB1 group (P < 0.05). In the antioxidant enzymes, the activity of SOD is obviously increased in the other two treatment groups except that the CB2 group and the Control group have no obvious difference; treatment group MDA activity was significantly lower than Control group, but there was no significant difference between the three treatment groups (fig. 10); compared with Control, CAT activity is not obviously different from that of CB1 group, and the CAT activity is obviously increased along with the increase of the concentration of bacteria. The CB1 group and the CB2 group of digestive enzymes are obviously higher than the Control group, and the trypsin and the lipase activities are CB1 group > CB2 group except the alpha amylase activity CB2 group > CB1 group, which is shown in figure 11. The embodiment demonstrates that clostridium butyricum B3 can be used as a feed additive to rapidly condition intestinal tracts of pelteobagrus fulvidraco, promote the rapid growth of pelteobagrus fulvidraco, remarkably improve the growth performance of pelteobagrus fulvidraco, the liver digestive enzyme activity and regulate the liver nonspecific immunity and the antioxidation capability.
Example 5
Clostridium butyricum B3 short chain fatty acid production capability
The method comprises the following specific steps:
1) Preparing RCM culture medium and clostridium butyricum growth culture medium
RCM medium plates: 10g of tryptone, 10g of beef extract, 3g of yeast extract powder, 5g of glucose, 1g of soluble starch, 5g of sodium chloride, 3g of anhydrous sodium acetate, 0.5g of L-cysteine hydrochloride, 20g of solid culture medium agar, 1000mL of distilled water, pH 6.9+/-0.1, sterilizing by high-pressure steam at 121 ℃ for 20min, and deoxidizing the mixed gas (H 2 5％、CO2 15％、N2%) for 30min.
Clostridium butyricum growth medium: 30g of yeast extract powder, 10g of glucose, 2g of sodium chloride, 0.25g of magnesium sulfate heptahydrate, 2g of dimethyl phosphate, 0.5g of L-cysteine hydrochloride, 1000mL of distilled water, pH 7.0+/-0.1, sterilizing by high-pressure steam at 121 ℃ for 20min, and deoxidizing the mixed gas (H 2 5％、CO2 15％、N2%) for 30min.
2) Purification culture of clostridium butyricum
Clostridium butyricum B3 was inoculated into RCM liquid medium, cultured at 37℃and 200rpm to logarithmic phase, diluted in sterile PBS, and 20. Mu.L of the diluted solution was spread on RCM solid medium, and anaerobically cultured at 37℃for 24 hours.
3) Fermentation preparation of short chain fatty acids
Picking the single bacterial colony of clostridium butyricum B3 prepared in the step 2), inoculating to a 15mL anaerobic tube containing 10mL clostridium butyricum growth medium (RCM), culturing at 37 ℃ for 1, 2,4, 6 and 8 days, and sampling. The GC-MS method is used for measuring the content of short chain fatty acids (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and caproic acid) in the fermentation broth, and the result is shown in FIG. 12.
The results show that: in the fermentation period of the clostridium butyricum B3 single colony, the acetic acid content continuously rises and reaches a peak value in 1d within 0-1 d, the highest yield is 672.67 +/-51.67 mg/L at the moment, the acetic acid content slowly drops within 1-2 d, and the acetic acid content slowly rises after 2 d;
The propionic acid content continuously rises in 0-1 d and reaches a peak value in 1d, the highest yield is 14.10+/-0.30 mg/L at the moment, the propionic acid content slowly decreases in 1-2 d, the propionic acid content slowly increases in 2-6 d, and the propionic acid content slightly decreases in 6-8 d;
The butyric acid content in 0-1 d continuously rises and reaches a peak value in 1d, the highest yield is 890.00 +/-23.00 mg/L at the moment, the butyric acid content is slowly decreased in 1-2 d, the butyric acid content dynamically changes in 2-8 d, and the butyric acid content is wholly increased;
The isobutyric acid content continuously rises within 0-1 d and reaches a peak value at 1d, the highest yield is 11.90+/-0.30 mg/L at the moment, the isobutyric acid content obviously decreases within 1-2 d, and the isobutyric acid content increases within 2-8 d;
the valeric acid content continuously rises in 0-1 d and reaches a peak value in 1d, the valeric acid content slowly decreases in 1-2 d, the valeric acid content increases in 2-8 d, and the valeric acid content reaches the highest value of 0.60+/-0.00 mg/L in 8 d;
The isovaleric acid content continuously rises in 0-1 d and reaches a peak value in 1d, the isovaleric acid content slowly decreases in 1-2 d, the isovaleric acid content increases in 2-8 d, and the isovaleric acid content reaches the highest value of 3.93+/-0.23 mg/L in 8 d;
the caproic acid content in 0-4 d continuously rises and reaches a peak value at 4d, at the moment, the highest yield is 0.45+/-0.05 mg/L, and the caproic acid content is obviously reduced after 4 d.
This example demonstrates that clostridium butyricum B3 has the properties of short-term high yield of SCFA, especially acetic acid and butyric acid, with great potential as a probiotic for fish.
In sum, the clostridium butyricum B3 of the pelteobagrus fulvidraco source has certain antibiotic resistance, strong survivability under high temperature conditions, large pH tolerance range and strong SCFA production capability, and the growth performance of the pelteobagrus fulvidraco can be obviously improved after the clostridium butyricum B3 is fed by mixing, the composition and the diversity index of intestinal dominant flora are changed, and a plurality of SCFA producing bacteria are enriched. These results show that the clostridium butyricum B3 of pelteobagrus fulvidraco source can be used as a feed additive of pelteobagrus fulvidraco, and plays roles in promoting growth and improving intestinal microorganism composition.
Claims (10)
1. Clostridium butyricum B3 which is a yellow catfish source and is high in yield of short-chain fatty acid is identified as clostridium butyricum (Clostridium butyricum) and is preserved in China center for type culture collection (China center for type culture collection), wherein the preservation time is 2023, 09 and 18 days, and the preservation number is CCTCCNO: M20231711.
2. Use of clostridium butyricum B3 of claim 1 in short-term high yield of short chain fatty acids.
3. Use according to claim 2, characterized in that the clostridium butyricum B3 is highly productive of short chain fatty acids including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid within 24 h.
4. Use according to claim 2, characterized in that the clostridium butyricum B3 is highly productive of short chain fatty acids, preferably comprising acetic acid and butyric acid, within 24 hours of the strain.
5. Use of clostridium butyricum B3 of claim 1 as a fish feed additive.
6. The use according to claim 4, wherein the fish is pelteobagrus fulvidraco.
7. The use according to claim 4, characterized in that clostridium butyricum B3 is used as a feed additive for rapidly conditioning the intestinal tracts of pelteobagrus fulvidraco and promoting the rapid growth of pelteobagrus fulvidraco.
8. The use according to claim 6, characterized in that clostridium butyricum B3 is used as a feed additive for specifically improving the growth performance of pelteobagrus fulvidraco, liver digestive enzyme activity and regulating the liver nonspecific immunity and antioxidant capacity.
9. A method for culturing Clostridium butyricum B3 according to claim 1, wherein the separation process is performed strictly in anaerobic environment (oxygen concentration < 0.5%) and the solid and liquid culture mediums are strictly deoxygenated.
10. The culture method according to claim 9, wherein the culture medium is an RCM anaerobic solid culture medium, and mainly comprises 8-12g of tryptone, 8-12g of beef extract, 2-4g of yeast extract powder, 4-6g of glucose, less than or equal to 1g of soluble starch, 4-6g of sodium chloride, 2-4g of anhydrous sodium acetate, less than or equal to 0.5g of L-cysteine salt, 15-25g of agar, 1000mL of ultra-pure water and pH of 6.9+ -0.5; the liquid medium does not contain the agar.
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