CN110218676B - Clostridium butyricum and application thereof - Google Patents
Clostridium butyricum and application thereof Download PDFInfo
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Abstract
The invention discloses Clostridium butyricum and application thereof, wherein the Clostridium butyricum is Clostridium butyricum (Clostridium butyricum) SCUT620 which is preserved in Guangdong province microorganism strain preservation center in 2019, 3 and 29 days, and the preservation number is GDMCC NO: 60623, deposit address: zhou 100 Dazhou 59, building 5, Guangzhou institute of microorganisms. The strain is separated from healthy dairy cow dung, can directly utilize cheap starch substrates such as corn starch and cassava starch to ferment and produce butyric acid, and has higher butyric acid conversion rate; the strain can produce butyric acid by starch fermentation and can also prepare a clostridium butyricum viable bacteria preparation, the method can simultaneously produce the butyric acid and the clostridium butyricum viable bacteria preparation by one-time fermentation, and the method has the advantages of simple process and low production cost and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a clostridium butyricum with a starch substrate efficiently utilized, wherein the strain is used for producing butyric acid and preparing a clostridium butyricum viable bacteria preparation by utilizing corn starch and cassava starch for fermentation.
Background
N-butyric acid (butyric acid) is a short-chain organic carboxylic acid containing four carbon atoms, and butyric acid and its derivatives have wide application in the fields of chemical industry, food, medicine and feed industry [ Dwidar M et al, the future of butyric acid in industry, Sci World J,2012,471417 ]. At present, two methods of chemical synthesis and microbial fermentation are mainly used for producing butyric acid, and the chemical synthesis method mainly adopts an oxidized n-butyraldehyde method to prepare butyric acid, has the advantages of simple process, easily controlled conditions and high product yield, is the main method for producing industrial butyric acid at present, but the raw materials are derived from nonrenewable fossil resources, and toxic metal pollutants are generated in the reaction process, so the sustainability and the environmental protection are restricted. The method for producing butyric acid by using the microbial fermentation method is a new butyric acid production method in recent years, has the advantages of low raw material cost, mild fermentation conditions and small environmental pollution, and is favored by food, medicine and feed industries, but the prior microbial fermentation method still has the problems of low butyric acid conversion rate, difficult separation of byproducts and the like, so that the production cost is high, and the industrial application is limited. Therefore, in recent years, the research on the production of butyric acid by a microbial fermentation method mainly focuses on the problems of reducing the cost of raw materials, improving the conversion rate and selectivity of butyric acid [ Jiang L et al. butyl acid: Applications and receivances in ics bioproduction. Biotechnology Advances, 2018, 36 (8): 2101-2117].
Butyric acid can be produced by fermentation of microorganisms of the genera Clostridium (Clostridium), butyricum (butyrobacterium), vibrio (Butyrivibrio), Eubacterium (Eubacterium), Sarcina (Sarcina), Megasphaera (Megasphaera), Clostridium (Fusobacterium), etc., wherein Clostridium butyricum (Clostridium butyricum) and Clostridium butyricum (Clostridium butyricum) of the genera have relatively good butyric acid production and butyric acid conversion rates, and thus are considered to be the butyric acid fermentation strains having the most commercial development potential. However, the carbon source utilization spectrum of clostridium tyrobutyricum is relatively narrow, mainly comprises glucose, fructose, xylose and the like, so that the direct utilization of cheap substrates is limited, and a complex substrate pretreatment process is often needed for fermentation. Clostridium butyricum has a wide substrate utilization range, including hexose, pentose, molasses, whey, starch, glycerol and the like, and clostridium butyricum has been approved as a new feed additive since 2009, so that the safety of strains is guaranteed, but the yield and product selectivity of butyric acid are slightly inferior to those of clostridium butyricum.
The development of a cheap biomass substrate fermentation process is one of important strategies for reducing the production cost of a microbial fermentation method, and starch serving as a renewable biomass resource has the advantages of simple production process, low price and the like, and can replace glucose serving as an industrial fermentation substrate, so that a strain which can efficiently utilize a starch substrate and has excellent butyric acid productivity and character is screened, and a novel technological route for producing butyric acid by the microbial fermentation method can be provided.
Disclosure of Invention
In view of the above, one of the purposes of the present invention is to provide a clostridium butyricum strain capable of efficiently utilizing starch substrate to produce butyric acid, which can directly utilize corn starch or tapioca starch as a carbon source, and has high utilization efficiency and high butyric acid conversion rate; the second object of the present invention is to provide a method for culturing Clostridium butyricum; the invention also aims to provide a fermentation method for producing butyric acid by using starch by using the clostridium butyricum and a preparation method for using fermented solid residues for a clostridium butyricum viable bacteria agent.
In order to achieve the above purpose, the invention provides the following technical scheme:
clostridium butyricum (Clostridium butyricum) SCUT620 is preserved in Guangdong province collection of microorganisms in 2019 at 29 months 3, with the preservation number GDMCC NO: 60623, deposit address: zhou 100 Dazhou 59, building 5, Guangzhou institute of microorganisms.
A method for producing butyric acid by fermentation, comprising the steps of:
(1) activating strains: diluting clostridium butyricum SCUT620 bacterial liquid, coating the liquid on a solid activation culture medium, culturing for 48 hours at 37 ℃, selecting a single colony, inoculating the single colony in a seed culture medium, standing at 35 +/-2 ℃ for anaerobic culture for 16-24 hours until OD is reached6001.5 to 2;
(2) seed culture; inoculating clostridium butyricum SCUT620 activated bacterial liquid into a seed culture medium, standing at 35 +/-2 ℃ for anaerobic culture for 8-16 h, and performing OD6001.5-2, which is a seed culture solution;
(3) fermentation culture: heating the fermentation medium to gelatinize starch, then sterilizing, and inoculating the clostridium butyricum SCUT620 seed culture solution into the fermentation medium for fermentation;
(4) and (4) centrifugally separating the fermentation liquor, and separating the supernatant to obtain butyric acid.
Preferably, the clostridium butyricum uses starch or starch processing residues as a carbon source to ferment and produce butyric acid.
Preferably, the starch is tapioca starch or corn starch.
Preferably, the activation medium is: tryptone 10 g/L; 10g/L of beef extract; 5g/L of glucose; 5g/L of sodium chloride; 3g/L of yeast powder; 3g/L of sodium acetate; 1g/L of soluble starch; 0.5g/L of L-cysteine hydrochloride; agar was also added at 15g/L during solid culture.
Preferably, the seed culture medium is: 20g/L of glucose; 5g/L of sodium chloride; 5g/L of yeast powder; 3g/L of ammonium sulfate; dipotassium phosphate is 1.5 g/L; MgSO (MgSO)4·7H2O 0.6g/L，FeSO4·7H2O 0.03g/L。
Preferably, the fermentation medium is: 2g/L of dipotassium phosphate; 1g/L of ammonium sulfate; 5g/L of yeast powder; MgSO (MgSO)4·7H2O 0.6g/L；FeSO4·7H2O0.03 g/L; 20-60 g/L of carbon source; 50g/L of calcium carbonate; initial pH 7.0.
Preferably, the fermentation conditions are: culturing for 48-72 h at 35 +/-2 ℃ and the rotating speed of a shaking table of 100-150 rpm.
Preferably, the fermentation liquid precipitate after centrifugal separation in the step (4) is directly frozen and dried to obtain the clostridium butyricum viable bacteria preparation.
Preferably, residual bacteria and soluble proteins in the supernatant are removed through ultrafiltration, the supernatant is concentrated to a butyric acid concentration of 40-60 g/L, trioctylamine is used as an extracting agent, octanol is used as a diluent, the extracting condition is that the concentration of trioctylamine is 40% (v/v), the pH value of fermentation liquor is 2-3, the organic phase is subjected to back extraction by NaOH after extraction at a temperature of 25 ℃ compared with 0.8, and finally pure butyric acid is obtained through separation.
The clostridium butyricum SCUT620 specifically comprises the following steps:
adding about 1g of fresh cow dung sample into 9mL of PBS, putting into 80 ℃ water bath for 10min to kill non-spore bacteria, reversing and uniformly mixing, inoculating 2mL of the mixture into an enrichment medium (tryptone 10 g/L; beef extract 10 g/L; glucose 5 g/L; sodium chloride 5 g/L; yeast extract 3 g/L; sodium acetate 3 g/L; soluble starch 1 g/L; L-cysteine hydrochloride 0.5g/L), and standing at 37 ℃ for anaerobic culture for 48 h; the culture broth was diluted to 10-4The strain is coated on a clostridium solid screening culture medium (tryptone 15g/L, yeast powder 10g/L, sodium sulfite 0.5g/L, neomycin 0.02g/L, polymyxin 0.05g/L, ferric citrate 0.5g/L, agar 15g/L and pH 7.2), after anaerobic culture at 37 ℃ for 48 hours, single colony is selected to be in an enrichment culture medium, after anaerobic culture at 37 ℃ for 24 hours, a small amount of sample is taken to be observed by gram stauroscopy, gram staining is selected to be positive, and the strain with the rod-shaped microscopic morphology and spores is subjected to subsequent 16S rDNA sequence strain identification and metabolic characteristic identification.
The clostridium butyricum SCUT620 has the following characteristics:
(1) colony characteristics: the bacterial colony is round, smooth in surface, irregular in edge, convex, white or milky white, and the diameter of the bacterial colony is 1.5-2 mm;
(2) the morphological characteristics of the cells are as follows: the cells are rod-shaped, and the top ends of the cells are round and blunt; gram positive staining;
(3) physiological and biochemical characteristics: strictly anaerobic growth, which can utilize glucose, fructose, mannose, xylose, arabinose, sucrose, maltose, trehalose, cellobiose, starch, etc., and can not utilize cellulose; the metabolites are mainly butyric acid and acetic acid.
(4) The 16S rRNA gene sequence of Clostridium butyricum (Clostridium butyricum) SCUT620 has the length of 1,427bp, and the nucleotide sequence is shown as SEQ ID NO: 1 is shown.
The clostridium butyricum SCUT620 culture method comprises the following steps:
the anaerobic environment required by the culture medium of the invention is filled with 100 percent N2Typically, the injection pressure is 0.5 atm.
Activation medium composition (/ L): 10g of tryptone; 10g of beef extract; 5g of glucose; 5g of sodium chloride; 3g of yeast powder; 3g of sodium acetate; 1g of soluble starch; 0.5g of L-cysteine hydrochloride; agar 15g was added for solid culture.
Seed medium composition (/ L): 20g of glucose; 5g of sodium chloride; 5g of yeast powder; 3g of ammonium sulfate; dipotassium phosphate 1.5 g; MgSO (MgSO)4·7H2O 0.6g，FeSO4·7H2O 0.03g。
Shake flask fermentation medium composition (/ L): 2g of dipotassium phosphate; 1g of ammonium sulfate; 5g of yeast powder; MgSO (MgSO)4·7H2O 0.6g；FeSO4·7H20.03g of O; 20-60 g of corresponding carbon source; 50g of calcium carbonate; the initial pH was 7.0.
The method for producing butyric acid by fermenting the starch substrate by the clostridium butyricum SCUT620 comprises the following steps:
(1) activating strains: diluting clostridium butyricum SCUT620 bacterial liquid to 10-4Spread on solid activation medium, and cultured at 37 ℃ for 48 hours. Selecting a single colony, inoculating the single colony in a centrifugal tube filled with 5-10 mL of a sub-culture medium, and performing static culture at 37 ℃ for 16-24 hours until the colony reaches OD600About 1.5 to about 2.
(2) Seed culture; inoculating 2-5 mL of clostridium butyricum SCUT620 activated bacteria liquid into a serum bottle filled with 50-100 mL of seed culture medium by using an aseptic syringe, standing at 37 ℃ for anaerobic culture for 8-16 h, and performing OD600About 1.5-2 to obtain a seed culture solution.
(3) And (3) shaking flask fermentation: preparing shake flask fermentation culture medium with corn starch and cassava starch as carbon source, gelatinizing starch in culture medium with final concentration of 40g/L before sterilization in boiling water bath for 10min, and sterilizing at 115 deg.C for 20 min. 2.5mL of Clostridium butyricum SCUT620 seed culture medium was inoculated into 50mL of the corresponding carbon source in a shake flask fermentation medium at 37 ℃ and 150rpm for 72 hours using a sterile syringe.
(4) And (3) performing solid-liquid separation on fermentation liquor obtained after starch fermentation, collecting precipitates, and performing freeze drying to obtain the clostridium butyricum viable bacteria preparation.
Compared with the prior art, the invention has the beneficial effects that:
the provided clostridium butyricum SCUT620 has a wider substrate utilization spectrum, particularly has better catabolism capacity on a starch-based biomass substrate, can utilize corn starch and cassava starch and even residues in the starch processing process to biologically ferment and convert the butyric acid, has higher butyric acid conversion rate, and the butyric acid conversion rate reaches 0.33g/g of corn starch and 0.37g/g of cassava starch8More than cfu/g, good storage stability, and viable count of more than 1 × 10 after being placed at room temperature for 21 days7cfu/g, can be directly used as a clostridium butyricum viable bacteria preparation and is used for feed addition in the livestock breeding industry. Compared with the method for producing butyric acid by fermenting glucose as a carbon source, the fermentation method has the advantages of lower raw material cost, simple process steps, strong operability, no production waste, suitability for industrial production and better economic benefit.
Drawings
FIG. 1 phylogenetic tree.
FIG. 2 Clostridium butyricum SCUT620 colony morphology.
Fig. 3 clostridium butyricum SCUT620 cell morphology map.
FIG. 4 is a graph showing results of shake flask fermentation under different carbon source conditions.
FIG. 5 is a graph of shake flask fermentation results using corn starch and tapioca starch.
FIG. 6 is a graph of a test of the stability of a live Clostridium butyricum preparation.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Example 1 Clostridium butyricum isolation screening
Adding about 1g of fresh cow dung sample into 9mL of PBS, putting into 80 ℃ water bath for 10min to kill non-spore bacteria, reversing and uniformly mixing, inoculating 2mL of the mixture into an enrichment medium (tryptone 10 g/L; beef extract 10 g/L; glucose 5 g/L; sodium chloride 5 g/L; yeast extract 3 g/L; sodium acetate 3 g/L; soluble starch 1 g/L; L-cysteine hydrochloride 0.5g/L), and standing at 37 ℃ for anaerobic culture for 48 h; the culture broth was diluted to 10-4The strain is coated on a solid screening culture medium (tryptone 15 g/L; yeast powder 10 g/L; sodium sulfite 0.5 g/L; neomycin 0.02 g/L; polymyxin 0.05 g/L; ferric citrate 0.5 g/L; agar 15 g/L; pH 7.0), is subjected to standing anaerobic culture at 37 ℃ for 48 hours, a single colony is selected in an enrichment culture medium, a small amount of samples are taken after anaerobic culture at 37 ℃ for 24 hours, gram staining microscopic examination observation is carried out, the strains which are positive in gram staining and rod-shaped in microscopic form and have spores are selected for subsequent strain identification.
Example 2 identification of bacterial species
First, cell morphological feature identification
The single colony obtained in the separation in the example 1 is selected and inoculated in a centrifuge tube containing an enrichment medium, anaerobic culture is carried out at 37 ℃ for 24h, and then a small amount of bacterial liquid is taken for gram staining microscopic observation.
Gram stain test:
(1) dyeing: and (3) dropwise adding an appropriate amount of bacteria onto the glass slide, fixing for 2-3 times by flame, dropwise adding ammonium oxalate crystal violet liquid, and dyeing the fixed smear for 1 min.
(2) Washing with water: the staining solution on the smear was slowly rinsed with water and blotted dry with absorbent paper. The cell morphology can be observed after simple staining.
(3) Mordant dyeing: dripping 1 drop of iodine solution, dyeing for 1min, and washing with water.
(4) And (3) decoloring: absorbing residual water, continuously dropwise adding 95% ethanol for decoloring for 20-30 s until effluent liquid is purple, and immediately washing with water.
(5) Absorbing residual water, continuously dropwise adding 95% ethanol for decoloring for 20-30 s until effluent liquid is purple, and immediately washing with water.
Similar strains were screened for subsequent 16S rDNA gene sequence analysis and identification by gram-stained microscopy, according to Bergey' S Manual of Systematic Bacteriology (second edition), and Clostridium morphological characterization.
Secondly, 16S rDNA gene sequence analysis and identification:
the strain separated in the cell morphological characteristic identification of the embodiment 2 is picked up and inoculated in a centrifuge tube containing an enrichment medium, and a small amount of sample is taken for PCR amplification of a 16S rDNA sequence after anaerobic culture at 37 ℃ for 24 h. With 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5 'GGTTACCTTGTTACGACTT-3' as forward and reverse primers, respectively, and the PCR reaction system (25. mu.L) was: takara PrimerSTARmix 12.5. mu.L; ddH2O9.5 mu L; 1 mu L of forward and reverse primers respectively; bacterial suspension 1. mu.L.
PCR amplification procedure: pre-denaturation at 98 deg.C for 10min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 5s, extension at 72 deg.C for 2min, performing 30 cycles, extension at 72 deg.C for 10min, and storing at 4 deg.C. The PCR product is detected by 1% agarose gel electrophoresis, and the positive product with the fragment of about 1500bp is purified and then subjected to sequence determination. The sequencing result shows that the length of the 16SrRNA gene sequence of the selected strain SCUT620 is 1,427bp, and the nucleotide sequence is shown as SEQ ID NO: 1 is shown. The determined gene sequence was compared with the GenBank database by using BLAST on NCBI for homology, the Clostridium butyricum phylogenetic tree was constructed using MEGA 6.0, the identification result is shown in fig. 1, and the selected SCUT620 strain had the highest homology with Clostridium butyricum strain.
Thirdly, colony morphology and cell morphology characteristics
Diluting the culture solution of the selected SCUT620 strain to 10-4Coated on solid enrichment mediumAfter anaerobic culture at 37 ℃ for 48 hours, the colony morphology was observed. As shown in FIG. 2, the colonies of the SCUT620 strain are round, smooth in surface, irregular in edge, convex, white or milky white. In addition, gram staining was identified as positive, and as shown in FIG. 3, the cells were rod-shaped, blunt-ended, and have spores.
As described above, the selected SCUT620 strain was identified as Clostridium butyricum (Clostridium butyricum) according to the results of cell morphology, colony morphology characterization, and 16S rRNA gene sequence analysis and identification thereof in Bergey' S Manual of Systematic Bacteriology (second edition).
Fourth, shake flask fermentation characteristic
Inoculating the separated Clostridium butyricum (Clostridium butyricum mCUT 620) into a liquid activation culture medium (10 g/L tryptone, 10g/L beef extract, 5g/L glucose, 5g/L sodium chloride, 3g/L yeast powder, 3g/L sodium acetate, 1g/L soluble starch and 0.5 g/L-cysteine hydrochloride), standing for anaerobic culture at 37 ℃ for 48 hours, and then inoculating 2.5mL of Clostridium butyricum SCUT620 activation bacterial liquid into a seed culture medium (20 g/L glucose, 5g/L sodium chloride, 5g/L yeast powder, 3g/L ammonium sulfate, 1.5g/L dipotassium phosphate and MgSO 620) filled with 50mL of seed culture medium4·7H2O 0.6g/L；FeSO4·7H2O0.03 g/L), standing at 37 ℃ for anaerobic culture for 8-16 h, and performing OD600About 1.5-2 to obtain a seed culture solution. Inoculating 2.5mL of Clostridium butyricum SCUT620 seed culture solution into 50mL of fermentation medium (dipotassium hydrogen phosphate 2 g/L; ammonium sulfate 1 g/L; yeast powder 5 g/L; MgSO)4·7H2O 0.6g/L；FeSO4·7H2O0.03 g/L; 40g/L of corresponding carbon source; 50g/L of calcium carbonate; initial pH is 7.0), the fermentation medium respectively takes glucose, fructose, mannose, xylose, arabinose, sucrose, maltose, trehalose, cellobiose, soluble starch and cellulose with final concentration of 40g/L as carbon source, 50mL of prepared fermentation liquor is filled in a 120mL serum bottle, the vacuum pumping and nitrogen charging are carried out, the high temperature and high pressure sterilization is carried out for 20min at the temperature of 115 ℃, and the cooling is carried out for standby application. And in the fermentation process, standing at 37 ℃ for anaerobic culture for 60 hours for culture, sampling at regular time, and monitoring the fermentation product.
The result is shown in fig. 4, the separated Clostridium butyricum (Clostridium butyricum spec 620) has a wide substrate spectrum, and tests show that glucose, fructose, mannose, xylose, arabinose, sucrose, maltose, trehalose, cellobiose and starch can be used as carbon sources to produce butyric acid, and the yield reaches 10-14 g/L; but cannot directly utilize cellulose as a carbon source.
Example 3 fermentation of corn starch to butyric acid and Clostridium butyricum inoculum
Seed broth was prepared according to the procedure described in the shake flask fermentation characteristics of example 2. Directly using corn starch as a carbon source to prepare a shake flask fermentation medium, wherein the final concentration of the carbon source is 40g/L, gelatinizing the starch in the medium in boiling water bath for 10min before sterilization, and then sterilizing the starch for 20min at 115 ℃. 2.5mL of Clostridium butyricum SCUT620 seed culture was inoculated into 50mL of fermentation medium using a sterile syringe and fermented at 37 ℃ for 72 hours at 150 rpm. The fermentation results are shown in FIG. 5, 40g/L corn starch can be converted into 13.23g/L butyric acid product at the shake flask fermentation level, the butyric acid conversion rate reaches 0.33g/g corn starch, and the by-product is only acetic acid (see FIG. 5). The results show that the clostridium butyricum SCUT620 has strong corn starch utilization capacity and good butyric acid selectivity and conversion rate. Centrifuging the fermented fermentation liquor at room temperature under non-anaerobic conditions, removing residual bacteria and soluble protein from supernatant through ultrafiltration, concentrating to the concentration of about 50g/L butyric acid, taking trioctylamine as an extracting agent with the concentration of 40% (v/v), octanol as a diluent, and keeping the pH value of the fermentation liquor at 2-3, wherein the temperature is 25 ℃ compared with 0.8. And (3) carrying out back extraction on the organic phase by using 0.8M NaOH after extraction, and finally separating to obtain pure butyric acid. Directly freeze-drying the fermentation liquor precipitate to obtain the clostridium butyricum viable bacteria preparation. The prepared clostridium butyricum viable bacteria preparation is continuously placed for 3 weeks at room temperature, and the viable bacteria quantity is detected every week. As shown in FIG. 6, the viable bacteria count of the Clostridium butyricum viable bacteria preparation prepared from the fermentation liquid waste residue can reach 2 × 108More than cfu/g, the number of viable bacteria can still be maintained at 1 x 10 after continuously placing for 3 weeks7More than cfu/g, better storage stability, simple preparation process and strong operability.
Example 4 fermentation of cassava starch to butyric acid and Clostridium butyricum microbial inoculum
Seed broth was prepared according to the procedure described in the shake flask fermentation characteristics of example 2. The cassava starch is directly used as a carbon source to prepare a shake flask fermentation medium, the final concentration of the carbon source is 40g/L, the medium is gelatinized by boiling water bath for 10min before sterilization, and then the sterilization is carried out for 20min at 115 ℃. 2.5mL of Clostridium butyricum SCUT620 seed culture medium was inoculated into 50mL of the corresponding carbon source in a shake flask fermentation medium at 37 ℃ and 150rpm for 72 hours using a sterile syringe. The fermentation result is shown in figure 5, 40g/L cassava starch can be converted into 14.65g/L butyric acid product at the shake flask fermentation level, the butyric acid conversion rate reaches 0.37g/g cassava starch, and the by-product is only acetic acid. The results show that the clostridium butyricum SCUT620 has strong cassava starch substrate utilization capacity and good butyric acid selectivity and conversion rate. Centrifuging the fermented fermentation liquor at room temperature under non-anaerobic conditions, removing residual bacteria and soluble protein from supernatant through ultrafiltration, concentrating to the concentration of about 50g/L butyric acid, taking trioctylamine as an extracting agent with the concentration of 40% (v/v) and octanol as a diluent, and keeping the pH value of the fermentation liquor at 2-3, which is 0.8 compared with that of the fermentation liquor at 25 ℃. Carrying out back extraction on the organic phase by using 0.8M NaOH after extraction, and finally separating to obtain pure butyric acid; directly freeze-drying the fermentation liquor precipitate to obtain the clostridium butyricum viable bacteria preparation. The prepared clostridium butyricum viable bacteria preparation is continuously placed for 3 weeks at room temperature, and the viable bacteria quantity is detected every week. As shown in FIG. 6, the viable bacteria count of the Clostridium butyricum viable bacteria preparation prepared from the fermentation liquid waste residue can reach 4 × 108More than cfu/g, the number of viable bacteria can still be maintained at 1 x 10 after continuously placing for 3 weeks7More than cfu/g, better storage stability, simple preparation process and strong operability.
Sequence listing
<110> university of southern China's science
<120> clostridium butyricum and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
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<211> 1427
<212> DNA
<213> Clostridium butyricum (Clostridium butyricum)
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acgaactttg ggtattgcca actctcatgg tgtgacgggc ggtgtgtaca aggcccggga 120
acgtattcac cgcgacattc tgattcgcga ttactagcaa ctccagcttc atgtaggcga 180
gtttcagcct acaatccgaa ctgagatcgg ttttatagtt ttgctcactc tcgcgaggtt 240
gcatctcatt gtaccgacca ttgtagcacg tgtgtagccc tagacataag gggcatgatg 300
atttgacgtc atccccacct tcctcccggt taacccgggc agtctcgcta gagtgctcaa 360
ctaaatggta gcaactaaca ataagggttg cgctcgttgc gggacttaac ccaacatctc 420
acgacacgag ctgacgacaa ccatgcacca cctgtcttcc tgccaccgaa gtggcttcct 480
ccattacaga gtaattcagg agatgtcaag tctaggtaag gttcttcgcg ttgcttcgaa 540
ttaaaccaca tgctccgctg cttgtgcggg cccccgtcaa ttcctttgag ttttaatctt 600
gcgaccgtac tccccaggcg gaatacttaa tgcgttagcg gcggcacaga ggtcatgaca 660
acccctacac ctagtattca tcgtttacgg cgtggactac cagggtatct aatcctgttt 720
gctccccacg ctttcgagcc tcagtgtcag ttacagtcca gaaaggcgcc ttcgccactg 780
gtattcttcc taatctctac gcatttcacc gctacactag gaattctcct ttcctctcct 840
gcactctaga tatccagttt ggaatgcagc acccaggtta agcccgggta tttcacatcc 900
cacttaaata tccacctacg ctccctttac gcccagtaaa tccggacaac gcttgccacc 960
tacgtattac cgcggctgct ggcacgtagt tagccgtggc ttcctcctta ggtaccgtca 1020
ttatcgtccc taaagacaga gctttacaat ccgaagaccg tcatcactca cgcggcgttg 1080
ctgcatcagg gtttccccca ttgtgcaata ttccccactg ctgcctcccg taggagtctg 1140
ggccgtgtct cagtcccaat gtggccgatc accctctcag gtcggctacg catcgtcgcc 1200
ttggtgagcc gttacctcac caactagcta atgcgacgcg ggtccatctc atagcggatt 1260
actcctttaa ttgctgtacc atgcggtact acaatcttat gcggtattaa tcttcctttc 1320
gaaaggctat tcccctctat gaggcaggtt acccacgtgt tactcacccg tccgccgcta 1380
atccactccc gaaggagctt catcgctcga cttgcatgtg tagcacc 1427
Claims (10)
1. The Clostridium butyricum is Clostridium butyricum (Clostridium butyricum) SCUT620, which is preserved in 29 months in 2019 in the culture collection of microorganisms in Guangdong province, wherein the preservation number is GDMCC NO: 60623, deposit address: zhou 100 Dazhou 59, building 5, Guangzhou institute of microorganisms.
2. The use of Clostridium butyricum of claim 1, wherein the Clostridium butyricum produces butyric acid by fermentation using starch or starch processing residues as a carbon source.
3. Use according to claim 2, wherein the starch is tapioca starch or corn starch.
4. Use according to claim 2 or 3, wherein the fermentation conditions are: culturing for 48-72 h at 35 +/-2 ℃ and the rotating speed of a shaking table of 100-150 rpm.
5. Use according to claim 2 or 3, wherein the fermentation medium is: 2g/L of dipotassium phosphate; 1g/L of ammonium sulfate; 5g/L of yeast powder; MgSO (MgSO)4·7H2O0.6g/L；FeSO4·7H2O0.03g/L; 20-60 g/L of carbon source; 50g/L of calcium carbonate; initial pH 7.0.
6. Use according to claim 2 or 3, characterized in that it comprises the following steps:
(1) activating strains: diluting clostridium butyricum SCUT620 bacterial liquid, coating the liquid on a solid activation culture medium, culturing for 48 hours at 37 ℃, selecting a single colony, inoculating the single colony in a seed culture medium, standing at 35 +/-2 ℃ for anaerobic culture for 16-24 hours until OD is reached6001.5 to 2;
(2) seed culture; inoculating clostridium butyricum SCUT620 activated bacterial liquid into a seed culture medium, standing at 35 +/-2 ℃ for anaerobic culture for 8-16 h, and performing OD6001.5-2, which is a seed culture solution;
(3) fermentation culture: heating the fermentation medium to gelatinize starch, then sterilizing, and inoculating the clostridium butyricum SCUT620 seed culture solution into the fermentation medium for fermentation;
(4) and (4) centrifugally separating the fermentation liquor, and separating the supernatant to obtain butyric acid.
7. The use according to claim 6,
the activation medium is as follows: tryptone 10 g/L; 10g/L of beef extract; 5g/L of glucose; 5g/L of sodium chloride; 3g/L of yeast powder; 3g/L of sodium acetate; 1g/L of soluble starch; 0.5g/L of L-cysteine hydrochloride; agar is added for solid culture at a ratio of 15 g/L;
the seed culture medium is as follows: 20g/L of glucose; 5g/L of sodium chloride; 5g/L of yeast powder; 3g/L of ammonium sulfate; dipotassium phosphate is 1.5 g/L; MgSO (MgSO)4·7H2O0.6g/L，FeSO4·7H2O0.03g/L。
8. The use of claim 6, wherein the fermentation liquid precipitate obtained after the centrifugal separation in the step (4) is directly freeze-dried to obtain the live clostridium butyricum preparation.
9. The application of claim 6, wherein residual bacteria and soluble proteins in the supernatant are removed through ultrafiltration, the supernatant is concentrated to a butyric acid concentration of 40-60 g/L, trioctylamine is used as an extractant, octanol is used as a diluent for extraction, the organic phase is subjected to back extraction through NaOH after extraction, and pure butyric acid is finally obtained through separation.
10. The use of claim 9, wherein the extraction conditions are a trioctylamine concentration of 40% (v/v) and a fermentation broth pH of 2-3 at a temperature of 25 ℃ compared to 0.8.
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| CN112358986B (en) * | 2020-11-09 | 2022-10-21 | 华南理工大学 | Clostridium butyricum and application thereof in production of 1,3-propylene glycol through immobilized fermentation |
| CN112626135A (en) * | 2021-01-15 | 2021-04-09 | 驻马店华中正大有限公司 | Method for producing butyric acid by fermenting corn starch |
| CN117305381B (en) * | 2023-11-28 | 2024-02-02 | 广东容大生物股份有限公司 | Application of clostridium butyricum in producing butyrate by fermentation and method for producing butyrate |
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| CN104560779A (en) * | 2014-11-26 | 2015-04-29 | 厦门大学 | Clostridium butyricum and application thereof |
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