CN110218676A - A kind of clostridium butyricum and its application - Google Patents
A kind of clostridium butyricum and its application Download PDFInfo
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Abstract
The invention discloses a kind of clostridium butyricum and its applications, the bacterium is clostridium butyricum (Clostridium butyricum) SCUT620, Guangdong Province's Culture Collection is preserved on March 29th, 2019, its deposit number is GDMCC NO:60623, preservation address: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, Guangdong Microbes Inst.The bacterial strain is isolated from healthy cow excrement, can directly utilize low cost starch matrix such as cornstarch, tapioca fermenting and producing butyric acid, butyric acid conversion ratio with higher；The bacterial strain can also prepare clostridium butyricum active bacteria preparation while producing butyric acid using amylofermentation, and this method can produce butyric acid and clostridium butyricum active bacteria preparation simultaneously by one time fermentation, have the advantages that simple process, production cost are low, have broad application prospects.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to plant height effect utilizes the clostridium butyricum of starchiness matrix, the bacterium
Strain is utilizing cornstarch and tapioca fermenting and producing butyric acid and the method for preparing clostridium butyricum active bacteria preparation.
Background technique
N-butyric acie (butyric acid) is a kind of short chain organic carboxyl acid (short-chain containing four carbon atom
Fatty acids), butyric acid and its derivative have extensive purposes in fields such as chemical industry, food, medicine and feedstuff industries
[Dwidar M et al.The future of butyric acid in industry.Sci World J,2012,
471417].Currently, the production of butyric acid mainly has two methods of chemical synthesis and microbial fermentation, chemical synthesis is main at present
Butyric acid is prepared using oxidation n-butanal method, this method has simple process, and condition is easily controllable, and the high advantage of product yield is
It is currently to industrialize the main method of butyric acid production, but raw material is derived from non-renewable fossil resource, and reaction process can generate
Toxic metallic contaminants, therefore its sustainability and the feature of environmental protection are restricted.Production by Microorganism Fermentation butyric acid is new in recent years
Emerging butyric acid production method, have cost of material it is low, fermentation condition is mild, and environmental pollution is small, and biology base butyric acid more by food,
Medicine and feedstuff industry favor, but for microbe fermentation method there are still butyric acid conversion ratio is low, by-product is difficult to the problems such as separating at present,
Lead to its high production cost, industrial applications are restricted.Therefore the research for Production by Microorganism Fermentation butyric acid in recent years
The problems such as focusing primarily upon reduction cost of material, promoting butyric acid conversion ratio and selectivity [Jiang L et al.Butyric
acid:Applications and recent advances in its bioproduction.Biotechnology
Advances, 2018,36 (8): 2101-2117].
Butyric acid can be by fusobacterium (Clostridium), Butyribacterium (Butyribacterium), Butyrivibrio
(Butyrivibrio), Eubacterium (Eubacterium), Sarcina (Sarcina), Megasphaera category
(Megasphaera), the microbial fermentations such as Fusobacterium (Fusobacterium) produce, wherein the clostridium tyrobutyricum of fusobacterium
(Clostridium tyrobutyricum) and clostridium butyricum (Clostridium butyricum) have relatively good fourth
Acid yield, butyric acid conversion ratio, therefore be considered as the butyric fermentation bacterial strain of most commercial development potentiality.But clostridium tyrobutyricum carbon
Source limits its direct utilization to cheap substrates, often using spectrum relative narrower, predominantly glucose, fructose and xylose etc.
Need complicated substrate preprocessing process that could be used to ferment.And clostridium butyricum then has extensive substrate utilization scope, including
Hexose, pentose, molasses, whey, starch and glycerol etc., furthermore clostridium butyricum was approved for new feedstuff and has added from 2009
Add agent, therefore have in terms of strain safety and ensure, but is slightly inferior to clostridium tyrobutyricum in terms of its butyric acid yield and selectivity of product.
Developing cheap biomass fermenting substrate technique is to reduce microbe fermentation method to produce one of cost Critical policies, and form sediment
Powder has many advantages, such as simple production process as a kind of renewable biomass resources, cheap, and alternative glucose is as work
Industry fermentation substrate, therefore screen and a kind of can efficiently utilize starch matrix and the excellent strain of butyric acid production capacity character, it is possible to provide one
Novel microbial fermentation method produces butyric acid process route.
Summary of the invention
In view of this, one of the objects of the present invention is to provide a strain capable of high-efficiency to utilize the butyric acid of starch matrix production butyric acid
Clostridium bacterial strain, can be directly using cornstarch and tapioca as carbon source, and utilization efficiency is high, butyric acid high conversion rate；This hair
The bright second purpose is to provide the cultural method of the clostridium butyricum；The third object of the present invention is to provide clostridium butyricum benefit
The preparation method of clostridium butyricum active bacteria microbial inoculum is used for solid residue after the fermentation process of Starch Production butyric acid and fermentation.
For achieving the above object, the invention provides the following technical scheme:
A kind of clostridium butyricum, which is clostridium butyricum (Clostridium butyricum) SCUT620, in March, 2019
It is preserved within 29th Guangdong Province's Culture Collection, deposit number is GDMCC NO:60623, preservation address: Guangzhou
5 building, the building of compound the 59th of martyr Road 100, Guangdong Microbes Inst.
A kind of method of fermenting and producing butyric acid, includes the following steps:
(1) after the dilution of clostridium butyricum SCUT620 bacterium solution, solid activation medium, 37 DEG C of cultures actication of culture: are coated on
48h, picking single colonie are inoculated in seed culture medium, and 35 ± 2 DEG C of standing Anaerobic culturels 16~for 24 hours, until OD600It is 1.5~2；
(2) seed culture；Clostridium butyricum SCUT620 activation bacterium solution is taken to be inoculated in seed culture medium, 35 ± 2 DEG C of standings are detested
Oxygen culture 8~16h, OD600It is 1.5~2, this is seed culture fluid；
(3) fermented and cultured: then fermentation medium Heat Gelatinization starch sterilizes, and clostridium butyricum SCUT620 seed is trained
Nutrient solution, which is inoculated in fermentation medium, ferments；
(4) fermentation liquid is centrifuged, the isolated butyric acid from supernatant.
Preferably, the clostridium butyricum produces butyric acid using starch or starch process residues as carbon source through fermentation.
Preferably, the starch is tapioca or cornstarch.
Preferably, the activation medium are as follows: tryptone 10g/L；Beef extract 10g/L；Glucose 5g/L；Sodium chloride
5g/L；Yeast powder 3g/L；Sodium acetate 3g/L；Soluble starch 1g/L；L- cysteine ??acid hydrochloride 0.5g/L；In solid culture
Also add agar 15g/L.
Preferably, the seed culture medium are as follows: glucose 20g/L；Sodium chloride 5g/L；Yeast powder 5g/L；Ammonium sulfate 3g/L；
Dipotassium hydrogen phosphate 1.5g/L；MgSO4·7H2O 0.6g/L, FeSO4·7H2O 0.03g/L。
Preferably, fermentation medium are as follows: dipotassium hydrogen phosphate 2g/L；Ammonium sulfate 1g/L；Yeast powder 5g/L；MgSO4·
7H2O0.6g/L；FeSO4·7H2O 0.03g/L；20~60g/L of carbon source；Calcium carbonate 50g/L；Initial pH=7.0.
Preferably, the fermentation condition are as follows: 35 ± 2 DEG C, 100~150rpm of shaking speed, cultivate 48~72h.
Preferably, the fermentation liquid sediment directly freezed after step (4) centrifuge separation is dried to obtain clostridium butyricum active bacteria
Preparation.
Preferably, the supernatant removes residual thallus and soluble protein by ultrafiltration, is concentrated into 40~60g/L butyric acid
After concentration, using trioctylamine as extractant, for octanol as diluent, extraction conditions is that trioctylamine concentration is 40% (v/v), hair
Zymotic fluid pH value 2~3 is compared 0.8, is stripped with NaOH to organic phase after 25 DEG C of temperature extractions, final separation obtains pure
Butyric acid.
Above-mentioned clostridium butyricum SCUT620, specific screening technique include the following steps:
About 1g milk cow fresh excreta sample is taken, is added in 9mLPBS, is put into 80 DEG C of water-bath 10min to kill non-gemma
Bacterium takes 2mL to be inoculated in enriched medium (tryptone 10g/L after being mixed by inversion；Beef extract 10g/L；Glucose 5g/L；Chlorination
Sodium 5g/L；Yeast extract 3g/L；Sodium acetate 3g/L；Soluble starch 1g/L；L- cysteine ??acid hydrochloride 0.5g/L) it is stood in 37 DEG C
Anaerobic culturel 48h；Culture solution is diluted to 10-4, it is coated on clostridium solid screening and culturing medium (tryptone 15g/L；Yeast powder
10g/L；Sodium sulfite 0.5g/L；Ovobiocin 0.02g/L；Polymyxins 0.05g/L；Citric acid iron 0.5g/L；Agar 15g/
L；PH=7.2), after 37 DEG C of Anaerobic culturel 48h, picking single bacterium is fallen in enriched medium, and 37 DEG C of Anaerobic culturels take on a small quantity afterwards for 24 hours
Sample carries out gram stain microscopy observation, selects Gram's staining as the positive, microscopic morphology is the rod-shaped bacterial strain with gemma
Carry out subsequent 16S rDNA sequence strain idenfication and metabolic characteristics identification.
Above-mentioned clostridium butyricum SCUT620 has the feature that
(1) colony characteristics: bacterium colony is rounded, and surface is smooth, and edge is irregular, protrusion, white or milky white, colony diameter
1.5-2mm；
(2) cell morphological characteristic: cell is rod-shaped, top round blunt；Gram-positive；
(3) physiological and biochemical property: strictly anaerobic growth can utilize glucose, fructose, mannose, xylose, Arab
Sugar, sucrose, maltose, trehalose, cellobiose, starch etc. cannot utilize cellulose；Metabolite is mainly butyric acid, acetic acid.
(4) the 16S rRNA gene order length of clostridium butyricum (Clostridium butyricum) SCUT620 is 1,
427bp, nucleotide sequence is as shown in SEQ ID NO:1.
Above-mentioned clostridium butyricum SCUT620 cultural method:
Anaerobic environment needed for culture medium of the present invention is by being filled with 100%N2It obtains, general injection reaches 0.5 atmosphere
Pressure.
Activation medium forms (/L): tryptone 10g；Beef extract 10g；Glucose 5g；Sodium chloride 5g；Yeast powder 3g；
Sodium acetate 3g；Soluble starch 1g；L- cysteine ??acid hydrochloride 0.5g；Agar 15g is added when solid culture.
Seed culture medium forms (/L): glucose 20g；Sodium chloride 5g；Yeast powder 5g；Ammonium sulfate 3g；Dipotassium hydrogen phosphate
1.5g；MgSO4·7H2O 0.6g, FeSO4·7H2O 0.03g。
Medium of shaking flask fermentation forms (/L): dipotassium hydrogen phosphate 2g；Ammonium sulfate 1g；Yeast powder 5g；MgSO4·7H2O
0.6g；FeSO4·7H2O 0.03g；20~60g of corresponding carbon source；Calcium carbonate 50g；Initial pH is 7.0.
The method of the clostridium butyricum SCUT620 fermentation starch substrate production butyric acid:
(1) actication of culture: clostridium butyricum SCUT620 bacterium solution is diluted to 10-4, it is coated on solid activation medium, 37 DEG C of trainings
Support 48h.Picking single colonie is inoculated in equipped in the sub- culture medium centrifuge tube of 5~10mL, and 37 DEG C of stationary cultures 16~for 24 hours, until OD600
About 1.5~2.
(2) seed culture；With asepsis injector take 2~5mL clostridium butyricum SCUT620 activation bacterium solution be inoculated in equipped with 50~
In the serum bottle of 100mL seed culture medium, 37 DEG C of standing Anaerobic culturels 8~16h, OD600About 1.5~2, to obtain seed training
Nutrient solution.
(3) shake flask fermentation: using cornstarch, and tapioca prepares Medium of shaking flask fermentation as carbon source, and carbon source is dense eventually
Degree is culture medium boiling water bath 10min gelatinized starch before 40g/L sterilizes, then 115 DEG C of sterilizing 20min.It is taken with asepsis injector
2.5mL clostridium butyricum SCUT620 seed culture fluid is inoculated in the Medium of shaking flask fermentation equipped with the corresponding carbon source of 50mL, and 37 DEG C,
150rpm, 72h.
(4) amylofermentation post-fermentation liquid collects precipitating, after freeze-drying, it is living to obtain clostridium butyricum by being separated by solid-liquid separation
Bacteria preparation.
Compared with prior art, the beneficial effects of the present invention are:
Provided clostridium butyricum SCUT620 has wide substrate using spectrum, especially to starch-based bio matter substrate
With preferable catabolism ability, sent out using the residue biology in cornstarch and tapioca even starch process
Ferment converts butyric acid, butyric acid conversion ratio with higher, and butyric acid conversion ratio reaches 0.33g/g cornstarch and 0.37g/g para arrowroot
Powder, furthermore the present invention also provides clostridium butyricum amylofermentation methods, clostridium butyricum after the solid sediment freeze-drying after fermentation
Number of viable is up to 2 × 108Cfu/g or more, and there is preferable storage-stable, viable count is greater than 1 after being placed at room temperature for 21 days
×107Cfu/g, can feed addition directly as clostridium butyricum active bacteria preparation, for livestock-raising industry.The fermentation process phase
It is compared to for using glucose as carbon source through fermentation production butyric acid, cost of material is lower, and processing step is simple, operability
By force, no production waste material generates, and is suitble to industrialized production, has preferable economic benefit.
Detailed description of the invention
Fig. 1 phylogenetic tree.
Fig. 2 clostridium butyricum SCUT620 colonial morphology figure.
Fig. 3 clostridium butyricum SCUT620 cellular morphology figure.
Shake flask fermentation result figure under the conditions of Fig. 4 different carbon source.
Fig. 5 utilizes cornstarch and tapioca shake flask fermentation result figure.
Fig. 6 clostridium butyricum active bacteria preparation stability test chart.
Specific embodiment
Below in conjunction with attached drawing, a preferred embodiment of the present invention will be described in detail.
Embodiment 1, clostridium butyricum separation screening
About 1g milk cow fresh excreta sample is taken, is added in 9mLPBS, is put into 80 DEG C of water-bath 10min to kill non-gemma
Bacterium takes 2mL to be inoculated in enriched medium (tryptone 10g/L after being mixed by inversion；Beef extract 10g/L；Glucose 5g/L；Chlorination
Sodium 5g/L；Yeast extract 3g/L；Sodium acetate 3g/L；Soluble starch 1g/L；L- cysteine ??acid hydrochloride 0.5g/L) it is stood in 37 DEG C
Anaerobic culturel 48h；Culture solution is diluted to 10-4, it is coated on solid screening and culturing medium (tryptone 15g/L；Yeast powder 10g/L；
Sodium sulfite 0.5g/L；Ovobiocin 0.02g/L；Polymyxins 0.05g/L；Citric acid iron 0.5g/L；Agar 15g/L；PH=
7.0), 37 DEG C of standing Anaerobic culturel 48h, picking single bacterium are fallen in enriched medium, and 37 DEG C of Anaerobic culturels take a small amount of sample afterwards for 24 hours
Gram stain microscopy observation is carried out, selects Gram's staining as the positive, microscopic morphology is that the rod-shaped bacterial strain with gemma carries out
Subsequent strain idenfication.
Embodiment 2, strain idenfication
One, cell morphological characteristic is identified
Isolated single colonie in picking embodiment 1 is inoculated in containing in the centrifuge tube in enriched medium, and 37 DEG C are detested
Oxygen culture takes a small amount of bacterium solution to carry out gram stain microscopy observation afterwards for 24 hours.
Gram stain test:
(1) it dyes: appropriate bacterium being taken to be added drop-wise on glass slide, by 2~3 fixations of flame, ammonium oxalate crystal violet liquid is added dropwise,
It contaminates in the smear staining 1min fixed.
(2) it washes: slowly rinsing the dyeing liquor for applying on piece with water, blotted with blotting paper.It is thin that simple dyeing terminates observable
Born of the same parents' form.
(3) mordant dyeing: 1 drop iodine solution is added dropwise, contaminates 1min, washing.
(4) it decolourizes: sucking residual water, continuous 95% 20~30s of ethanol decolorization to be added dropwise to efflux without purple, water immediately
It washes.
(5) residual water is sucked, it is continuous that 95% 20~30s of ethanol decolorization is added dropwise to efflux without purple, it washes immediately.
It is observed by gram stain microscopy, referring to " Bergey ' s Manual of Systematic
Bacteriology " (second edition), the description of fusobacterium morphological feature, the subsequent 16S rDNA gene order of screening similar strain progress
It analyzes and identifies.
Two, 16S rDNA gene sequencing is identified:
Isolated bacterial strain, is inoculated in containing in enriched medium in the identification of 2 cell morphological characteristic of picking embodiment
In centrifuge tube, 37 DEG C of Anaerobic culturels take a small amount of sample to carry out PCR amplification 16S rDNA sequence afterwards for 24 hours.Using 27F:5 '-
AGAGTTTGATCCTGGCTCAG-3 ' and 1492R:5 ' GGTTACCTTGTTACGACTT-3 ' draw respectively as forward and reverse
Object, PCR reaction system (25 μ L) are as follows: Takara PrimerSTARmix12.5 μ L；ddH2O 9.5μL；Forward and reverse each 1 μ of primer
L；1 μ L of bacterium solution.
PCR amplification program: 98 DEG C of initial denaturations 10min, 98 DEG C of denaturation 10s, 55 DEG C of annealing 5s, 72 DEG C of extension 2min are carried out
30 circulations, last 72 DEG C of extensions 10min, 4 DEG C of preservations.PCR product is detected using 1% agarose gel electrophoresis, and segment is
Sequencing is carried out after the positive products of 1500bp or so are purified.Sequencing result shows selected bacterial strain SCUT620's
16SrRNA gene order length is Isosorbide-5-Nitrae 27bp, and nucleotide sequence is as shown in SEQ ID NO:1.With the BLAST on NCBI
The gene order measured and GenBank database are subjected to tetraploid rice, clostridium butyricum system is constructed using MEGA 6.0 and sends out
Tree is educated, qualification result is as shown in Figure 1, the SCUT620 bacterial strain and Clostridium butyricum strain homology filtered out
Highest.
Three, colonial morphology and cell morphological characteristic
The culture solution of the SCUT620 bacterial strain filtered out is diluted to 10-4, it is coated on solids enrichment culture medium, 37 DEG C are detested
After oxygen culture 48h, its colonial morphology is observed.As a result as shown in Fig. 2, SCUT620 bacterial strain bacterium colony is rounded, surface is smooth, edge
It is irregular, protrusion, white or milky.Furthermore Gram's staining is accredited as the positive, as shown in figure 3, cellular morphology is rod-shaped, top
Round blunt is held, there is gemma.
In conclusion according to " Bergey ' s Manual of Systematic Bacteriology " (second edition), carefully
Born of the same parents' form, colony morphology characteristic description and its 16S rRNA gene sequencing qualification result, the SCUT620 bacterial strain filtered out
It is accredited as clostridium butyricum (Clostridium butyricum).
Four, shake flask fermentation feature
Above-mentioned isolated clostridium butyricum (Clostridium butyricumSCUT620) is inoculated in liquid activation
Culture medium is (tryptone 10g/L；Beef extract 10g/L；Glucose 5g/L；Sodium chloride 5g/L；Yeast powder 3g/L；Sodium acetate 3g/
L；Soluble starch 1g/L；L- cysteine ??acid hydrochloride 0.5g/L), after 37 DEG C of standing Anaerobic culturel 48h, taken with asepsis injector
2.5mL clostridium butyricum SCUT620 activation bacterium solution is inoculated in equipped with 50mL seed culture medium (glucose 20g/L；Sodium chloride 5g/L；
Yeast powder 5g/L；Ammonium sulfate 3g/L；Dipotassium hydrogen phosphate 1.5g/L；MgSO4·7H2O 0.6g/L；FeSO4·7H2O 0.03g/L)
Serum bottle in, 37 DEG C of standing Anaerobic culturels 8~16h, OD600About 1.5~2, to obtain seed culture fluid.Use aseptic injection
Device takes 2.5mL clostridium butyricum SCUT620 seed culture fluid to be inoculated in equipped with 50mL fermentation medium (dipotassium hydrogen phosphate 2g/L；Sulphur
Sour ammonium 1g/L；Yeast powder 5g/L；MgSO4·7H2O 0.6g/L；FeSO4·7H2O 0.03g/L；Corresponding carbon source 40g/L；Carbonic acid
Calcium 50g/L；7.0) initial pH is that fermentation medium is respectively with the glucose of final concentration 40g/L, fructose, mannose, xylose, Ah
Draw uncle's sugar, sucrose, maltose, trehalose, cellobiose, soluble starch and cellulose as carbon source, in the serum bottle of 120mL
Middle to be packed into prepared fermentation liquid 50ml, vacuum nitrogen gas carries out 115 DEG C, and 20min autoclave sterilization is cooling stand-by.Hair
Ferment process stands Anaerobic culturel 60h at 37 DEG C and is cultivated, and timing sampling, monitors tunning.
As a result as shown in figure 4, isolated clostridium butyricum (Clostridium butyricumSCUT620) has very
Wide substrate spectrum, after tested using glucose, fructose, mannose, xylose, arabinose, sucrose, maltose, trehalose, fibre
Two sugar and starches are tieed up as carbon source and produce butyric acid, yield reaches 10~14g/L；But DIRECT UTILIZATION OF CELLULOSE is unable to as carbon
Source.
Embodiment 3, cornstarch fermentation generate butyric acid and clostridium butyricum microbial inoculum
Seed culture fluid is prepared according to process described in 2 shake flask fermentation feature of embodiment.With cornstarch directly as
Carbon source prepares Medium of shaking flask fermentation, and the final concentration of 40g/L of carbon source, sterilize preceding culture medium boiling water bath 10min gelatinized starch, then
115 DEG C of sterilizing 20min.It takes 2.5mL clostridium butyricum SCUT620 seed culture fluid to be inoculated in asepsis injector to ferment equipped with 50mL
In culture medium, 37 DEG C, 150rpm, 72h.Fermentation results are as shown in figure 5,40g/L cornstarch can be in shake flask fermentation level
It is converted into 13.23g/L butyric acid product, butyric acid conversion ratio reaches 0.33g/g cornstarch, and furthermore by-product only has acetic acid (see figure
5).The above results show that clostridium butyricum SCUT620 has stronger cornstarch Utilization ability, and have the selection of preferable butyric acid
Property and conversion ratio.Fermentation liquid after above-mentioned fermentation is centrifuged under room temperature and non-anaerobic condition, supernatant is removed residual by ultrafiltration
Thallus and soluble protein are stayed, after being concentrated into about 50g/L butyric acid density, is used using trioctylamine as extractant, octanol is as dilute
Agent is released, extraction conditions is that trioctylamine concentration is 40% (v/v), fermentation liquid pH value 2~3, compared to 0.8,25 DEG C of temperature.It is used after extraction
0.8MNaOH is stripped organic phase, and final separation obtains pure butyric acid.After fermentation liquid sediment directly freezed is dry
To clostridium butyricum active bacteria preparation.The clostridium butyricum active bacteria preparation of preparation is continuously placed at room temperature 3 weeks, detection is lived weekly
Bacterium number amount.As a result as shown in fig. 6, the clostridium butyricum active bacteria preparation as prepared by fermentation liquid waste residue, quantity can reach quantity up to 2
×108Cfu/g or more, number of viable is still able to maintain 1 × 10 after continuous placement 3 weeks7Cfu/g or more has preferable storage-stable
Property, and preparation process is simple, strong operability.
Embodiment 4, tapioca fermentation generate butyric acid and clostridium butyricum microbial inoculum
Seed culture fluid is prepared according to process described in 2 shake flask fermentation feature of embodiment.With tapioca directly as
Carbon source prepares Medium of shaking flask fermentation, and the final concentration of 40g/L of carbon source, sterilize preceding culture medium boiling water bath 10min gelatinized starch, then
115 DEG C of sterilizing 20min.2.5mL clostridium butyricum SCUT620 seed culture fluid is taken to be inoculated in asepsis injector corresponding equipped with 50mL
In the Medium of shaking flask fermentation of carbon source, 37 DEG C, 150rpm, 72h.Fermentation results as shown in figure 5, in shake flask fermentation level 40g/
L tapioca can be converted into 14.65g/L butyric acid product, and butyric acid conversion ratio reaches 0.37g/g tapioca, furthermore by-product
Only acetic acid.The above results show that clostridium butyricum SCUT620 has stronger tapioca substrate Utilization ability, and have preferable
Butyric acid selectivity and conversion ratio.Fermentation liquid after above-mentioned fermentation is centrifuged under room temperature and non-anaerobic condition, supernatant passes through
Ultrafiltration removal residual thallus and soluble protein after being concentrated into about 50g/L butyric acid density, is used using trioctylamine as extractant,
Octanol is as diluent, and extraction conditions is that trioctylamine concentration is 40% (v/v), and fermentation liquid pH value 2~3 compares 0.8, temperature 25
℃.Organic phase is stripped with 0.8MNaOH after extraction, final separation obtains pure butyric acid；Fermentation liquid sediment is directly cold
Be lyophilized it is dry after obtain clostridium butyricum active bacteria preparation.The clostridium butyricum active bacteria preparation of preparation is placed into continuous placement at room temperature
3 weeks, number of viable was detected weekly.As a result as shown in fig. 6, clostridium butyricum active bacteria preparation, quantity as prepared by fermentation liquid waste residue
Quantity be can reach up to 4 × 108Cfu/g or more, number of viable is still able to maintain 1 × 10 after continuous placement 3 weeks7Cfu/g or more, table
It is bright that there is preferable storage-stable, and preparation process is simple, strong operability.
Sequence table
<110>South China Science & Engineering University
<120>a kind of clostridium butyricum and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1427
<212> DNA
<213>clostridium butyricum (Clostridium butyricum)
<400> 1
ttgtcgactc accccaatcg ctgaccctac cttaggtcgc tgcctccttg cggttagctc 60
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. a kind of clostridium butyricum, which is characterized in that the bacterium is clostridium butyricum (Clostridium butyricum) SCUT620, in
On March 29th, 2019, it is preserved in Guangdong Province's Culture Collection, deposit number is GDMCC NO:60623, preservation
Address: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, Guangdong Microbes Inst.
2. the application of clostridium butyricum described in claim 1, which is characterized in that the clostridium butyricum is residual with starch or starch processing
Slag is that carbon source through fermentation produces butyric acid.
3. application according to claim 2, which is characterized in that the starch is tapioca or cornstarch.
4. application according to claim 2 or 3, which is characterized in that the fermentation condition are as follows: 35 ± 2 DEG C, shaking speed
100~150rpm cultivates 48~72h.
5. application according to claim 2 or 3, which is characterized in that fermentation medium are as follows: dipotassium hydrogen phosphate 2g/L；Sulfuric acid
Ammonium 1g/L；Yeast powder 5g/L；MgSO4·7H2O0.6g/L；FeSO4·7H2O0.03g/L；20~60g/L of carbon source；Calcium carbonate
50g/L；Initial pH=7.0.
6. application according to claim 2 or 3, which comprises the steps of:
(1) actication of culture: after the dilution of clostridium butyricum SCUT620 bacterium solution, being coated on solid activation medium, 37 DEG C of culture 48h,
Picking single colonie is inoculated in seed culture medium, and 35 ± 2 DEG C of standing Anaerobic culturels 16~for 24 hours, until OD600It is 1.5~2；
(2) seed culture；Clostridium butyricum SCUT620 activation bacterium solution is taken to be inoculated in seed culture medium, 35 ± 2 DEG C of standing anaerobism trainings
Support 8~16h, OD600It is 1.5~2, this is seed culture fluid；
(3) fermented and cultured: then fermentation medium Heat Gelatinization starch sterilizes, by clostridium butyricum SCUT620 seed culture fluid
It is inoculated in fermentation medium and ferments；
(4) fermentation liquid is centrifuged, the isolated butyric acid from supernatant.
7. application according to claim 6, which is characterized in that
The activation medium are as follows: tryptone 10g/L；Beef extract 10g/L；Glucose 5g/L；Sodium chloride 5g/L；Yeast powder
3g/L；Sodium acetate 3g/L；Soluble starch 1g/L；L- cysteine ??acid hydrochloride 0.5g/L；Agar is also added in solid culture
15g/L；
The seed culture medium are as follows: glucose 20g/L；Sodium chloride 5g/L；Yeast powder 5g/L；Ammonium sulfate 3g/L；Dipotassium hydrogen phosphate
1.5g/L；MgSO4·7H2O0.6g/L, FeSO4·7H2O0.03g/L。
8. application according to claim 6, which is characterized in that the fermentation liquid sediment after step (4) centrifuge separation is straight
It connects freeze-drying and obtains clostridium butyricum active bacteria preparation.
9. application according to claim 6, which is characterized in that the supernatant removes residual thallus and solvable by ultrafiltration
Property albumen, after being concentrated into 40~60g/L butyric acid density, using trioctylamine as extractant, octanol is extracted as diluent
It takes, organic phase is stripped with NaOH after extraction, final separation obtains pure butyric acid.
10. application according to claim 9, which is characterized in that the extraction conditions is that trioctylamine concentration is 40% (v/
V), fermentation liquid pH value 2~3, compared to 0.8,25 DEG C of temperature.
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| CN111334532A (en) * | 2020-04-27 | 2020-06-26 | 驻马店华中正大有限公司 | Method for continuously fermenting butyric acid |
| CN112358986A (en) * | 2020-11-09 | 2021-02-12 | 华南理工大学 | Clostridium butyricum and application thereof in immobilized fermentation production of 1, 3-propylene glycol |
| 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 |
| CN117305381A (en) * | 2023-11-28 | 2023-12-29 | 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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