CN107227273A - Bacillus coagulans and its application for preparing L lactic acid - Google Patents
Bacillus coagulans and its application for preparing L lactic acid Download PDFInfo
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- CN107227273A CN107227273A CN201710472366.2A CN201710472366A CN107227273A CN 107227273 A CN107227273 A CN 107227273A CN 201710472366 A CN201710472366 A CN 201710472366A CN 107227273 A CN107227273 A CN 107227273A
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- bacillus
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- bacillus coagulans
- lactic acid
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- 241000193749 Bacillus coagulans Species 0.000 title claims abstract description 39
- 229940054340 bacillus coagulans Drugs 0.000 title claims abstract description 39
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 title description 10
- 238000000855 fermentation Methods 0.000 claims abstract description 69
- 230000004151 fermentation Effects 0.000 claims abstract description 67
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 24
- 239000001913 cellulose Substances 0.000 claims abstract description 9
- 229920002678 cellulose Polymers 0.000 claims abstract description 9
- 239000002994 raw material Substances 0.000 claims abstract description 8
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 85
- 239000004310 lactic acid Substances 0.000 claims description 43
- 235000014655 lactic acid Nutrition 0.000 claims description 43
- 239000001963 growth medium Substances 0.000 claims description 33
- 240000008042 Zea mays Species 0.000 claims description 32
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 32
- 239000010902 straw Substances 0.000 claims description 30
- 230000007062 hydrolysis Effects 0.000 claims description 25
- 238000006460 hydrolysis reaction Methods 0.000 claims description 25
- 239000007788 liquid Substances 0.000 claims description 23
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 18
- 235000005822 corn Nutrition 0.000 claims description 18
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 14
- 235000009973 maize Nutrition 0.000 claims description 14
- 108090000790 Enzymes Proteins 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 13
- 239000000413 hydrolysate Substances 0.000 claims description 11
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- 238000012545 processing Methods 0.000 claims description 8
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- 241000894006 Bacteria Species 0.000 claims description 6
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- 241000193375 Bacillus alcalophilus Species 0.000 description 1
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- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 description 1
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- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
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- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
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- 238000005374 membrane filtration Methods 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12R2001/07—Bacillus
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
- C12P7/56—Lactic acid
Abstract
The present invention provides a kind of bacillus coagulans BC-TJ or the bacillus produced by its passage, wherein described aggegation bacillus BC-TJ deposit number is CGMCC NO.14044.The bacterial strain is amphimicrobian bacterial strain, can be grown under aerobic and oxygen free condition, and fermentation temperature is high, and in fermentation process, living contaminants probability is small, without being sterilized to equipment etc., carries out open type fermented, reduces the costs such as the energy, manpower.The bacterial strain is that fermenting raw materials produce Pfansteihl using ligno-cellulose hydrolysate, and its yield is up to 160 ± 1.5g/l, and optical purity is up to more than 100%, and fermenter productivity is 2.2g/l/h.It without supplementing reducing sugar, can further reduce production cost during the fermentation, and it has larger potentiality in large-scale industrial production.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of bacillus coagulans and its utilizes lignocellulose for fermentation
Produce the application of Pfansteihl.
Background technology
Lactic acid is a kind of multiduty organic acid for being widely used in the industries such as food, medicine, weaving and chemical industry.It is used as one
Kind of multifunction green platform chemicals, lactic acid is considered as a kind of fine chemicals for having very much prospect and a good development potentiality.
Traditional lactic acid bacteria production method is broadly divided into chemical synthesis and microbe fermentation method.Compared with traditional chemical synthesis,
Optically pure Pfansteihl or D-ALPHA-Hydroxypropionic acid can be produced by different bacterial strains according to the need for difference using microbe fermentation method.
Two bottleneck problems are primarily present using Production by Microorganism Fermentation lactic acid at present.On the one hand, fermentation method use can
The sugar fermentation such as cost such as glucose, starch is higher；On the other hand, sterilizing, downstream separation purifying cost are higher in fermentation process.Pin
To first bottleneck problem, the starch or wooden fibre in more cheap renewable raw material, such as agricultural wastes can be used
Cellulosic material, and industry and urban waste etc. are tieed up as raw material fermentation lactic acid producing, the production cost of lactic acid is not only substantially reduced,
Component environment can also be solved the problems, such as, is turned waste into wealth.For second bottleneck problem, can with seed selection some thermophilic bacillus
To be fermented.Due to bacillus alcalophilus, environmental resistance is strong to external world, and fermentation temperature is high, and living contaminants probability is low, therefore nothing
Sterilizing need to be carried out to equipment etc. and carries out open type fermented, the costs such as the energy, manpower are reduced.Therefore, asked from above-mentioned two bottleneck
From the point of view of the solution of topic, seed selection is resistant to elevated temperatures, can be simultaneously solution for the bacterial strain of fermentation substrate using pentose and hexose
The certainly key of problem.
For above mentioned problem, application publication number discloses a kind of for preparing for CN102690764 Chinese invention patent
The bacillus coagulans and its application process of Pfansteihl, the bacterial strain can be using pentoses or hexose as raw material, after being fermented
Pfansteihl is obtained, and its yield is also higher, but the bacterial strain is mainly the accessory substance produced using in xylitol production process as original
Material, the xylose especially extracted using in Corncob hydrolysate is raw material, and it needs to add carbon source during the fermentation, because
This, its application is greatly limited.
Application publication number discloses bacillus for CN102643874 Chinese invention patent and utilizes corn stalk hydrolysis
The method for producing polymer grade Pfansteihl, specifically disclosing the bacillus can send out using corn stalk hydrolysis as sole carbon source
Ferment obtains Pfansteihl, and its Pfansteihl yield is up to 80g/l (every liter of zymotic fluid produces 80gL- lactic acid), although it can also be utilized
Corn stalk hydrolysis produces Pfansteihl, but its yield has much room for improvement.
The content of the invention
In view of this, the bacillus produced the invention provides a kind of bacillus coagulans BC-TJ or by its passage,
Wherein described bacillus BC-TJ deposit number is CGMCC NO.14044.Present invention also offers above-mentioned condensation gemma
Bacillus BC-TJ is passed on and application of the bacillus of generation in lactic acid is prepared by it, especially prepares answering in Pfansteihl
With.
The application of lactic acid is prepared in the bacillus produced above by bacillus coagulans BC-TJ or by its passage
In, its raw material can be ligno-cellulose hydrolysate.
Exemplarily, lignocellulosic is in maize straw, wheat stalk, rice straw or other agricultural crop straws
It is one or more.Present invention also offers a kind of culture medium, the culture medium is comprising above-mentioned bacillus coagulans BC-TJ or by its biography
Generation and produce bacillus.
Exemplary, the bacillus that the culture medium produces for above-mentioned bacillus coagulans BC-TJ or by its passage
Culture medium after fermentation.
Further, ligno-cellulose hydrolysate is also included in the culture medium.
Exemplary, ligno-cellulose hydrolysate is corn stalk hydrolysis, wheat stalk hydrolyzate, rice straw hydrolysis
Liquid or other agricultural crop straw hydrolyzates.Preferably, the maize straw is by sour water solution or enzyme hydrolysis or sour enzyme-linked and water
Hydrolyzate obtained by solution.
The bacillus that the present invention also provides above-mentioned bacillus coagulans BC-TJ or produced by its passage is repaiied by heredity
The bacillus for changing and preparing.
The present invention also offer prepares the preparation method of lactic acid, and it is included by above-mentioned bacillus coagulans BC-TJ or by it
The fermentation of bacillus lignocellulosic for passing on and producing, preferably ligno-cellulose hydrolysate.Exemplary, wood fibre water
Solution liquid is corn stalk hydrolysis, wheat stalk hydrolyzate, rice straw hydrolyzate or other agricultural crop straw hydrolyzates.
Specifically, the method for the present invention for preparing lactic acid includes：Bacillus coagulans BC-TJ or its passage are produced
Raw bacillus is inoculated in containing ligno-cellulose hydrolysate (such as corn stalk hydrolysis, wheat stalk hydrolyzate, straw
Stalk hydrolyzate or other agricultural crop straw hydrolyzates) processing culture medium in, under preference temperature ferment.The stalk hydrolysis
Liquid is the acid hydrolysis liquid or enzyme hydrolyzate or sour enzyme united hydrolysis liquid of stalk.
Exemplary, above-mentioned preparation method includes：By the bacillus coagulans BC-TJ seed liquors after activation, inoculation is everywhere
Manage in culture medium, at 30 DEG C -60 DEG C, quiescent culture is up to total reducing sugars and Pfansteihl in culture medium under the conditions of preferably 50 DEG C
Terminate fermentation during stable content.
Exemplary, acid described above is enzyme-linked and hydrolyzate preparation method includes：
1) stalk (such as maize straw, wheat stalk, rice straw or other agricultural crop straws) is immersed in dilute sulfuric acid
It is middle to be pre-processed, and optionally, treated stalk is washed, filter, be used to digest after drying；
2) cellulase solution and beta-glucosidase enzyme liquid enzymolysis processing are added in above-mentioned pretreated stalk；With
3) product after enzymolysis is stood or centrifuged, collect supernatant, as stalk hydrolyzate.
The present invention also provides the culture for the bacillus comprising above-mentioned bacillus coagulans BC-TJ or passed on by it and produced
Thing or its machining object.
The present invention has filtered out a kind of bacillus coagulans BC-TJ, and its deposit number is CGMCC NO.14044.The bacterial strain
For amphimicrobian bacterial strain, it can be grown under aerobic and oxygen free condition, and fermentation temperature is high, in fermentation process, living contaminants is several
Rate is small, without being sterilized to equipment etc., carries out open type fermented, reduces the costs such as the energy, manpower.The bacterial strain utilizes wooden
Cellulosic hydrolysate is that fermenting raw materials produce Pfansteihl, and its yield is up to 160 ± 1.5g/l, and optical purity is up to 100%, and fermentation is given birth to
Production capacity power is 2.2g/l/h.It without supplementing reducing sugar, can further reduce production cost during the fermentation, its
There are larger potentiality in large-scale industrial production.
Embodiment
The technical scheme in the embodiment of the present invention is clearly and completely described below, it is clear that described embodiment
Only it is a part of embodiment of the invention, rather than whole embodiments.Based on the embodiment in the present invention, ordinary skill
The every other embodiment that personnel are obtained under the premise of creative work is not made, belongs to the scope of protection of the invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
The separation and screening of the bacterial strain of embodiment 1
Process of the test used medium is as follows：
Enriched medium：Contain xylose 10g, dusty yeast 10g in every liter of culture medium, remaining is distilled water, pH 6.0,115
DEG C sterilizing 20min.
Fermentation medium：Contain xylose 50g, dusty yeast 10g in every liter of culture medium, remaining is distilled water, pH 6.0,115
DEG C sterilizing 20min.
Growth medium：Contain xylose 30g, dusty yeast 10g, CaCO in every liter of culture medium310g, 15~20g of agar powder,
6.5,115 DEG C of sterilizing 20min of pH.
Seed culture medium：Contain glucose 50g, dusty yeast 10g, CaCO in every liter of culture medium320g, remaining is distilled water,
6.5,115 DEG C of sterilizing 20min of pH.
Handle culture medium：Gas explosion 100~150ml of corn stalk hydrolysis, dusty yeast 10g, CaCO330g, pH6~7,
115 DEG C of sterilizing 20min.
Hebei rural area compost sample is gathered, 1g is weighed and is placed in 20ml enriched mediums, 50 DEG C of enrichment culture 12h.It is placed in
20min is heated in 80 DEG C of water-baths, the thalline for not forming gemma is killed, gradient dilution separation is carried out after taking-up.Single bacterium to be grown
Fall behind, select fast growth and the big bacterium colony of production transparent circle area carries out purifying culture.Bacterial strain after purification is accessed into fermentation
50 DEG C of quiescent culture 48h in culture medium, L-lactic acid is determined using bio-sensing analyzer SBA-40D.Bio-sensing is analyzed
Instrument SBA-40D is, using Pfansteihl as substrate, H to be generated under the catalytic action of enzyme with oxygen gas and water2O2。H2O2Through the internal layer of enzyme membrane
Electron reduction is lost into H on platinum electrode surface2O, platinum electrode obtains electronics and produces current signal, the current signal and H2O2's
Concentration is proportional, and H2O2Concentration is linearly proportional with concentration of substrate.Produced by comparing measured object and standard sample
H2O2Amount, so that it may calculate the content of substrate in sample.
By screening is repeated several times, a kind of lactic acid production highest bacterial strain BC-TJ is selected, is preserved and carried out follow-up
Experiment.
The bacterial strain BC-TJ of embodiment 2 colony morphology characteristic and physiological and biochemical property
Bacterial strain BC-TJ is inoculated on growth medium and grown after 24h, observes that its bacterium colony is rounded, surface is smooth, in breast
White, edge-smoothing.Gram's staining is positive, and cellular morphology is shaft-like, and thalline size is (0.7~0.9) × (3~5) μm,
Endogenous spore.The suitable growth temperature of the bacterial strain is 30 DEG C~60 DEG C, and the most suitable growth pH is 5.5~6.5.In physiological and biochemical test
In, gelatin liquefaction, indoles experiment and glucose fermentation aerogenesis are feminine gender；Starch Hydrolysis, V.P experiments are the positive, 60 DEG C of aerobic bars
It can be grown under part and anaerobic condition.Specific experiment result is referring to table 1.
The bacterial strain BC-TJ of table 1 bio-chemical characteristics result
Note："+" represents positive；"-" represents negative.
The bacterial strain BC-TJ of embodiment 3 16S rDNA sequence analyses
In order to more accurately identify isolated bacterial strain BC-TJ, the 16S rDNA sequences to bacterial strain BC-TJ expand
Increase and compare.Bacterial strain BC-TJ genomic DNA is extracted first, the template for 16S rDNA sequence amplifications.
Bacterial strain BC-TJ, 12000rpm room temperature the centrifugation 1min that quiescent culture is stayed overnight collect thalline, are carried using DNA of bacteria
Kit is taken to carry out DNA extractions (TIANGEN Biotech (Beijing) Co., Ltd.), method is described to specifications to be carried out.
16S rDNA sequence amplification primers：
27f：5’-AGAGTTTGATCCTGGCTCAG-3’
1492r：5’-GGYTACCTTACGACTT-3’
Reaction condition：95 DEG C of pre-degeneration 5min；5 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 1min, totally 35 are followed
Ring；72 DEG C extend 10min afterwards.
Reaction system：15 μ l, Fast-pfu Taq enzyme of μ l, Buffer of template 1,4 μ l of μ l, dNTP (2.5mM each), just
Reverse primer (100mM) each 0.4 μ l, ddH2O complement to 50 μ l reaction systems.
Sequencing is sent after purpose fragment glue reclaim.Sequencing results (http in NCBI：//
Www.ncbi.nlm.nih.gov BLASTn comparisons) are carried out.As a result show, gained sequence and bacillus coagulans (Bacillus
Coagulans) homology is up to 99%, in conjunction with morphology, physiological and biochemical test result, with reference to Bergey ' s manual of
Determinative bacteriology (Holt JG etc. 1994) determine that bacterial strain BC-TJ is bacillus coagulans (Bacillus
Coagulans) BC-TJ, and given in depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation (address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), preservation date：On April 18th, 2017, preserving number：CGMCC
NO.14044。
The bacillus coagulans BC-TJ fermented maize stalk fermentations liquid of embodiment 4 produces Pfansteihl
1. the acquisition of gas explosion corn stalk hydrolysis
Gas explosion maize straw is handled by steam blasting equipment.Reaction condition is as follows：By maize straw be immersed in 3% it is dilute
2h in sulfuric acid, 170 DEG C of processing 5min.Then by washing 10 time of the treated maize straw with 10 times of volumes, drying after filtering
It is directly used in enzymolysis.Gas explosion corn stalk is handled using trichoderma reesei cellulase enzyme liquid and aspergillus niger beta-glucosidase enzyme liquid
Stalk.Enzyme digestion reaction system and condition are as follows：Substrate loading capacity is 10% (w/v), and cellulase is 15FPU/g substrates, β-grape
Glycosidase is 30CBU/g substrates, reacts in 50mM, pH 4.8 citrate buffer solution and carries out 48h.Hydrolysate after enzymolysis
8000rpm centrifuges 10min, and collection supernatant, as gas explosion corn stalk hydrolysis add during the fermentation as medium component
Plus.
2. the measure of lactic acid content
Lactic acid content determines and uses liquid chromatography.Using waters600 high performance liquid chromatograph systems, chromatographic column is used
Chiral column Chirex3126 (D)-penicillami, 4.6mmID × 250mmL.Mobile phase 2mmol/L CuSO4Solution, wherein
Solvent is 5% aqueous isopropanol, and flow rate of mobile phase is 0.7ml/min, and 30 DEG C of column temperature, UV-detector wavelength is 254nm, is entered
Sample amount is 2pl, is integrated with Empower integral softwares.Testing sample and the equal use of flowing are preceding after 0.22 μm of membrane filtration
Ultrasound degassing.Standard curve is made according to standard items, the content of lactic acid in sample is then calculated further according to standard curve.
3. the measure of total reducing sugars
Total reducing sugars is determined using DNS methods (3,5- dinitrosalicylic Acid Colorimetry).Glucose is diluted different first
Gradient prepares glucose standard curve, and concentration calculating is carried out further according to standard curve.Specific reaction is as follows：
Addition 0.5ml glucose solution sample in test tube, adds 0.05mM, pH 4.8 citrate buffer solution 1ml, 50 DEG C
Water-bath 60min；3ml DNS reaction solutions are added, are mixed, boiling water bath heating 5min is cooled to room temperature；Under 540nm wavelength,
The absorbance of determination sample.
4. optical purity is calculated
The optical purity of Pfansteihl=(Pfansteihl yield-D-ALPHA-Hydroxypropionic acid yield)/(Pfansteihl yield+D-ALPHA-Hydroxypropionic acid yield) ×
100%
5.L- lactic fermentations production capacity is calculated
Pfansteihl production capacity (g/l/h)=Pfansteihl yield (g/l)/fermentation time (h)
6. bacillus coagulans BC-TJ fermentation production of L-lactic acid
By the bacterial strain BC-TJ streak inoculations being stored in -80 DEG C of glycerine on growth medium, 50 DEG C of culture 24h are activated
Strain；Aseptically the ring strain of oese 1 is connected in the 250ml triangular flasks containing 30ml seed culture mediums, 30 DEG C quiet
Put after culture 20h as seed；Seed is by 10% inoculum concentration access processing culture medium, and 50 DEG C of quiescent cultures are until culture medium
Terminate fermentation when middle total reducing sugars and stable L-lactic acid.According to above-mentioned assay method after fermentation ends, to each index
It is measured and calculates.Experimental result refers to table 2.
The situation of the bacillus coagulans BC-TJ L-lactic acid fermentations of table 2
| Hydrolyzate (ml/l) | 100 | 125 | 150 |
| The time required to fermentation is completed (h) | 72h | 72h | 72h |
| Pfansteihl (g/l) | 112±2.1 | 143±3.1 | 160±1.5 |
| Conversion ratio (g/ml) | 1.12 | 1.14 | 1.07 |
| Production capacity (g/l/h) | 1.56 | 1.99 | 2.22 |
Bacillus coagulans BC-TJ mass produces the application of Pfansteihl
During the bacillus coagulans BC-TJ fermentation large-scale production Pfansteihls provided using the present invention, by seed culture medium
Fermentation tank is accessed by 5%~20% inoculum concentration of volume ratio, fermentation tank can select 1m according to needs of production3、2m3、3m3
Or higher volume of fermentation tank, fermentation tank charge weight is 70%~80% (V/V), and obstructed gas agitating, stir speed (S.S.) is 80-
100r/min。
Large-scale fermentation medium is made up of carbon source, nitrogen source and inorganic salts, the bacillus coagulans BC- that the present invention is provided
TJ carbon source and nitrogen source selection it is quite varied, carbon source can for glucose, sucrose, maltose, fructose, arabinose or corn,
The starchy material such as rice, sweet potato, cassava or cornstarch, tapioca, wheaten starch, starch from sweet potato, farina or
One or more in maize straw, wheat stalk, rice straw or other agricultural crop straw class materials, wherein starchy material
Hydrolysis sugar is prepared into through enzymatic liquefaction, saccharification, agricultural crop straw class material is through sour water solution or enzyme hydrolysis or smoked plum united hydrolysis system
It is standby into hydrolyzate.Nitrogen source can be peptone, dusty yeast, yeast extract, beef extract, Mai Gen, wheat bran, rice bran, corn steep liquor, soybean
One or more in peptide, cottonseed protein etc..
During the fermentation CaCO is added according to mass volume ratio3, 4.5- is maintained with the pH of control process culture medium
6.5.Fermentation temperature is 40 DEG C~60 DEG C, preferably 50 DEG C.
Environment of the bacillus coagulans BC-TJ in oxygen deprivation that the preserving number that the present invention is provided is CGMCC NO.14044
Under, the Pfansteihl of high-optical-purity is produced by semicontinuous batch fermentation mode or middle feeding glucose fermentation mode.
Illustrate semicontinuous batch fermentation mode of the invention or middle feeding glucose fermentation side by taking corn stalk hydrolysis as an example below
Formula mass produces the process of Pfansteihl.
The semicontinuous batch fermentation mode of application example 1 mass produces Pfansteihl
By the bacillus coagulans BC-TJ streak inoculations being stored in -80 DEG C of glycerine in growth medium, 40 DEG C~50
DEG C culture 18~24h activated spawns；Aseptically the ring strain of oese 1 is connected in seed culture medium, 50 DEG C stand training
Seed is used as after supporting 20h；Seed is by 5%~20% fermentation tank of the inoculum concentration access equipped with processing culture medium, and fermentation tank can
1m is selected according to needs of production3、2m3、3m3Or higher volume of fermentation tank, temperature control is at 40 DEG C~60 DEG C, preferably
For 45 DEG C~55 DEG C, more preferably 50 DEG C, 24~48h of anaerobic fermentation, obstructed gas agitating, speed of agitator is 80~100r/
Min, as glucose residual≤0.3g/L in zymotic fluid, terminates fermentation, turns the 5%~20% of this zymotic fluid as seed liquor
The fermentation of next batch is carried out in the fresh processing culture medium of access, remaining zymotic fluid enters abstraction process.
Remaining lactic fermentation liquid is filtered to remove solid insoluble matter, calcium lactate solution is obtained；Then into calcium lactate solution
Sulfuric acid solution is added, the mass fraction of sulfuric acid is 30%, and treatment temperature is 70 DEG C, and the pH value for controlling final acidolysis system is 2.5.
Filtered after acidolysis, obtain lactic acid solution after acidolysis, heating concentration removes in lactic acid solution 80% moisture, obtain preliminary dense
The lactic acid solution of contracting.
The lactic acid solution temperature of initial concentration is risen to 40 DEG C, the activated carbon of lactic acid solution quality 1.5% is added, persistently stirs
Mix 25min and carry out decolorization, the lactic acid clear liquid after being decolourized adds the n-butanol of 0.02 times of volume in above-mentioned clear liquid,
Secondary concentration dehydration is carried out by using thin film evaporation, it is 80kPa to control vacuum, and lactic acid content is into lactic acid clear liquid
91%, lactic acid concentrate after being decolourized.Above-mentioned lactic acid concentrate is placed in ice-water bath, when temperature is down to 15 DEG C, Xiang Qi
It is middle to add the sodium acetate of concentrate quality 0.5%, and the ethanol of 3 times of volumes of concentrate is slowly added to while stirring, by 25min
Left and right, the temperature of system is down to 5 DEG C, stops cooling, is filtrated to get insoluble solid content and lactic acid filtrate.
Ethanol washing will be added in gained solid content, addition is 0.6 times of above-mentioned ethanol usage amount, is sunk after filtering
Starch and lactic acid filtrate.Two parts lactic acid filtrate is mixed, vacuum distillation is carried out under vacuum 90kPa, removing is therein to be had
Machine solvent (n-butanol, ethanol) and residual moisture.Finally, refined by molecular distillation, temperature is 70 DEG C, pressure is
20Pa.The yield of Pfansteihl is 94.6%, and optical purity is 100%.
The middle feeding glucose fermentation mode of application example 2 mass produces Pfansteihl
By the bacillus coagulans BC-TJ streak inoculations being stored in -80 DEG C of glycerine in growth medium, 40 DEG C~50
DEG C culture 18~24h activated spawns；Aseptically the ring strain of oese 1 is connected in seed culture medium, 50 DEG C stand training
Seed is used as after supporting 20h；Seed is by 5%~20% fermentation tank of the inoculum concentration access equipped with processing culture medium, and fermentation tank can
1m is selected according to needs of production3、2m3、3m3Or higher volume of fermentation tank, temperature control is at 40 DEG C~60 DEG C, preferably
For 45 DEG C~55 DEG C, more preferably 50 DEG C, obstructed gas agitating, speed of agitator is 80~100r/min, anaerobic fermentation 12~
24h, adds corn stalk hydrolysis, continues to ferment, reacts and terminate after 60-72h, zymotic fluid enters abstraction process.
Above-mentioned lactic fermentation liquid is filtered to remove solid insoluble matter, calcium lactate solution is obtained；Then into calcium lactate solution
Sulfuric acid solution is added, the mass fraction of sulfuric acid is 30%, and treatment temperature is 70 DEG C, and the pH value for controlling final acidolysis system is 2.5.
Filtered after acidolysis, obtain lactic acid solution after acidolysis, heating concentration removes in lactic acid solution 80% moisture, obtain preliminary dense
The lactic acid solution of contracting.
The lactic acid solution temperature of initial concentration is risen to 40 DEG C, the activated carbon of lactic acid solution quality 1.5% is added, persistently stirs
Mix 25min and carry out decolorization, the lactic acid clear liquid after being decolourized adds the n-butanol of 0.02 times of volume in above-mentioned clear liquid,
Secondary concentration dehydration is carried out by using thin film evaporation, it is 80kPa to control vacuum, and lactic acid content is into lactic acid clear liquid
91%, lactic acid concentrate after being decolourized.Above-mentioned lactic acid concentrate is placed in ice-water bath, when temperature is down to 15 DEG C, Xiang Qi
It is middle to add the sodium acetate of concentrate quality 0.5%, and the ethanol of 3 times of volumes of concentrate is slowly added to while stirring, by 25min
Left and right, the temperature of system is down to 5 DEG C, stops cooling, is filtrated to get insoluble solid content and lactic acid filtrate.
Ethanol washing will be added in gained solid content, addition is 0.6 times of above-mentioned ethanol usage amount, is sunk after filtering
Starch and lactic acid filtrate.Two parts lactic acid filtrate is mixed, vacuum distillation is carried out under vacuum 90kPa, removing is therein to be had
Machine solvent (n-butanol, ethanol) and residual moisture.Finally, refined by molecular distillation, temperature is 70 DEG C, pressure is
20Pa.The yield of Pfansteihl is 93.2%, and optical purity is 100%.
The preserving number that the present invention is provided is a kind of new can be used for for CGMCC NO.14044 bacillus coagulans BC-TJ
The high temperature resistant microorganism of Pfansteihl fermenting and producing, can homofermentation production optical purity be 100% in the case of oxygen deprivation
Pfansteihl, its yield may be up to 160 ± 1.5g/l, and fermenter productivity is 2.2g/l/h.The strain fermentation maize straw ferments
Liquid produces Pfansteihl, and its fermentation process is simple, without oxygen supply, reduces stirring for the energy consumption needed for filtrated air supply and fermentation tank
Energy consumption is mixed, production cost has been saved.The most suitable fermentation temperature of the bacterial strain is 40 DEG C -60 DEG C, and its higher fermentation temperature can
Greatly reduce the chance of living contaminants.In addition, the fermentation substrate of the bacterial strain be maize straw, wheat stalk, rice straw or
One or more in the hydrolyzate of other agricultural crop straws, can give up the stalks such as maize straw, wheat stalk, rice straw
Gurry is fully utilized, and is turned waste into wealth, it is to avoid pollution environment.
Using the bacterial strain large scale fermentation corn stalk hydrolysis, the yield of Pfansteihl is more than 93%, and optical purity is
100%, therefore, the bacterial strain can be applied to large-scale industrial production.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God is with principle, and any modification, equivalent substitution for being made etc. should be included in the scope of the protection.
<110>Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences
<120>Bacillus coagulans and its application for preparing Pfansteihl
<130> 2017
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 16
<212> DNA
<213>It is artificial synthesized
<400> 2
ggytacctta cgactt 16
Claims (13)
1. bacillus coagulans BC-TJ or the bacillus produced by its passage, wherein described bacillus coagulans BC-
TJ deposit number is CGMCCNO.14044.
2. bacillus coagulans BC-TJ defined in claim 1 or the bacillus produced by its passage are preparing lactic acid
Application in (such as Pfansteihl).
3. application according to claim 2, the bacillus coagulans BC-TJ or the gemma bar produced by its passage
Bacterium is using ligno-cellulose hydrolysate as raw material.
4. application according to claim 3, the lignocellulosic is selected from by maize straw, wheat stalk and rice straw
The group of composition.
5. culture medium, its gemma produced comprising bacillus coagulans BC-TJ defined in claim 1 or by its passage
Bacillus.
6. culture medium according to claim 5, it is fermentation culture.
7. culture medium according to claim 6, it includes ligno-cellulose hydrolysate (for example：The sour water solution of maize straw
Liquid or enzyme hydrolyzate or sour enzyme united hydrolysis liquid).
8. the bacillus produced from bacillus coagulans BC-TJ defined in claim 1 or by its passage is by heredity
The bacillus changed and prepared.
9. the preparation method of lactic acid, it is including the use of bacillus coagulans BC-TJ defined in claim 1 or by its passage
And the fermentation of bacillus ligno-cellulose hydrolysate (such as corn stalk hydrolysis) produced.
10. preparation method according to claim 9, it is included bacillus coagulans BC- defined in claim 1
In the processing culture medium that TJ or the bacillus produced by its passage are inoculated in containing corn stalk hydrolysis, in preference temperature
Lower fermentation.
11. preparation method according to claim 10, the corn stalk hydrolysis for maize straw acid hydrolysis liquid or
Enzyme hydrolyzate or sour enzyme united hydrolysis liquid.
12. preparation method according to claim 10, the preference temperature is 30 DEG C -60 DEG C, preferably 50 DEG C.
13. culture or its machining object comprising the bacillus coagulans BC-TJ described in claim 1.
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| CN112694993A (en) * | 2020-12-31 | 2021-04-23 | 安徽丰原生物技术股份有限公司 | Bacillus coagulans and method for preparing L-lactic acid by using same |
| CN112694993B (en) * | 2020-12-31 | 2021-09-24 | 安徽丰原生物技术股份有限公司 | Bacillus coagulans and method for preparing L-lactic acid by using same |
| CN113186232A (en) * | 2021-06-15 | 2021-07-30 | 南京林业大学 | Method for producing lactic acid by co-culture fermentation of pseudomonas putida and bacillus coagulans |
| CN113186232B (en) * | 2021-06-15 | 2023-11-17 | 南京林业大学 | Method for producing lactic acid by co-culture fermentation of pseudomonas putida and bacillus coagulans |
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