CN101285047A - High-optical-purity D-lactic acid producing strain and process for producing D-lactic acid by fermentation of same - Google Patents
High-optical-purity D-lactic acid producing strain and process for producing D-lactic acid by fermentation of same Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 104
- 230000004151 fermentation Effects 0.000 title claims abstract description 104
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 title claims abstract description 79
- 229940022769 d- lactic acid Drugs 0.000 title claims abstract description 79
- 229930182843 D-Lactic acid Natural products 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 16
- 239000001963 growth medium Substances 0.000 claims abstract description 48
- 238000011218 seed culture Methods 0.000 claims abstract description 39
- 238000004519 manufacturing process Methods 0.000 claims abstract description 29
- 230000003287 optical effect Effects 0.000 claims abstract description 29
- 239000002253 acid Substances 0.000 claims abstract description 19
- 229940039696 lactobacillus Drugs 0.000 claims description 54
- 241000186660 Lactobacillus Species 0.000 claims description 51
- 239000007788 liquid Substances 0.000 claims description 49
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 32
- 239000008103 glucose Substances 0.000 claims description 31
- 238000005516 engineering process Methods 0.000 claims description 29
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 27
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- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 22
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a high-optical-purity D-lactic acid producing strain, namely a lactobacillus sp.Y2-8 strain and a process for producing D-lactic acid by fermenting the same. The whole fermentation process comprises plate culture, seed culture and fermentation acid production, anaerobic conditions are kept from the plate culture, the seed culture to the fermentation acid production, strict step-by-step regulation and control are not needed, and the operation is simple; the components of the culture medium of the fermentation process are easy to obtain, the formula is simple, and the production cost is low; the optical purity of the D-lactic acid produced by the strain and the fermentation process is as high as 99.1 percent, and the content of the D-lactic acid in the fermentation liquor is 9.1 to 16.5 percent, so the strain and the fermentation process have great social significance and economic value.
Description
Technical field
The present invention relates to the technology of a plant height optical purity D-lactic-acid-producing strain lactobacillus Sporolactobacillus sp.Y2-8 bacterial strain and fermentation production of D-lactic acid thereof, belong to the organic acid fermentation production field.
Background technology
Lactic acid (Lactic acid) has another name called alpha-hydroxypropionic acid (α-Hydroxypropionic acid), is one of three universally acknowledged big organic acids, can be divided into L-lactic acid, D-lactic acid and D according to its opticity character, L-lactic acid.
The series exploitation of purposes newly along with D-lactic acid also more and more is subjected to people's attention to the application of D-lactic acid and the research of production.At present, the purposes of D-lactic acid mainly shows:
(1) D-lactic acid is synthetic fine herbicide-galloping horse (Puma Super) of gramineous weeds and New-type wide-spectrum highy potent herbicide-Wei despot's the raw material prevented and kill off, and the while also is that methoxone propionic acid, fluorine are the raw material of novel agrochemical such as weedicide;
(2) by high-optical-purity (optical purity is more than 97%) D-lactic acid and methanol esterification synthetic D-methyl lactate at the raw material of medicine, agricultural chemicals, solvent and the synthetic precursor and the intermediate of other chipal compounds;
(3) be used for the production poly(lactic acid):
Poly(lactic acid) will progressively substitute existing degradability plastics, and have the ability of competing mutually with polyolefins.By 2010, the poly(lactic acid) annual requirement will break through 1,000,000 tons.Lactic acid makes rac-Lactide through cyclodehydration, and rac-Lactide makes poly(lactic acid) (PLA) through ring-opening polymerization.So because lactic acid be chiral molecules correspondingly, institute's synthetic poly(lactic acid) has poly (l-lactic acid) (PLLA), poly-D-lactic acid (PDLA) and poly-D, L-lactic acid (PDLLA).But the low and degradation speed of the fusing point of PLLA is relatively slow but to have become to limit the bottleneck that it further develops.Current research shows, the blended complex of PLLA and PDLA can be brought up to 225 ℃ from 177 ℃ with the fusing point of poly(lactic acid), adjust the degradation speed of the final material of adjustable ratio control of different components in PLLA and the PDLA blended complex, this has brought more wide prospect for the application of poly(lactic acid).High optical purity D-lactic acid polymerizes is prepared from and PDLA is just by adding.
The huge market potential of above product has expedited the emergence of carrying out of efficient high optical purity D-lactic acid-producing research.Directly utilize Production by Microorganism Fermentation chirality lactic acid, compare with chemical synthesis, chiral separation method and enzyme transforming process, fermentation method not only can obtain the D-lactic acid of optical purity by strain improvement or metabolic regulation, and fermentation raw material wide material sources, production cost are low, reusable edible, environmental friendliness, have become the method that the manufacturer production chirality lactic acid in the whole world nearly 80% adopts.1994, it was that substrate fermentation produces D-lactic acid with glucose that people such as Kosaki utilize synanthrin lactobacillus (Sporolactobacillus inulinus ATCC15538), and behind the fermentation 37h, the production peak of D-lactic acid reaches 98g/L, and optical purity reaches 99%; Japan Da Yisaier ( イ セ Le) chemical industrial company had the D-lactic acid-producing ability of annual 10 tons of optical purities more than 97% in 1997; 2003, people such as Remedios Yanez and Ana Belen Moldes are raw material with the Mierocrystalline cellulose, utilize Lactobacillus coryniformic subsp.torquens ATCC 25600 fermentations to produce D-lactic acid, add cellulase and beta-glucosidase during fermentation, carry out the fermentation of cellulosic saccharification and D-lactic acid simultaneously, at 39 ℃, the condition bottom fermentation 48h of pH5.4, the D-lactic acid yield reaches 0.5gL
-1H
-1, be 0.89g D-lactic acid/g Mierocrystalline cellulose to the Mierocrystalline cellulose productive rate, but the D-lactic acid production is lower; 2004, G.Bustos, people such as A.B.Moldes and J.L.Alonso utilizes the response surface optimized method that the substratum of Lactobacillus coryniformis fermentation production of D-lactic acid is optimized, the final corn steep liquor 5g/L that adopts, the substratum of yeast extract paste 3.6g/L and peptone 10g/L obtains D-lactic acid 58.9g/L behind the fermentation 96h; 2006, T.Tanaka, people such as M.Hoshina are raw material with defatted rice bran (bran), utilize Lactobacillus delbrueckii subsp.DelbrueckiiIFO 3202 fermentations to produce D-lactic acid, adding rice bran, amylase, cellulase carry out saccharification and fermentation simultaneously during fermentation, the mass yield of final D-lactic acid can reach rice bran 28% and enzymic hydrolysis liquid glucose 78%, and the optical purity of D-lactic acid is 95%.
Domestic open and report lessly to D-lactic-acid-producing strain and production technique.The Ding Zijian of this research department equals 2004 reported first and utilizes lactobacillus (Sporolactobacillus sp.) to produce the technology of D-lactic acid from glucose fermentation, fermenting, the D-lactic acid production is 40.7g/L after 72 hours, optical purity reaches 96.04%, the product optical purity of this technology is higher, but output is on the low side relatively; 2006, Yang Wenge etc. have reported and have utilized lactobacillus to adopt the combined fermentation technology to produce the technology of D-lactic acid, from glucose, fermented 25～38 hours, produce acid and can reach 75～131g/L lactic acid, optical purity reaches 99%, but this technology has been used the combined fermentation method, be that fermenting process needs to regulate and control to carry out stage by stage according to aerobic fermentation, micro-aerobe fermentation and last anaerobism acidogenic fermentation three steps, the relative difficulty of operation with control.Therefore, the D-lactic-acid-producing strain of seed selection high-optical-purity, and development and operation is simple, the high D-zymotechnique of lactic acid of the sour efficient of product is a D-lactic acid industrial expansion direction.
Summary of the invention
Technical purpose of the present invention provides the good D-lactic acid-producing bacterial strain of a strain, and utilize this strain fermentation to produce the technology of D-lactic acid, the D-lactic acid that makes this production bacterial strain be produced has high-optical-purity, and by suitable technological condition for fermentation is provided, the feasible technology of utilizing this production strain fermentation to produce D-lactic acid is not only simple and convenient, and raw material is easy to get, and is with low cost, reaches higher acid producing ability on the basis that guarantees the product high-optical-purity.
For realizing technical purpose of the present invention, the present invention takes following technical scheme:
One, high optical purity D-lactic acid is produced bacterium lactobacillus Sporolactobacillus sp.Y2-8 bacterial strain:
1, D-lactic acid original strain is produced in screening from soil
With the high sugar liquors substratum of glucose the sugared microorganism of anti-height in the pedotheque of area, Nanjing is carried out enrichment culture, again by the colour developing of high glucose-tetrabromo-mcresolsulfonphthalein dull and stereotyped under the anaerobism culture condition to the acid-producing bacteria strain of anti-high sugar carry out list from, select the big and fireballing bacterial strain of variable color of variable color loop diameter and carry out acidogenic fermentation research, obtain a strain D-lactic homofermentation bacterial strain, called after Sporolactobacillus DX12, the very low temperature preservation is standby.
Sporolactobacillus DX12 identifies through BIOLOG automatic bacterial assessing instrument, show that this bacterial strain and lactobacillus Sporolactobacillus similarity (SIM) are 0.60, show that through the 16SrDNA sequential analysis being higher than 99% bacterial strain with this sequence similarity degree is Sporolactobacillus and belongs to bacterial strain, be 99.8% wherein, be defined as a new bacterial strain of lactobacillus with bacterial strain Sporolactobacillusinulinus ATCC15538 similarity.
2, the selection of bacterial strain Y2-8
After adopting the low energy ion injection method that D-lactic homofermentation DX12 bacterial strain is carried out mutagenesis, on pH reagent type flat board, screen, with the bacterium variable color circle that sets out is contrast, is the forward mutation standard with the variable color loop diameter greater than starting strain 0.5cm, screens 18 strain output positive mutating strains altogether; At last, fermention medium is optimized, adopts the C18 post tunning of 16 strain output positive mutating strains to be carried out quantitative assay with the HPLC method.Through the fermentation test of going down to posterity, obtain a strain and stablize high yield D-lactobacillus strain, called after Sporolactobacillus sp.Y2-8.
The present invention identifies the biological characteristic of the mutagenesis mutant strain Y2-8 of lactobacillus Sporolactobacillus DX12, the gramstaining of this bacterium observed show that this bacterial strain is G
+Bacillus has gemma, and size is 0.5 μ m * 2～3 μ m.Behind the growth 36h, the bacterium colony size is 1.5～2mm in plate culture medium, and growth temperature range is 25～42 ℃, and optimum temperuture is 37 ℃, and growth pH is 4～7.5, and optimal pH is 6.0; Its physio-biochemical characteristics show that to the catalase reaction feminine gender, gelatin does not liquefy, and do not form indoles, do not reduce nitrate, the litmus milk nondiscoloration.The result is positive for Inositol nf12 99, maltose, D-sucrose, seminose and α-D-lactose utilization, and D-pectinose, D-semi-lactosi and citric acid utilize the result negative, grow under the amphimicrobian condition.
Two, utilize the technology of lactobacillus Y2-8 fermentation production of D-lactic acid, comprise dull and stereotyped cultivation, seed culture and fermentation and acid, it is characterized in that: after under anaerobic carrying out flat board cultivation and seed culture, with starch based saccharification liquid is carbon source, carry out fermentation and acid by the mode that in fermentor tank, feeds rare gas element, the omnidistance anaerobism of technological process is carried out.
(1) the dull and stereotyped cultivation: lactobacillus Y2-8 is seeded to the plate culture medium anaerobism cultivates 30～40 ℃ of culture temperature, incubation time 20～36h;
Plate culture medium weight percent proportioning is a glucose 10～15%, yeast extract paste 0.1～0.3%, anhydrous sodium acetate 0.2～0.5%, anhydrous magnesium sulfate 0.01～0.05%, potassium primary phosphate 0.1～0.3%, agar 1.8%, water surplus, pH7.0;
(2) seed culture: the lactobacillus Y2-8 that flat board is cultivated is inoculated into the cultivation of seed culture medium anaerobism, 30～40 ℃ of culture temperature, incubation time 12～18h.
The required carbon source of seed culture medium comprises that concentration is a kind of in 2%～5% glucose, the corn saccharification liquid or both mixtures;
The required nitrogenous source of seed culture medium comprises that concentration is one or more mixture of 0.2%～1.0% yeast extract paste, peptone, extractum carnis, corn steep liquor, wheat bran;
Mineral ion weight percent proportioning in the seed culture medium is an anhydrous magnesium sulfate 0.01～0.05%, ferrous sulfate 0.001～0.005%, manganous sulfate 0.001～0.005%, lime carbonate 1.5～3%, water surplus, pH7.0.
The seed culture stage is by the fluid-tight of 2～3cm whiteruss, or reduces rotating speed when increasing liquid amount and realize that anaerobism cultivates; Seed culture can be added granulated glass sphere in the stage in shaking bottle, to strengthen mass transfer effect.
(3) fermentation and acid: seed culture fluid is inoculated into carries out acidogenic fermentation in the fermention medium, be carbon source directly with starch based saccharification liquid, inoculum size is 5%～10% (v/v), 30～40 ℃ of leavening temperatures, feed rare gas element with every liter of substratum 0.6～2L/min amount of overall flow rate, adopt neutralizing agent that fermentation system pH is controlled at 5.0～7.0, fermentation 72～120h.
The carbon source of fermentation and acid substratum comprises that concentration is a kind of in 10%～18% glucose, the corn saccharification liquid or both mixtures;
The nitrogenous source of fermentation and acid substratum comprises that concentration is one or more the mixture in 0.2%～2.0% urea, ammonium sulfate, ammoniacal liquor, yeast extract paste, peptone, extractum carnis, corn steep liquor, the wheat bran;
Mineral ion weight percent proportioning in the fermentation and acid substratum is an anhydrous magnesium sulfate 0.01～0.1%, ferrous sulfate 0.001～0.01%, manganous sulfate 0.001～0.01%, water surplus, pH7.0.
The used neutralizing agent of fermentation and acid comprises KOH, NaOH, Na
2CO
3, CaCO
3, one or more the combination among the CaO, ammoniacal liquor, carbon ammonia.
Beneficial effect of the present invention has been to provide a strain to stablize the bacterial strain of the high optical purity D-of high yield lactic acid; And the present invention utilizes this strain fermentation to produce the technology of D-lactic acid, keeps anaerobic condition from flat board cultivation, seed culture to fermentation and acid always, need not strict substep regulation and control, and is simple to operate; The medium component that respectively goes on foot of this zymotechnique is easy to get, and prescription is simple, and production cost is lower; The D-lactic acid optical purity of utilizing the production of bacterial strain of the present invention and zymotechnique is up to 99.1%, and the D-lactic acid content reaches 9.1%～16.5% in the fermented liquid, has important social meaning and economic worth.
Microorganism classification called after lactobacillus Sporolactobacillus sp.Y2-8 of the present invention, preservation date is on April 10th, 2008, depositary institution's full name is Chinese typical culture collection center, is called for short CCTCC, deposit number: CCTCCNo:M 208052.
Embodiment
Embodiment 1
The selection of present embodiment explanation lactobacillus Y2-8.
1, D-lactic acid original strain is produced in screening from soil:
With the high sugar liquors substratum of 100g/L glucose the sugared microorganism of anti-height in the pedotheque of area, Nanjing is carried out enrichment culture, again by the colour developing of 100g/L glucose-tetrabromo-mcresolsulfonphthalein dull and stereotyped under the anaerobism culture condition to the acid-producing bacteria strain of anti-high sugar carry out list from, select the big and fireballing bacterial strain of variable color of variable color loop diameter and carry out acidogenic fermentation research.The optical purity of product detects and adopts high performance liquid chromatography chiral stationary phase method to detect, and ligand exchange post model is Sumichiral CA5000, and moving phase is the CuSO of 1mM
4Solution, ultraviolet detection wavelength are 254nm.Obtain a strain D-lactobacillus strain, called after Sporolactobacillus inulinus DX12, the very low temperature preservation is standby.
2, the selection of bacterial strain Y2-8 is:
(1) low energy ion injects mutagenesis: get the Sporolactobacillus inulinus DX12 bacterium mud that anaerobism was cultivated 3 days, make bacteria suspension with 0.85% physiological saline, carry out ion implantation after aseptic blank culture dish is made mycoderm.Ion energy is 10keV, and implantation dosage is 0.40 * 10
15, 2.4 * 10
15, 4.4 * 10
15, 6.60 * 10
15Ions/cm
2, injection length is 5s, be 15s pitch time.
(2) pH reagent type plate screening:, bacteria suspension is coated 150g/L glucose-tetrabromo-mcresolsulfonphthalein colour developing flat board be placed on 37 ℃ of anaerobism and cultivate with physiological saline wash-out mycoderm.With the bacterium variable color circle that sets out is contrast, is the forward mutation standard with the variable color loop diameter greater than starting strain 0.5cm, is sieved to 16 strain output positive mutating strains altogether.Adopt the C18 post with the HPLC method tunning to be carried out quantitative assay, moving phase is the NH of 2.5mM
4H
2PO
4Solution, ultraviolet detection wavelength are 230nm.The optical purity of product detects and adopts high performance liquid chromatography chiral stationary phase method to detect, and ligand exchange post model is Sumichiral CA5000, and moving phase is the CuSO of 1mM
4Solution, ultraviolet detection wavelength are 254nm.18 strain output positive mutating strain D-lactic fermentation results are as shown in table 1.
Table 1 output positive mutating strain D-lactic acid quantitative assay result
(3) genetic stability test: after genetic stability is investigated in the fermentation test of going down to posterity, obtain a strain and stablize high yield D-lactobacillus strain, called after Y2-8.The bacterial strain Y2-8 fermentation test result that goes down to posterity is as shown in table 2.
The genetic stability experimental result of table 2 bacterial strain Y2-8
3, fermention medium optimization:
Kind and concentration to initial fermention medium carbon source, nitrogenous source, mineral ion and neutralizing agent is optimized successively.Bacterial strain Y2-8 has obtained 16.5% lactic fermentation result under the optimization postcondition, D-lactic acid optical purity reaches 99.1%.
Embodiment 2
The technology that the present embodiment explanation utilizes lactobacillus Y2-8 fermentation to produce D-lactic acid.
Plate culture medium: glucose 10%, yeast extract paste 0.2%, anhydrous sodium acetate 0.2%, anhydrous magnesium sulfate 0.01%, potassium primary phosphate 0.2%, agar 1.8%, water surplus, pH7.0;
Seed culture medium: glucose 2%, yeast extract paste 0.3%, corn steep liquor 0.4%, wheat bran 0.3%, anhydrous magnesium sulfate 0.02%, ferrous sulfate 0.001%, manganous sulfate 0.001%, lime carbonate 1.5%, water surplus, pH7.0;
Fermention medium: glucose 10%, corn steep liquor 1.5%, anhydrous magnesium sulfate 0.05%, ferrous sulfate 0.001%, manganous sulfate 0.001%, water surplus, pH7.0.
Lactobacillus Y2-8 is inoculated in the plate culture medium, places the anaerobism incubator to cultivate, 30 ℃ of culture temperature, incubation time 36h.The cultured lactobacillus of flat board is inoculated in the seed culture medium that liquid amount is 60mL, adds 10 granulated glass spherees, and carry out cultivating after the fluid-tight with the thick whiteruss of 2～3cm, 37 ℃ of culture temperature, the cultivation rotating speed is 120rpm, behind the cultivation 12h, as seed liquor OD
660nmThe switchable fermentation culture that enters when reaching 5 left and right sides.
Select the 5.0L fermentor tank for use, preparation 3.5L fermention medium, inoculate with 5% inoculum size, feed nitrogen, 30 ℃ of leavening temperatures with overall flow rate 0.06L/min amount, the form fermentation system pH that adds ammoniacal liquor with stream is controlled at 5.0, mixing speed 150rpm, fermentation 96h adopts liquid chromatogram measuring, the D-lactic acid content reaches 9.1% in the fermented liquid, and optical purity is 99.03%.
Embodiment 3
The technology that the present embodiment explanation utilizes lactobacillus Y2-8 fermentation to produce D-lactic acid.
Plate culture medium: glucose 12%, yeast extract paste 0.1%, anhydrous sodium acetate 0.3%, anhydrous magnesium sulfate 0.03%, potassium primary phosphate 0.3%, agar 1.8%, water surplus, pH7.0;
Seed culture medium: glucose 5%, extractum carnis 0.6%, anhydrous magnesium sulfate 0.01%, ferrous sulfate 0.003%, manganous sulfate 0.005%, lime carbonate 2%, water surplus, pH7.0.
Fermention medium: corn saccharification liquid (glucose meter) 10%, yeast extract paste 0.2%, anhydrous magnesium sulfate 0.05%, ferrous sulfate 0.001%, manganous sulfate 0.001%, water surplus, pH7.0.
Lactobacillus Y2-8 is inoculated in the plate culture medium, places the anaerobism incubator to cultivate, 37 ℃ of culture temperature, incubation time 30h.The cultured lactobacillus of flat board is inoculated in the seed culture medium that liquid amount is 60mL, carries out cultivating after the fluid-tight with the thick whiteruss of 2～3cm, 40 ℃ of culture temperature, the cultivation rotating speed is 150rpm, incubation time 18h is as seed liquor OD
660nmThe switchable fermentation culture that enters when reaching 5 left and right sides.
Select the 5.0L fermentor tank for use, configuration 3.5L fermention medium, inoculate with 7.5% inoculum size, feed nitrogen, 40 ℃ of leavening temperatures with overall flow rate 0.06L/min amount, adding NaOH solution by stream makes fermentation system pH be controlled at 7.0, mixing speed 250rpm, fermentation 72h adopts liquid chromatogram measuring, the D-lactic acid content reaches 9.3% in the fermented liquid, and optical purity is 98.8%.
Embodiment 4
The technology that the present embodiment explanation utilizes lactobacillus Y2-8 fermentation to produce D-lactic acid.
Plate culture medium: glucose 15%, yeast extract paste 0.3%, anhydrous sodium acetate 0.5%, anhydrous magnesium sulfate 0.05%, potassium primary phosphate 0.1%, agar 1.8%, water surplus, pH7.0;
Seed culture medium: glucose and corn saccharification liquid mixed solution 5%, yeast extract paste 0.3%, peptone 0.2%, anhydrous magnesium sulfate 0.05%, ferrous sulfate 0.005%, manganous sulfate 0.003%, wheat bran 0.5%, lime carbonate 3%, water surplus, pH7.0.
Fermention medium: corn saccharification liquid (glucose meter) 10%, peptone 1.0%, anhydrous magnesium sulfate 0.05%, ferrous sulfate 0.001%, manganous sulfate 0.001%, lime carbonate 5%, water surplus, pH7.0.
Lactobacillus Y2-8 is inoculated in the plate culture medium, places the anaerobism incubator to cultivate, 40 ℃ of culture temperature, incubation time 20h.The cultured lactobacillus of flat board is inoculated in the seed culture medium that liquid amount is 60mL, adds the 10mL granulated glass sphere, and carry out cultivating after the fluid-tight with the thick whiteruss of 3cm, 37 ℃ of culture temperature, the cultivation rotating speed is 120rpm, and incubation time 18h is as seed liquor OD
660nmThe switchable fermentation culture that enters when reaching 5 left and right sides.
Select the 5.0L fermentor tank for use, configuration 3.5L fermention medium, inoculate with 10% inoculum size, feed nitrogen, 37 ℃ of leavening temperatures with overall flow rate 0.06L/min amount, pH is controlled at 5.0, mixing speed 200rpm, fermentation 120h adopts liquid chromatogram measuring, the D-lactic acid content reaches 9.3% in the fermented liquid, and optical purity is 99%.
Embodiment 5
The technology that the present embodiment explanation utilizes lactobacillus Y2-8 fermentation to produce D-lactic acid.
Plate culture medium: with embodiment 2.
Seed culture medium: glucose 3%, yeast extract paste 0.2%, anhydrous magnesium sulfate 0.02%, ferrous sulfate 0.001%, manganous sulfate 0.001%, lime carbonate 1.5%, water surplus, pH7.0.
Fermention medium: corn saccharification liquid adds part glucose to glucose concn and reaches 15%, extractum carnis 1.2%, anhydrous magnesium sulfate 0.05%, ferrous sulfate 0.001%, manganous sulfate 0.001%, calcium oxide 6%, water surplus, pH7.0.
Lactobacillus Y2-8 is inoculated in the plate culture medium, places the anaerobism incubator to cultivate, 30 ℃ of culture temperature, incubation time 36h.The cultured lactobacillus of flat board is inoculated in the seed culture medium that liquid amount is 120mL, adds 15 granulated glass spherees, 37 ℃ of culture temperature, the cultivation rotating speed is 100rpm, incubation time 18 h are as seed liquor OD
660nmThe switchable fermentation culture that enters when reaching 5 left and right sides.
Select the 5.0L fermentor tank for use, configuration 3.5L fermention medium, inoculate with 5% inoculum size, feed nitrogen, 37 ℃ of leavening temperatures with overall flow rate 0.06L/min amount, adding NaOH by stream makes fermentation system pH be controlled at 6.0, mixing speed 300rpm, fermentation 120h adopts liquid chromatogram measuring, the D-lactic acid content reaches 13.6% in the fermented liquid, and optical purity is 99%.
Embodiment 6
The technology that the present embodiment explanation utilizes lactobacillus Y2-8 fermentation to produce D-lactic acid.
Plate culture medium: with embodiment 3.
Seed culture medium: corn saccharification liquid 3%, corn steep liquor 1.0%, anhydrous magnesium sulfate 0.02%, ferrous sulfate 0.001%, manganous sulfate 0.001%, lime carbonate 1.5%, water surplus, pH7.0.
Fermention medium: glucose 15%, yeast extract paste 1.0%, wheat bran 0.4%, anhydrous magnesium sulfate 0.05%, ferrous sulfate 0.001%, manganous sulfate 0.001%, lime carbonate 4%, water surplus, pH7.0.
Lactobacillus Y2-8 is inoculated in the plate culture medium, places the anaerobism incubator to cultivate, 37 ℃ of culture temperature, incubation time 30h.The cultured lactobacillus of flat board is inoculated in the seed culture medium that liquid amount is 60mL, adds 10 granulated glass spherees, and carry out cultivating after the fluid-tight with the thick whiteruss of 2cm, 37 ℃ of culture temperature, the cultivation rotating speed is 110rpm, and incubation time 13h is as seed liquor OD
660nmThe switchable fermentation culture that enters when reaching 5 left and right sides.
Select the 5.0L fermentor tank for use, configuration 3.5L fermention medium, inoculate with 10% inoculum size, feed nitrogen, 35 ℃ of leavening temperatures with overall flow rate 1L/min amount, intermittent stream adds sodium carbonate solution to be kept fermentation system pH and is controlled at 6.0, mixing speed 200rpm, fermentation 120h adopts liquid chromatogram measuring, the D-lactic acid content reaches 14.5% in the fermented liquid, and optical purity is 99.1%.
Embodiment 7
The technology that the present embodiment explanation utilizes lactobacillus Y2-8 fermentation to produce D-lactic acid.
Plate culture medium: with embodiment 4.
Seed culture medium: corn saccharification liquid 5%, wheat bran 1.0%, anhydrous magnesium sulfate 0.02%, ferrous sulfate 0.001%, manganous sulfate 0.001%, lime carbonate 1.5%, water surplus, pH7.0.
Fermention medium: corn saccharification liquid (glucose meter) 15%, yeast extract paste 0.5%, peptone 0.5%, ammonium sulfate 0.4%, anhydrous magnesium sulfate 0.05%, ferrous sulfate 0.001%, manganous sulfate 0.001%, lime carbonate 4%, water surplus, pH7.0.
Lactobacillus Y2-8 is inoculated in the plate culture medium, places the anaerobism incubator to cultivate, 40 ℃ of culture temperature, incubation time 20h.The cultured lactobacillus of flat board is inoculated in the seed culture medium that liquid amount is 60mL, carries out cultivating after the fluid-tight with the thick whiteruss of 2cm, 37 ℃ of culture temperature, the cultivation rotating speed is 110rpm, incubation time 18h is as seed liquor OD
660nmThe switchable fermentation culture that enters when reaching 5 left and right sides.
Select the 5.0L fermentor tank for use, configuration 3.5L fermention medium, inoculate with 10% inoculum size, feed nitrogen, 42 ℃ of leavening temperatures with overall flow rate 0.06L/min amount, stream adds carbon ammonia makes fermentation system pH be controlled at 5.0, mixing speed 200rpm, fermentation 96h adopts liquid chromatogram measuring, the D-lactic acid content reaches 14.2% in the fermented liquid, and optical purity is 99.1%.
Embodiment 8
The technology that the present embodiment explanation utilizes lactobacillus Y2-8 fermentation to produce D-lactic acid.
Plate culture medium: with embodiment 2.
Seed culture medium: corn saccharification liquid 2%, peptone 0.6%, anhydrous magnesium sulfate 0.02%, ferrous sulfate 0.001%, manganous sulfate 0.001%, lime carbonate 1.5%, water surplus, pH7.0.
Fermention medium: glucose 18%, yeast extract paste 0.4%, extractum carnis 0.4%, corn steep liquor 0.4%, urea 0.4%, anhydrous magnesium sulfate 0.05%, ferrous sulfate 0.001%, manganous sulfate 0.001%, water surplus, pH7.0.
Lactobacillus Y2-8 is inoculated in the plate culture medium, places the anaerobism incubator to cultivate, 30 ℃ of culture temperature, incubation time 36h.The cultured lactobacillus of flat board is inoculated in the seed culture medium that liquid amount is 60mL, carries out cultivating after the fluid-tight with the thick whiteruss of 2cm, 37 ℃ of culture temperature, the cultivation rotating speed is 120rpm, incubation time 18h is as seed liquor OD
660nmThe switchable fermentation culture that enters when reaching 5 left and right sides.
Select the 5.0L fermentor tank for use, configuration 3.5L fermention medium, inoculate with 10% inoculum size, feed nitrogen, 35 ℃ of leavening temperatures with overall flow rate 0.2L/min amount, stream adds carbon ammonia makes fermentation system pH be controlled at 6.0, mixing speed 300rpm, fermentation 96h adopts liquid chromatogram measuring, the D-lactic acid content reaches 15.9% in the fermented liquid, and optical purity is 99.0%.
Embodiment 9
The technology that the present embodiment explanation utilizes lactobacillus Y2-8 fermentation to produce D-lactic acid.
Plate culture medium: with embodiment 3.
Seed culture medium: glucose and corn saccharification liquid mixed solution 5%, yeast extract paste 0.3%, peptone 0.2%, anhydrous magnesium sulfate 0.02%, ferrous sulfate 0.001%, manganous sulfate 0.001%, wheat bran 0.5%, lime carbonate 1.5%, water surplus, pH7.0.
Fermention medium: corn saccharification liquid (glucose meter) 18%, yeast extract paste 0.5%, peptone 0.4%, wheat bran 0.7%, ammoniacal liquor 0.4%, anhydrous magnesium sulfate 0.05%, ferrous sulfate 0.001%, manganous sulfate 0.001%, lime carbonate 5%, water surplus, pH7.0.
Lactobacillus Y2-8 is inoculated in the plate culture medium, places the anaerobism incubator, 37 ℃ of culture temperature, incubation time 30h.The cultured lactobacillus of flat board is inoculated in the seed culture medium that liquid amount is 60mL, adds 10 granulated glass spherees, and carry out cultivating after the fluid-tight with the thick whiteruss of 3cm, 37 ℃ of culture temperature, the cultivation rotating speed is 120rpm, and incubation time 18h is as seed liquor OD
660nmThe switchable fermentation culture that enters when reaching 5 left and right sides.
Select the 5.0L fermentor tank for use, configuration 3.5L fermention medium, inoculate with 10% inoculum size, feed nitrogen, 37 ℃ of leavening temperatures with overall flow rate 0.2L/min amount, stream adds yellow soda ash makes fermentation system pH be controlled at 5.0, mixing speed 200rpm, fermentation 120h adopts liquid chromatogram measuring, the D-lactic acid content reaches 15.8% in the fermented liquid, and optical purity is 99%.
Embodiment 10
The technology that the present embodiment explanation utilizes lactobacillus Y2-8 fermentation to produce D-lactic acid.
Plate culture medium: with embodiment 4.
Seed culture medium: glucose and corn saccharification liquid mixed solution 5%, yeast extract paste 0.3%, peptone 0.2%, anhydrous magnesium sulfate 0.04%, ferrous sulfate 0.001%, manganous sulfate 0.001%, wheat bran 0.5%, lime carbonate 1.5%, water surplus, pH7.0.
Fermention medium: glucose and corn saccharification liquid mixed solution (glucose meter) 18%, yeast extract paste 0.6%, peptone 0.4%, extractum carnis 0.4%, anhydrous magnesium sulfate 0.05%, ferrous sulfate 0.001%, manganous sulfate 0.001%, lime carbonate 5%, water surplus, pH7.0.
Lactobacillus Y2-8 is inoculated in the plate culture medium, places under the anaerobic condition, 40 ℃ of culture temperature, incubation time 20h.The cultured lactobacillus of flat board is inoculated in the seed culture medium that liquid amount is 60mL, adds the 10mL granulated glass sphere, and carry out cultivating after the fluid-tight with the thick whiteruss of 3cm, 37 ℃ of culture temperature, the cultivation rotating speed is 120rpm, and incubation time 18h is as seed liquor OD
660nmThe switchable fermentation culture that enters when reaching 5 left and right sides.
Select the 5.0L fermentor tank for use, configuration 3.5L fermention medium, inoculate with 10% inoculum size, feed nitrogen, 37 ℃ of leavening temperatures with overall flow rate 0.2L/min amount, stream adds ammoniacal liquor makes fermentation system pH be controlled at 6.0, mixing speed 200rpm, fermentation 120h adopts liquid chromatogram measuring, the D-lactic acid content reaches 16.3% in the fermented liquid, and optical purity is 99.1%.
Claims (9)
1, a plant height optical purity D-lactic-acid-producing strain, classification called after lactobacillus Sporolactobacillussp.Y2-8, its preservation registration number is CCTCC No:M 208052.
2, utilize the technology of the described lactobacillus Sporolactobacillus of claim 1 sp.Y2-8 fermentation production of D-lactic acid, comprise dull and stereotyped cultivation, seed culture and fermentation and acid, it is characterized in that: after under anaerobic carrying out flat board cultivation and seed culture, carry out fermentation and acid by the mode that in fermentor tank, feeds rare gas element, the omnidistance anaerobism of technological process is carried out.
3, according to the technology of the described lactobacillus Sporolactobacillus of claim 2 sp.Y2-8 fermentation production of D-lactic acid, it is characterized in that:
(1) the dull and stereotyped cultivation: lactobacillus Y2-8 is seeded to the plate culture medium anaerobism cultivates 30～40 ℃ of culture temperature, incubation time 20～36h;
(2) seed culture: the lactobacillus Y2-8 that flat board is cultivated is inoculated into and carries out anaerobism in the seed culture medium and cultivate 30～40 ℃ of culture temperature, incubation time 12～18h;
(3) fermentation and acid: seed culture fluid is inoculated into carries out acidogenic fermentation in the fermention medium, be carbon source directly with starch based saccharification liquid, inoculum size is 5%～10% (v/v), 30～40 ℃ of leavening temperatures, feed rare gas element with every liter of substratum 0.6～2L/min amount of overall flow rate, adopt neutralizing agent that fermentation system pH is controlled at 5.0～7.0, fermentation 72～120h.
4, according to the technology of claim 2 and 3 described lactobacillus Sporolactobacillus sp.Y2-8 fermentation production of D-lactic acid, it is characterized in that described plate culture medium weight percent proportioning is a glucose 10～15%, yeast extract paste 0.1～0.3%, anhydrous sodium acetate 0.2～0.5%, anhydrous magnesium sulfate 0.01～0.05%, potassium primary phosphate 0.1～0.3%, agar 1.8%, water surplus, pH7.0.
5, according to the technology of claim 2 and 3 described lactobacillus Sporolactobacillus sp.Y2-8 fermentation production of D-lactic acid, the carbon source that it is characterized in that described seed culture medium comprises that concentration is a kind of in 2%～5% glucose, the corn saccharification liquid or both mixtures; Nitrogenous source and somatomedin comprise that concentration is one or more the mixture in 0.2%～1.0% yeast extract paste, peptone, extractum carnis, corn steep liquor, the wheat bran; Mineral ion weight percent proportioning is an anhydrous magnesium sulfate 0.01～0.05%, ferrous sulfate 0.001～0.005%, manganous sulfate 0.001～0.005%, lime carbonate 1.5～3%, water surplus, pH7.0.
6, according to the technology of claim 2 and 3 described lactobacillus Sporolactobacillus sp.Y2-8 fermentation production of D-lactic acid, the carbon source that it is characterized in that described fermentation and acid substratum comprises that concentration is a kind of in 10%～18% glucose, the corn saccharification liquid or both mixtures; Nitrogenous source and somatomedin comprise that concentration is one or more the mixture in 0.2%～2.0% urea, ammonium sulfate, ammoniacal liquor, yeast extract paste, peptone, extractum carnis, corn steep liquor, the wheat bran; Mineral ion weight percent proportioning is an anhydrous magnesium sulfate 0.01～0.1%, ferrous sulfate 0.001～0.01%, manganous sulfate 0.001～0.01%, water surplus, pH7.0.
7, according to the technology of claim 2 and 3 described lactobacillus Sporolactobacillus sp.Y2-8 fermentation production of D-lactic acid, it is characterized in that the described seed culture stage with the fluid-tight of 2～3cm whiteruss, or reduce rotating speed when increasing liquid amount and realize that anaerobism cultivates.
8,, it is characterized in that described seed culture can add granulated glass sphere in the stage in shaking bottle, to strengthen mass transfer effect according to the technology of claim 2 and 3 described lactobacillus Sporolactobacillus sp.Y2-8 fermentation production of D-lactic acid.
9,, it is characterized in that described used neutralizing agent of fermentation and acid stage comprises NaOH, KOH, Na according to the technology of claim 2 and 3 described lactobacillus Sporolactobacillus sp.Y2-8 fermentation production of D-lactic acid
2CO
3, CaCO
3, one or more combination such as CaO, ammoniacal liquor, carbon ammonia.
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| CN102329824A (en) * | 2011-09-28 | 2012-01-25 | 安徽丰原发酵技术工程研究有限公司 | Method for producing D-lactic acid by using fiber hydrolysate |
| CN103114053A (en) * | 2012-12-28 | 2013-05-22 | 张健鹏 | Lactobacilluscoryniformis subsp.shaanxiensis capable of producing high-optical-purity D-lactic acid and application thereof |
| US8492127B2 (en) | 2010-05-20 | 2013-07-23 | Shanghai Jiao Tong University | Bacillus coagulans strains and their applications in L-lactic acid production |
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| CN114645006A (en) * | 2020-12-21 | 2022-06-21 | 中国科学院微生物研究所 | High-sugar-resistant lactic acid producing strain and application thereof in D-lactic acid production |
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| CN103173501A (en) * | 2013-03-20 | 2013-06-26 | 中国科学院微生物研究所 | Method for producing D-lactic acid by using sodium hydroxide as neutralizer |
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| US8492127B2 (en) | 2010-05-20 | 2013-07-23 | Shanghai Jiao Tong University | Bacillus coagulans strains and their applications in L-lactic acid production |
| CN102329824A (en) * | 2011-09-28 | 2012-01-25 | 安徽丰原发酵技术工程研究有限公司 | Method for producing D-lactic acid by using fiber hydrolysate |
| CN103589654A (en) * | 2012-08-14 | 2014-02-19 | 湖南普菲克生物科技有限公司 | Bacillus coagulans strain and fast identification of anaerobic metabolism and lactic acid production of the strain in animal digestive tract hindgut segment simulator |
| CN103114053A (en) * | 2012-12-28 | 2013-05-22 | 张健鹏 | Lactobacilluscoryniformis subsp.shaanxiensis capable of producing high-optical-purity D-lactic acid and application thereof |
| CN104419657A (en) * | 2013-09-11 | 2015-03-18 | 中国石油化工股份有限公司 | D-lactic acid producing strain with high growth rate and acid producing velocity and application thereof |
| WO2015106627A1 (en) * | 2014-01-17 | 2015-07-23 | 上海交通大学 | Sporolactobacillus terrae and uses thereof |
| CN105315442A (en) * | 2015-12-01 | 2016-02-10 | 仇颖超 | Preparation method of starch modified polycaprolactone plastic material |
| CN109906268A (en) * | 2016-11-01 | 2019-06-18 | Ptt全球化学股份有限公司 | For producing the fermentation process of D-ALPHA-Hydroxypropionic acid or its salt |
| CN106589327A (en) * | 2016-12-11 | 2017-04-26 | 闫博文 | Polylactic acid and preparation method thereof |
| CN106754551A (en) * | 2017-01-20 | 2017-05-31 | 国家海洋局第三海洋研究所 | A kind of bacterium amount lactobacillus preparation high and preparation method and application |
| CN111378698A (en) * | 2020-06-01 | 2020-07-07 | 中粮营养健康研究院有限公司 | Method for preparing D-lactic acid by fermentation |
| CN114645006A (en) * | 2020-12-21 | 2022-06-21 | 中国科学院微生物研究所 | High-sugar-resistant lactic acid producing strain and application thereof in D-lactic acid production |
| CN114645006B (en) * | 2020-12-21 | 2023-09-01 | 中国科学院微生物研究所 | High sugar-tolerant lactic acid production strain and application thereof in D-lactic acid production |
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