CN100485027C - Method for producing D-lactic acid and spore lactobacillus special for the same - Google Patents
Method for producing D-lactic acid and spore lactobacillus special for the same Download PDFInfo
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Abstract
The invention relates to a method for producing D-lactic acid and the special bacterial strain, in particular to a method for producing D-lactic acid and the special Sporolactobacillus. The Sporolactobacillus of the invention is Sporolactobacillus inulinus CASD and the preserved number is CGMCC NO2185. The method for producing D-lactic acid utilizes Sporolactobacillus inulinus CASD CGMCC NO2185 to produce D-lactic acid by fermenting hemicontinuously. The concentration of the D-lactic acid obtained by the invention can reach 162 g/L, the percent conversion can reach 94.7 to 96.0 percent and the optical purity can reach above 98.0 percent, further, the optical purity can still keep 98 percent after circular fermentation repeatedly and the pollution of impurity can be avoided. The invention has the advantages of enabling to be used for producing D-lactic acid, saving the cost, improving productivity efficiency, therefore being suitable for application in manufacturing production.
Description
Technical field
The present invention relates to a kind of method and special strain therefore thereof of the D-of production lactic acid, particularly relate to a kind of method and special-purpose lactobacillus thereof of the D-of production lactic acid.
Background technology
Lactic acid (Lactic Acid) is a kind of organic acid simple in structure, extensively is present in nature as metabolism intermediate and meta-bolites.The poly(lactic acid) that is generated by lactic acid polymerizes has excellent biological compatibility and biodegradability, can be used for producing operating sutures, biology is planted sheet, Biodegradable fibers, wrapping material and agricultural film etc.Having a chiral centre in the lactic acid molecules, have opticity, is a kind of chiral molecules.According to the difference of opticity, lactic acid can be divided into L-lactic acid, D-lactic acid and racemic lactic acid.Wherein, high purity D-lactic acid is mainly used in aspects such as biodegradable plastic, pesticide herbicide and medicine intermediate.Germany HOECHST company is " prestige despot's weedicide " of raw material production with optical purity D-lactic acid, can prevent and kill off the gramineous weeds in the farmland, broad weed-killing spectrum, use flexibly, can be mixed with multiple other weedicide, also can use with, can on beet, rape, flax, Soybean and Other Crops, use with sterilant (as Decamethrin etc.).The weedicide S-2-chloropropionic acid that Germany BASF AG produces is to be raw material with the D-isopropyl lactate.In addition, to reduce medicine, picolinic acid derivative, dimethyl tetrachloro propionic acid and fluorine be that weedicide etc. all needs high purity D-lactic acid as raw material to calcium antagonist.
The method of producing lactic acid mainly contains three kinds of chemical synthesis, enzyme catalysis method and fermentation methods.Wherein, the direct synthetic lactic acid of chemical method is DL-lactic acid, if will obtain D-lactic acid or L-lactic acid then need carry out optical resolution, and synthetic used raw material is acetaldehyde and violent in toxicity prussic acid, can not be widely accepted so the lactic acid that synthesizes is applied to food, its production cost is also higher in addition.Enzyme catalysis method can obtain to specificity optical purity lactic acid, but the research difficulty of its reaction process is bigger, is not applied to suitability for industrialized production as yet.Production by Microorganism Fermentation lactic acid, can decompose resulting glucose etc. with renewable resourcess such as starch, Mierocrystalline celluloses is raw material, selection fermenting lactic acid by bacterial classification and culture condition, can obtain D-lactic acid, L-lactic acid or both a certain proportion of mixtures, its production cost is low, the Product Safety height is the main method of scale operation lactic acid.
Ding Zijian etc. adopt lactobacillus (Sporolactobacillus sp) to prepare D-lactic acid from glucose fermentation at report in 2004, can produce the D-lactic acid of 40.7g/L.Chinese patent CN 200610097453.6 discloses a kind of technology of combined fermentation production of D-lactic acid, produces acid 7.5%～13.1%.
In the method for Production by Microorganism Fermentation D-lactic acid, the output of D-lactic acid is still waiting further raising at present.Produce in the process of D-lactic acid at microbial fermentation, compare with batch fermentation, semicontinuous fermentation can shorten fermentation period, improve plant factor, raise labour productivity, reduce labour intensity, simplify the operation course, help controlling the cost of fermentative Production D-lactic acid.But semi-continuous type is produced D-lactic acid and is existed degradation problem under bacterial strain degeneration, pollution microbes, the D-lactic acid optical purity.
Summary of the invention
An object of the present invention is to provide a strain and can produce the lactobacillus of D-lactic acid.
Lactobacillus provided by the present invention is synanthrin lactobacillus (Sporolactobacillus inulinus) CASD, be from the soil in orchard, Pinggu, Beijing, to separate to obtain, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 24th, 2007 and (be called for short CGMCC, the address is: the Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), its deposit number is CGMCC № 2185.It has following biological property:
1) morphological specificity: cell straight-bar.Single or paired.Form statospore.A small amount of peritrichous, motion.On the agar plate that contains glucose, yeast powder and peptone, form tiny tip-like bacterium colony.
2) physiological and biochemical property: Gram-positive.Little aerobic.Do not contain the protoheme compound.Do not form indoles.Liquefy gelatin not.Do not reduce nitrate.Litmus milk is constant.Can utilize fructose, glucose, synanthrin, raffinose, maltose, seminose, trehalose, sucrose, N.F,USP MANNITOL to produce acid, can not utilize wood sugar, semi-lactosi, pectinose to produce acid.Do not grow 40～42 ℃ of optimum growth temperatures below 10 ℃ He more than 50 ℃.
Another object of the present invention provides a kind of method of the D-of production lactic acid.
The method of production D-lactic acid provided by the present invention is that fermentation synanthrin lactobacillus (Sporolactobacillusinulinus) CASD CGMCC № 2185 obtains D-lactic acid.The mode of fermentation culture specifically can be semicontinuous fermentation.
Wherein, contain in every liter of the fermention medium: glucose 150～180 grams, corn steep liquor 10～40 grams, soybean meal hydrolysate 100～200 grams are used to regulate and control the neutralizing agent of medium pH, and surplus is a water; The pH of described fermention medium is 5.5～7.
The described neutralizing agent that is used to regulate and control medium pH is specially lime carbonate, contains lime carbonate 75～90 grams in every liter of described fermention medium.
Described soybean meal hydrolysate can obtain from commercial channels, also can oneself make.Make according to following method among the present invention: with the sulphuric acid soln of bean cake powder (Beijing Kang Mingwei substratum technology limited liability company) and 2% (volumn concentration), with the 1g bean cake powder: the ratio of 5ml sulphuric acid soln is carried out abundant mixing, 90 ℃ of water-baths 15 hours obtain soybean meal hydrolysate.
Fermentation condition is 35 ℃～45 ℃ to be cultivated 48～75 hours, cultivated 48～65 hours as 37～42 ℃.
Also can stir in the fermentation culture process, the rotating speed of stirring is 30～100 rev/mins, and rotation radius is 33 or 42mm, and churning time is 20～40 minutes; Wherein, mixing speed is preferably 50 rev/mins, and churning time is preferably 30 minutes.
In the inventive method, before fermentation, earlier synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 is inserted in the seed culture medium, cultivated 10～24 hours, and then insert described fermention medium for 37～42 ℃.
Wherein, the aqueous solution that seed culture medium is made up of following material: glucose 40 grams per liters, yeast extract 5 grams per liters, peptone 10 grams per liters, extractum carnis 10 grams per liters, dipotassium hydrogen phosphate 2 grams per liters, dibasic ammonium citrate 2 grams per liters, sodium acetate 5 grams per liters, 1 milliliter/liter of tween 80, bitter salt 0.58 grams per liter, four anhydrous manganeses, 0.25 grams per liter, lime carbonate 15 grams per liters; The pH of described seed culture medium is 6～7.
The ability that synanthrin lactobacillus provided by the present invention (Sporolactobacillus inulinus) CASD CGMCC № 2185 produces D-lactic acid is strong, with glucose is that fermenting substrate is produced D-lactic acid, D-lactic acid production peak 162 grams per liters, transformation efficiency 94.7～96.0%, D-lactic acid optical purity is more than 98.0%, and do not degenerate through bacterial strain behind the circulating fermentation repeatedly, still can keep higher vigor; Utilize its fermentation production of D-lactic acid, can carry out repeatedly circulating fermentation, D-concentration of lactic acid height, transformation efficiency height that fermentation obtains, and also the purity of D-lactic acid also can keep original higher level, can also avoid the pollution of assorted bacterium.Therefore, utilize the inventive method to produce D-lactic acid, can save cost, enhance productivity, be suitable for applying in the industrial production.
Description of drawings
Fig. 1 is a high-efficient liquid phase chromatogram.
A is L-lactic acid standard substance high-efficient liquid phase chromatograms
B is D-lactic acid standard substance high-efficient liquid phase chromatograms
C is the high-efficient liquid phase chromatogram of the fermented liquid of synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 fermentation production of D-lactic acid
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
The step that relates in synanthrin lactobacillus (Sporolactobacillus inulinus) the CASD CGMCC № 2185 fermentation production of D-lactic acid methods of utilizing of the present invention is as follows:
(1) slant culture: synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 bacterial classification inoculations on the solid slant culture base that contains 1.5g/100ml agar, under 37～42 ℃ of conditions, were cultivated 24～48 hours.
(2) seed culture: with the slant culture of step (1), be inoculated under aseptic condition in the 30ml seed culture medium, under 37～42 ℃ of conditions, static cultivation 10～24 hours adds neutralizing agent control fermented liquid pH, makes seed culture fluid.
(3) fermentation culture: with the inoculum size of 10% volume ratio, seed culture fluid is inoculated in the fermention medium, cultivated 48～75 hours under 35～45 ℃ of conditions, wherein, manually vibration is adopted in fermentation in the triangular flask, every vibration in 12 hours once, fermentor tank adopts intermittently stir culture, stirred once every 12 hours, 30～100 rev/mins of stirring velocitys, rotation radius are 33 or 42mm, churning time 20～40 minutes, add neutralizing agent control fermented liquid pH, make the fermentation culture (circulating fermentation for the first time) that contains D-lactic acid; Then will be wherein 60～90% fermented liquid replace with fresh fermention medium with volume, cultivated 48～75 hours under 35～45 ℃ of conditions, wherein mode of oscillation is with consistent described in the circulating fermentation (circulating fermentation for the second time) for the first time; Again will be wherein 60～90% fermented liquid replace with fresh fermention medium with volume, cultivated 48～75 hours under 35～45 ℃ of conditions, wherein mode of oscillation is with the unanimity (circulating fermentation for the third time) described in the fermentation circulation for the first time; Circulating fermentation like this.
(4) handle sample: get fermented liquid centrifugal 5 minutes, get supernatant liquor with 6,000 rev/mins speed.
(5) sample detection: get the supernatant liquor dilution, detect glucose content, D-lactic acid content, L-lactic acid content.
Wherein, the yeast culture temperature described in the step (1) specifically can be 40～42 ℃.
Wherein, the yeast culture temperature described in the step (2) specifically can be 40～42 ℃.
Wherein, the yeast culture temperature described in the step (3) specifically can be 37～42 ℃.
Wherein, the yeast culture time described in the step (1) specifically can be 24～36 hours.
Wherein, the yeast culture time described in the step (2) specifically can be 18～20 hours.
Wherein, the yeast culture time described in the step (3) specifically can be 48～65 hours.
Wherein, the neutralizing agent that adds in step (2), (3) described culturing process is a lime carbonate, and control pH is 5.5～7.
Wherein, the determination of glucose method is that fermented liquid centrifugal back dilution adopts bio-sensing analyser SBA-40C (Shandong Province academy sciences Biology Research Institute) to measure.
Bio-sensing analyser SBA-40C is to be the analytical instrument of transmitter with the immobilized enzyme, and glucose and oxygen, water generate gluconic acid and hydrogen peroxide under the catalysis of glucose oxidase.The hydrogen peroxide that reaction is emitted contacts with platinum-silver electrode, and produces current signal, and this current signal and glucose concn are linearly proportional, can draw glucose concn by measuring current signal strength.
L-lactic acid and D-lactic acid content adopt Agilent 1100 liquid chromatographs (Anjelen Sci. ﹠ Tech. Inc) to measure, chiral separation post (Mitsubishi chemical company, MCI GEL-CRS10 W (3 μ) 4.6 ID * 50mm, the optics allosome separates to be used), 0.002mol/L copper sulfate is moving phase, flow 0.5ml/min, sample size 20 μ L, UV-detector detects wavelength 254nm, 25 ℃ of service temperatures.D-lactic acid standard substance are Sigma-Aldrich company product, and article No. is L0625, and L-lactic acid standard substance are Sigma-Aldrich company product, and article No. is L1750.With initial fermention medium in contrast.
Under above chromatographic condition, the high performance liquid chromatography detected result of standard substance is seen accompanying drawing 1.Wherein, A is L-lactic acid standard substance high-efficient liquid phase chromatograms, and B is D-lactic acid standard substance high-efficient liquid phase chromatograms.Under this chromatographic condition, the retention time of L-lactic acid standard substance is 11.566 minutes, and the retention time of D-lactic acid standard substance is 9.724 minutes.
D-lactic acid optical purity method of calculation: D-lactic acid optical purity (%)=[D-lactic acid concn (grams per liter)-L-lactic acid concn (grams per liter)] ÷ [D-lactic acid concn (grams per liter)+L-lactic acid concn (grams per liter)] * 100%
Transformation efficiency method of calculation: transformation efficiency (%)=D-lactic acid concn (grams per liter) ÷ glucose consumption amount (grams per liter) * 100%
Further set forth the present invention below by embodiment.
Separation, mutagenesis screening and the evaluation of embodiment 1, synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185
One, separation, the mutagenesis screening of bacterial strain synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185
Strains separation screening: gather Pinggu, Beijing orchard soil, take by weighing 5 grams and be dissolved in 100ml physiological saline, fully coat on the MRS solid medium 37 ℃ of cultivations for 100,000 times with the sterilized water dilution behind the mixing; After bacterium colony grows, the single bacterium colony of part is owing to produce acid with the dissolution of calcium carbonate in the periphery of bacterial colonies substratum, produce transparent circle, the tangible single bacterium colony of picking transparent circle, be inoculated in the 10ml fermention medium, 37 ℃ of static cultivations according to the detection method described in the above-mentioned embodiment, detected D-lactic acid content in the fermented liquid after 72 hours.Through repeatedly screening, the highest bacterial strain of picking one strain D-lactic acid production.
Ion beam mutagenesis screening: the inoculation that the D-lactic acid production of above-mentioned acquisition is the highest in the MRS liquid nutrient medium, 37 ℃ of static mid-log phases that are cultured to, centrifugal, thalline is suspended with physiological saline washing back, make bacteria suspension.Draw 100 μ L bacteria suspensions and coat on the aseptic blank culture dish that diameter is 3.5cm, coating diameter about 1.5cm, carry out behind the drying one-tenth mycoderm of aseptic wind ion implantation, ion energy 30keV, implantation dosage is respectively 1 * 10
14Ions/cm
2, 5 * 10
14Ions/cm
2, 10 * 10
14Ions/cm
2, 50 * 10
14Ions/cm
2, 100 * 10
14Ions/cm
2Plate after ion implantation 1mL physiological saline wash-out.Variant implantation dosage is drawn 100 μ L respectively, is coated on after the dilution on the MRS solid medium, wait to grow single bacterium colony after, select the bigger bacterium colony of transparent circle, be inoculated in the 10ml fermention medium 37 ℃ of static cultivations 48 hours; Then will be wherein 90% fermented liquid replace with fresh fermention medium, 37 ℃ of static cultivations 48 hours with volume; Again will be wherein 90% fermented liquid replace with fresh fermention medium, 37 ℃ of static cultivations 48 hours with volume; So circulating fermentation is 10 times.According to detection and the method for calculation described in the above-mentioned embodiment, detect the 1st time and the 10th circulating fermentation fermented liquid in glucose concn, D-lactic acid concn and L-lactic acid concn, calculate D-lactic acid optical purity and inversion rate of glucose.Found that through 10 circulating fermentations, the acid producing ability of most single bacterium colonies and D-lactic acid optical purity all have decline in various degree, have only the single bacterium colony of minority repeatedly keeping original acid producing ability and D-lactic acid optical purity substantially behind the circulating fermentation.Through repeatedly screening, it is active to obtain a plant height, that the D-lactic acid production is significantly improved before than ion beam mutagenesis, through keeping D-lactic acid production height, D-lactic acid optical purity more than 98%, the bacterial strain of transformation efficiency more than 95% behind the circulating fermentation repeatedly.
With the cultivation of repeatedly going down to posterity of this bacterial strain, and then carry out 10 round-robin semicontinuous fermentations and test, D-lactic acid production, D-lactic acid optical purity and transformation efficiency that its 10th circulating fermentation produces keep previous level substantially, prove that this bacterial strain promptly is the purpose bacterial strain, and name is called CASD.
Wherein, consisting of of MRS solid medium: glucose 20 grams per liters, yeast extract 5 grams per liters, peptone 10 grams per liters, extractum carnis 10 grams per liters, dipotassium hydrogen phosphate 2 grams per liters, dibasic ammonium citrate 2 grams per liters, sodium acetate 5 grams per liters, 1 milliliter/liter of tween 80, bitter salt 0.58 grams per liter, four anhydrous manganeses, 0.25 grams per liter, lime carbonate 15 grams per liters, agar powder 15 grams per liters, solvent are water; The pH of described MRS solid medium is 6.5.Sterilized 20 minutes for 115 ℃.
Consisting of of MRS liquid nutrient medium: glucose 20 grams per liters, yeast extract 5 grams per liters, peptone 10 grams per liters, extractum carnis 10 grams per liters, dipotassium hydrogen phosphate 2 grams per liters, dibasic ammonium citrate 2 grams per liters, sodium acetate 5 grams per liters, 1 milliliter/liter of tween 80, bitter salt 0.58 grams per liter, four anhydrous manganeses, 0.25 grams per liter, solvent is a water; The pH of described MRS liquid nutrient medium is 6.5.Sterilized 20 minutes for 115 ℃.
Consisting of of fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 150 grams per liters, soybean meal hydrolysate 100 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 10 grams per liters, lime carbonate 75 grams per liters, solvent are water; The pH of described fermention medium is 6.5.Sterilized 20 minutes for 115 ℃.
Wherein, soybean meal hydrolysate preparation method: with the sulphuric acid soln of bean cake powder (Beijing Kang Mingwei substratum technology limited liability company) and 2% (volumn concentration), with the 1g bean cake powder: the ratio of 5ml sulphuric acid soln is carried out abundant mixing, and 90 ℃ of water-baths 15 hours obtain soybean meal hydrolysate.
Two, the evaluation of bacterial strain synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185
According to " common bacteria system identification handbook " (eastern elegant pearl, Cai Miaoying etc. write, Beijing: Science Press, 2001.2) and method of describing in " uncle outstanding Bacteria Identification handbook " (the 8th edition), this bacterial strain is carried out morphological specificity is observed and the physio-biochemical characteristics evaluation.
Qualification result is as follows:
1) morphological specificity: cell straight-bar.Single or paired.Form statospore.A small amount of peritrichous, motion.On the agar plate that contains glucose, yeast powder and peptone, form tiny tip-like bacterium colony.
2) physiological and biochemical property: Gram-positive.Little aerobic.Do not contain the protoheme compound.Do not form indoles.Liquefy gelatin not.Do not reduce nitrate.Litmus milk is constant.Can utilize fructose, glucose, synanthrin, raffinose, maltose, seminose, trehalose, sucrose, N.F,USP MANNITOL to produce acid, can not utilize wood sugar, semi-lactosi, pectinose to produce acid.Do not grow 40～42 ℃ of optimum growth temperatures below 10 ℃ He more than 50 ℃.
Based on above feature, this strain D-lactic-acid-producing strain is accredited as synanthrin lactobacillus (Sporolactobacillusinulinus) CASD, and with its preservation, preserving number is CGMCC № 2185.
Synanthrin lactobacillus and synanthrin lactobacillus CASD form and physiological and biochemical property are more as shown in table 1 in " the outstanding Bacteria Identification handbook of uncle " (the 8th edition).
Table 1
| Synanthrin lactobacillus form and physiological and biochemical property in " the outstanding Bacteria Identification handbook of uncle " (the 8th edition) | Synanthrin lactobacillus CASD form and physiological and biochemical property among the present invention |
| Straight-bar, single, paired | Shaft-like, single, in pairs a small amount of |
| Borrow peritrichous motion few in number | A small amount of peritrichous, motion |
| The bacterium colony tip-like | Form tiny tip-like bacterium colony on the agar plate |
| Statospore | Statospore |
| Gram-positive | Gram-positive |
| Do not contain the protoheme compound | Do not contain the protoheme compound |
| Do not form indoles | Do not form indoles |
| Gelatin does not liquefy | Liquefy gelatin not |
| Do not reduce nitrate | Do not reduce nitrate |
| Litmus milk is constant | Litmus milk is constant |
| Little aerobic | Little aerobic |
| Do not grow below 10 ℃ He more than 45 ℃ | Do not grow below 10 ℃ He more than 50 ℃ |
| 35 ℃ of optimum growth temperatures | 40～42 ℃ of optimum growth temperatures |
| Strict homofermentation produces D-lactic acid | D-lactic acid optical purity is more than 98% |
| Produce acid from fructose, glucose, synanthrin, raffinose, maltose, seminose, trehalose, sucrose, N.F,USP MANNITOL etc. | Can utilize fructose, glucose, synanthrin, raffinose, maltose, seminose, trehalose, sucrose, N.F,USP MANNITOL to produce acid |
| From the acid of little product such as wood sugar, semi-lactosi, pectinose or do not produce acid | Can not utilize wood sugar, semi-lactosi, pectinose to produce acid |
Embodiment 2, utilize synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 fermentation production of D-lactic acid in triangular flask, circulating fermentation 2 times, each fermentation culture 48 hours, 35 ℃ of leavening temperatures replace with fresh fermention medium with volume with 90% fermented liquid wherein
Employed each substratum is composed as follows in the present embodiment:
Slant medium: glucose 20 grams per liters, yeast extract 5 grams per liters, peptone 10 grams per liters, extractum carnis 10 grams per liters, dipotassium hydrogen phosphate 2 grams per liters, dibasic ammonium citrate 2 grams per liters, sodium acetate 5 grams per liters, 1 milliliter/liter of tween 80, bitter salt 0.58 grams per liter, four anhydrous manganeses, 0.25 grams per liter, lime carbonate 15 grams per liters, agar powder 15 grams per liters, solvent are water; The pH of described slant medium is 6.5.Sterilized 20 minutes for 115 ℃.
Seed culture medium: glucose 40 grams per liters, yeast extract 5 grams per liters, peptone 10 grams per liters, extractum carnis 10 grams per liters, dipotassium hydrogen phosphate 2 grams per liters, dibasic ammonium citrate 2 grams per liters, sodium acetate 5 grams per liters, 1 milliliter/liter of tween 80, bitter salt 0.58 grams per liter, four anhydrous manganeses, 0.25 grams per liter, lime carbonate 15 grams per liters, solvent are water; The pH of described seed culture medium is 6.5.Sterilized 20 minutes for 115 ℃.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 150 grams per liters, soybean meal hydrolysate (the soybean meal hydrolysate preparation method is with embodiment 1) 100 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 30 grams per liters, lime carbonate 75 grams per liters, surplus is a water; The pH of described fermention medium is 5.5.Sterilized 20 minutes for 115 ℃.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 is inoculated on the slant medium, cultivated 24 hours for 40 ℃;
(2) seed culture: the bacterial strain with step (1) is cultivated, under aseptic condition, encircle in the 100ml triangular flask that the 30ml seed culture medium is housed with inoculating articulating 2,40 ℃ of static cultivations 20 hours make seed culture fluid;
(3) fermentation culture: the seed culture fluid access that 10ml step (2) makes is equipped with in the 300ml triangular flask of 90ml fermention medium, and 35 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 48 hours; Therefrom take out the 90ml fermented liquid then, add the fresh fermention medium of 90ml to wherein replenishing again, adding glucose to its final concentration simultaneously is 150 grams per liters, and 35 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 48 hours, finish fermentation.
After the fermentation ends,, detect D-lactic acid concn, L-lactic acid concn, glucose concn in the last circulating fermentation fermented liquid, calculate inversion rate of glucose and D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.
3 repetitions are established in this experiment altogether.The result shows: the D-lactic acid concn is 136 ± 3 grams per liters, and transformation efficiency is 95.0 ± 0.4%, and D-lactic acid optical purity is 98.3 ± 0.2%.
Embodiment 3, utilize synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 fermentation production of D-lactic acid in triangular flask, circulating fermentation 2 times, each fermentation culture 65 hours, 40 ℃ of leavening temperatures replace with fresh fermention medium with volume with 70% fermented liquid wherein
Employed each substratum is composed as follows in the present embodiment:
Slant medium and seed culture medium are with embodiment 2.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 160 grams per liters, soybean meal hydrolysate (the soybean meal hydrolysate preparation method is with embodiment 1) 150 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 40 grams per liters, lime carbonate 80 grams per liters, surplus is a water; The pH of described fermention medium is 6.Sterilized 20 minutes for 115 ℃.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 2.
(2) seed culture: with embodiment 2.
(3) fermentation culture: the seed culture fluid access that 10ml step (2) makes is equipped with in the 300ml triangular flask of 90ml fermention medium, and 40 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 65 hours; Therefrom take out the 70ml fermented liquid then, add the fresh fermention medium of 70ml to wherein replenishing again, adding glucose to its final concentration simultaneously is 160 grams per liters, and 40 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 65 hours, finish fermentation.
After the fermentation ends,, detect D-lactic acid concn, L-lactic acid concn, glucose concn in the last circulating fermentation fermented liquid, calculate inversion rate of glucose and D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.
3 repetitions are established in this experiment altogether.The result shows: the D-lactic acid concn is 146 ± 5 grams per liters, and transformation efficiency is 94.7 ± 0.4%, and D-lactic acid optical purity is 98.0 ± 0.3%.
Embodiment 4, utilize synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 fermentation production of D-lactic acid in triangular flask, circulating fermentation 4 times, each fermentation culture 75 hours, 45 ℃ of leavening temperatures replace with fresh fermention medium with volume with 60% fermented liquid wherein
Employed each substratum is composed as follows in the present embodiment:
Slant medium and seed culture medium are with embodiment 2.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 150 grams per liters, soybean meal hydrolysate (the soybean meal hydrolysate preparation method is with embodiment 1) 100 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 10 grams per liters, lime carbonate 75 grams per liters, surplus is a water; The pH of described fermention medium is 5.5.Sterilized 20 minutes for 115 ℃.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 2;
(3) fermentation culture: the seed culture fluid access that 10ml step (2) makes is equipped with in the 300ml triangular flask of 90ml fermention medium, and 45 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 75 hours; Therefrom take out the 60ml fermented liquid then, add the fresh fermention medium of 60ml to wherein replenishing again, adding glucose to its final concentration simultaneously is 150 grams per liters, and 45 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 75 hours; Therefrom take out the 60ml fermented liquid, add the fresh fermention medium of 60ml to wherein replenishing again, adding glucose to its final concentration simultaneously is 150 grams per liters, and 45 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 75 hours; This circulating fermentation is 1 time for another example, finishes fermentation.
After the fermentation ends,, detect D-lactic acid concn, L-lactic acid concn, glucose concn in the last circulating fermentation fermented liquid, calculate inversion rate of glucose and D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.
3 repetitions are established in this experiment altogether.The result shows: the D-lactic acid concn is 140 ± 3 grams per liters, and transformation efficiency is 95.3 ± 0.3%, and D-lactic acid optical purity is 98.5 ± 0.2%.
Embodiment 5, utilize synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 fermentation production of D-lactic acid in triangular flask, circulating fermentation 10 times, each fermentation culture 72 hours, 37 ℃ of leavening temperatures replace with fresh fermention medium with volume with 75% fermented liquid wherein
Employed each substratum is composed as follows in the present embodiment:
Slant medium and seed culture medium are with embodiment 2.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 160 grams per liters, soybean meal hydrolysate (the soybean meal hydrolysate preparation method is with embodiment 1) 200 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 40 grams per liters, lime carbonate 80 grams per liters, surplus is a water; The pH of described fermention medium is 6.Sterilized 20 minutes for 115 ℃.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 2;
(3) fermentation culture: the seed culture fluid access that 10ml step (2) makes is equipped with in the 300ml triangular flask of 90ml fermention medium, and 37 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 72 hours; Therefrom take out the 75ml fermented liquid, add the fresh fermention medium of 75ml to wherein replenishing again, adding glucose to its final concentration simultaneously is 160 grams per liters, and 37 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 72 hours; Therefrom take out the 75ml fermented liquid, add the fresh fermention medium of 75ml to wherein replenishing again, adding glucose to its final concentration simultaneously is 160 grams per liters, and 37 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 72 hours; So the recirculation fermentation is 7 times, finishes fermentation.
After the fermentation ends,, detect D-lactic acid concn, L-lactic acid concn, glucose concn in the last circulating fermentation fermented liquid, calculate inversion rate of glucose and D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.
3 repetitions are established in this experiment altogether.The result shows: the D-lactic acid concn is 145 ± 4 grams per liters, and transformation efficiency is 95.7 ± 0.5%, and D-lactic acid optical purity is 98.1 ± 0.2%.
Embodiment 6, utilize synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 fermentation production of D-lactic acid in triangular flask, circulating fermentation 10 times, each fermentation culture 60 hours, 40 ℃ of leavening temperatures replace with fresh fermention medium with volume with 80% fermented liquid wherein
Employed each substratum is composed as follows in the present embodiment:
Slant medium and seed culture medium are with embodiment 2.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 180 grams per liters, soybean meal hydrolysate (the soybean meal hydrolysate preparation method is with embodiment 1) 160 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 30 grams per liters, lime carbonate 90 grams per liters, surplus is a water; The pH of described fermention medium is 6.5.Sterilized 20 minutes for 115 ℃.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 2;
(3) fermentation culture: the seed culture fluid access that 10ml step (2) makes is equipped with in the 300ml triangular flask of 90ml fermention medium, and 40 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 60 hours; Therefrom take out the 80ml fermented liquid, add the fresh fermention medium of 80ml to wherein replenishing again, adding glucose to its final concentration simultaneously is 180 grams per liters, and 40 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 60 hours; Therefrom take out the 80ml fermented liquid, add the fresh fermention medium of 80ml to wherein replenishing again, adding glucose to its final concentration simultaneously is 180 grams per liters, and 40 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 60 hours; So the recirculation fermentation is 7 times, finishes fermentation.
After the fermentation ends,, detect D-lactic acid concn, L-lactic acid concn, glucose concn in the last circulating fermentation fermented liquid, calculate inversion rate of glucose and D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.
3 repetitions are established in this experiment altogether.The result shows: the D-lactic acid concn is 160 ± 2 grams per liters, and transformation efficiency is 95.7 ± 0.4%, and D-lactic acid optical purity is 98.3 ± 0.2%.
Embodiment 7, utilize synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 fermentation production of D-lactic acid in triangular flask, circulating fermentation 10 times, each fermentation culture 48 hours, 42 ℃ of leavening temperatures replace with fresh fermention medium with volume with 70% fermented liquid wherein
Employed each substratum is composed as follows in the present embodiment:
Slant medium and seed culture medium are with embodiment 2.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 170 grams per liters, soybean meal hydrolysate (the soybean meal hydrolysate preparation method is with embodiment 1) 200 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 20 grams per liters, lime carbonate 85 grams per liters, surplus is a water; The pH of described fermention medium is 7.Sterilized 20 minutes for 115 ℃.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 2;
(3) fermentation culture: the seed culture fluid access that 10ml step (2) makes is equipped with in the 300ml triangular flask of 90ml fermention medium, and 42 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 48 hours; Therefrom take out the 70ml fermented liquid, add the fresh fermention medium of 70ml to wherein replenishing again, adding glucose to its final concentration simultaneously is 170 grams per liters, and 42 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 48 hours; Therefrom take out the 70ml fermented liquid, add the fresh fermention medium of 70ml to wherein replenishing again, adding glucose to its final concentration simultaneously is 170 grams per liters, and 42 ℃ of static cultivations are stirred once every vibration in 12 hours, cultivate 48 hours; So the recirculation fermentation is 7 times, finishes fermentation.
After the fermentation ends,, detect D-lactic acid concn, L-lactic acid concn, glucose concn in the last circulating fermentation fermented liquid, calculate inversion rate of glucose and D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.
3 repetitions are established in this experiment altogether.The result shows: the D-lactic acid concn is 150 ± 2 grams per liters, and transformation efficiency is 95.2 ± 0.2%, and D-lactic acid optical purity is 98.5 ± 0.2%.
Embodiment 8, utilize synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 fermentation production of D-lactic acid in 5 liters of automatic fermenters, circulating fermentation 3 times, each fermentation culture 60 hours, 40 ℃ of leavening temperatures replace with fresh fermention medium with volume with 80% fermented liquid wherein
Employed each substratum is composed as follows in the present embodiment:
Slant medium and seed culture medium are with embodiment 2.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 180 grams per liters, soybean meal hydrolysate (the soybean meal hydrolysate preparation method is with embodiment 1) 180 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 30 grams per liters, lime carbonate 90 grams per liters, surplus is a water; The pH of described fermention medium is 6.5.Sterilized 20 minutes for 115 ℃.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: the bacterial strain that step (1) is cultivated is encircling in the 100ml triangular flask that the 30ml seed culture medium is housed with inoculating articulating 2 under the aseptic condition, and 40 ℃ of static cultivations 20 hours make seed culture fluid 1; 30ml seed culture fluid 1 is inserted under aseptic condition in the 500ml triangular flask that the 300ml seed culture medium is housed, and 40 ℃ of static cultivations 20 hours make seed culture fluid 2;
(3) fermentation culture: the seed culture fluid 2 that 300ml step (2) is made inserts under aseptic condition in 5 liters of automatic fermenters (emerging BIOTECH is protected in Shanghai) that 3.7 liters of fermention mediums are housed, 40 ℃ of static cultivations, stirred once every 12 hours, each rotating speed with 30 rev/mins stirred 20 minutes, rotation radius is 33mm, cultivates 60 hours (the 1st circulation); Therefrom take out 3.2 liters of fermented liquids, add 3.2 liters of fresh fermention mediums to wherein replenishing again, adding glucose to its final concentration simultaneously is 180 grams per liters, 40 ℃ of static cultivations, stirred once every 12 hours, each rotating speed with 30 rev/mins stirred 20 minutes, and rotation radius is 33mm, cultivated 60 hours (the 2nd circulation); Therefrom take out 3.2 liters of fermented liquids, add 3.2 liters of fresh fermention mediums to wherein replenishing again, adding glucose to its final concentration simultaneously is 180 grams per liters, 40 ℃ of static cultivations, stirred once every 12 hours, each rotating speed with 30 rev/mins stirred 20 minutes, and rotation radius is 33mm, cultivate 60 hours (the 3rd circulation), finish fermentation.
After the fermentation ends,, detect D-lactic acid concn, L-lactic acid concn, glucose concn in the 1st, 2,3 circulating fermentation liquid respectively, calculate inversion rate of glucose and D-lactic acid optical purity according to detection and the method for calculation described in the above-mentioned embodiment.
3 repetitions are established in this experiment altogether.3 fermentations the results are shown in Table 2.
Table 2
| Batch fermentation | D-lactic acid concn (grams per liter) | Transformation efficiency (%) | D-lactic acid optical purity (%) |
| The 1st circulating fermentation | 152±6 | 95.6±0.3 | 98.2±0.1 |
| The 2nd circulating fermentation | 161±4 | 95.0±0.3 | 98.0±0.3 |
| The 3rd circulating fermentation | 160±4 | 96.0±0.2 | 98.0±0.2 |
Embodiment 9, utilize synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 fermentation production of D-lactic acid in 5 liters of automatic fermenters, circulating fermentation 7 times, each fermentation culture 55 hours, 40 ℃ of leavening temperatures replace with fresh fermention medium with volume with 75% fermented liquid wherein
Employed each substratum is composed as follows in the present embodiment:
Slant medium and seed culture medium are with embodiment 2.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 170 grams per liters, soybean meal hydrolysate (the soybean meal hydrolysate preparation method is with embodiment 1) 170 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 25 grams per liters, lime carbonate 85 grams per liters, surplus is a water; The pH of described fermention medium is 6.5.Sterilized 20 minutes for 115 ℃.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 8;
(3) fermentation culture: the seed culture fluid 2 that 300ml step (2) is made inserts under aseptic condition in 5 liters of automatic fermenters (emerging BIOTECH is protected in Shanghai) that 3.7 liters of fermention mediums are housed, 40 ℃ of static cultivations, stirred once every 12 hours, each rotating speed with 50 rev/mins stirred 30 minutes, rotation radius is 33mm, cultivates 55 hours (circulation for the first time); Therefrom take out 3.0 liters of fermented liquids, add 3.0 liters of fresh fermention mediums to wherein replenishing again, adding glucose to its final concentration simultaneously is 170 grams per liters, 40 ℃ of static cultivations, stirred once every 12 hours, each rotating speed with 50 rev/mins stirred 30 minutes, and rotation radius is 33mm, cultivated 55 hours (circulation for the second time); Therefrom take out 3.0 liters of fermented liquids, add 3.0 liters of fresh fermention mediums to wherein replenishing again, adding glucose to its final concentration simultaneously is 170 grams per liters, 40 ℃ of static cultivations, stirred once every 12 hours, each rotating speed with 50 rev/mins stirred 30 minutes, and rotation radius is 33mm, cultivated 55 hours (circulation for the third time); This circulating fermentation is 4 times for another example, finishes fermentation.
Detect D-lactic acid concn, L-lactic acid concn, the glucose concn of the 1st to the 7th circulating fermentation fermented liquid, and calculate inversion rate of glucose and D-lactic acid optical purity.
3 repetitions are established in this experiment altogether.7 fermentations the results are shown in Table 3.The 6th time circulating fermentation fermented liquid high performance liquid chromatography detected result is seen accompanying drawing 1.Wherein, C is the fermented liquid high-efficient liquid phase chromatogram.
Table 3
| Batch fermentation | D-lactic acid concn (grams per liter) | Transformation efficiency (%) | D-lactic acid optical purity (%) |
| The 1st circulating fermentation | 145±2 | 95.2±0.3 | 98.3±0.3 |
| The 2nd circulating |
150±4 | 95.4±0.4 | 98.0±0.4 |
| The 3rd circulating fermentation | 158±6 | 95.5±0.1 | 98.5±0.4 |
| The 4th circulating fermentation | 156±2 | 95.0±0.3 | 99.0±0.2 |
| The 5th circulating fermentation | 160±5 | 95.0±0.5 | 98.8±0.5 |
| The 6th circulating fermentation | 162±3 | 95.3±0.4 | 98.0±0.1 |
| The 7th circulating fermentation | 157±2 | 95.6±0.4 | 98.3±0.1 |
Employed each substratum is composed as follows in the present embodiment:
Slant medium and seed culture medium are with embodiment 2.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 170 grams per liters, soybean meal hydrolysate (the soybean meal hydrolysate preparation method is with embodiment 1) 150 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 20 grams per liters, lime carbonate 85 grams per liters, surplus is a water; The pH of described fermention medium is 6.5.Sterilized 20 minutes for 115 ℃.
The method of this fermentation production of D-lactic acid may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: the bacterial strain that step (1) is cultivated is encircling in the 100ml triangular flask that the 30ml seed culture medium is housed with inoculating articulating 2 under the aseptic condition, and 40 ℃ of static cultivations 20 hours make seed culture fluid 1; 30ml seed culture fluid 1 is inoculated under aseptic condition in the 500ml triangular flask that the 300ml seed culture medium is housed, and 40 ℃ of static cultivations 20 hours make seed culture fluid 2; With 300ml seed culture fluid 2, under aseptic condition, be inoculated in 5 liters of triangular flasks that 2 liters of seed culture mediums are housed, 40 ℃ of static cultivations 20 hours make seed culture fluid 3;
(3) fermentation culture: the seed culture fluid 3 that 2 liters of steps (2) are made, under aseptic condition, insert and be equipped with in 30 liters of automatic fermenters (emerging BIOTECH is protected in Shanghai) of 18 liters of fermention mediums, 42 ℃ of static cultivations, stirred once every 12 hours, each rotating speed with 100 rev/mins stirred 40 minutes, rotation radius is 42mm, cultivates 48 hours (the 1st circulation); Therefrom take out 17 liters of fermented liquids, add 17 liters of fresh fermention mediums to wherein replenishing again, adding glucose to its final concentration simultaneously is 170 grams per liters, 42 ℃ of static cultivations, stirred once every 12 hours, each rotating speed with 100 rev/mins stirred 40 minutes, and rotation radius is 42mm, cultivated 48 hours (the 2nd circulation); This circulating fermentation is 2 times for another example, and note is done the 3rd circulation, the 4th circulation respectively, finishes fermentation.
Detect D-lactic acid concn, L-lactic acid concn, glucose concn in the 1st, 2,3,4 circulating fermentation liquid respectively, and calculate each inversion rate of glucose and D-lactic acid optical purity.
3 repetitions are established in this experiment altogether.4 fermentations the results are shown in Table 4.
Table 4
| Batch fermentation | D-lactic acid concn (grams per liter) | Transformation efficiency (%) | D-lactic acid optical purity (%) |
| The 1st circulating fermentation | 143±4 | 95.2±0.3 | 98.0±0.1 |
| The 2nd circulating |
150±2 | 95.0±0.2 | 98.3±0.3 |
| The 3rd circulating fermentation | 158±3 | 95.5±0.4 | 98.0±0.1 |
| The 4th circulating fermentation | 156±2 | 96.0±0.3 | 98.1±0.2 |
Claims (11)
1, synanthrin lactobacillus (Sporolactobacillus inulinus) CASD, its deposit number is CGMCC № 2185.
2, a kind of method of producing D-lactic acid is that fermentation synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 obtains D-lactic acid.
3, method according to claim 2 is characterized in that: the mode of described fermentation culture is a semicontinuous fermentation.
4, according to claim 2 or 3 described methods, it is characterized in that: contain in every liter of the fermention medium of described synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185: glucose 150～180 grams, corn steep liquor 10～40 grams, soybean meal hydrolysate 100～200 grams, be used to regulate and control the neutralizing agent of medium pH, surplus is a water; The pH of described fermention medium is 5.5～7.
5, method according to claim 4 is characterized in that: described neutralizing agent is a lime carbonate, contains lime carbonate 75～90 grams in every liter of described fermention medium.
6, according to claim 2 or 3 described methods, it is characterized in that: the fermentation condition of described synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 is 35 ℃～45 ℃ and cultivated 48～75 hours.
7, method according to claim 6 is characterized in that: described fermentation culture temperature is 37 ℃～42 ℃.
8, method according to claim 6 is characterized in that: described fermented incubation time is 48～65 hours.
9, according to claim 2 or 3 described methods, it is characterized in that: stir in the described fermentation culture process, the rotating speed of described stirring is 30～100 rev/mins, and rotation radius is 33mm or 42mm, and churning time is 20～40 minutes.
10, method according to claim 9 is characterized in that: described mixing speed is 50 rev/mins, and churning time is 30 minutes.
11, according to claim 2 or 3 described methods, it is characterized in that: in the described method, insert synanthrin lactobacillus (Sporolactobacillus inulinus) CASD CGMCC № 2185 in the seed culture medium earlier, cultivated 10～24 hours at 37～42 ℃, insert described fermention medium again;
The aqueous solution that described seed culture medium is made up of following material: glucose 40 grams per liters, yeast extract 5 grams per liters, peptone 10 grams per liters, extractum carnis 10 grams per liters, dipotassium hydrogen phosphate 2 grams per liters, dibasic ammonium citrate 2 grams per liters, sodium acetate 5 grams per liters, 1 milliliter/liter of tween 80, bitter salt 0.58 grams per liter, four anhydrous manganeses, 0.25 grams per liter, lime carbonate 15 grams per liters; The pH of described seed culture medium is 6～7.
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| CN101886095B (en) * | 2010-06-13 | 2013-10-30 | 天津工业生物技术研究所 | Method for producing high-concentration D-lactic acid by adopting synchronous enzymolysis and fermentation on peanut meal and special culture medium thereof |
| CN101974447B (en) * | 2010-07-30 | 2012-07-25 | 上海新立工业微生物科技有限公司 | Sporolactobacillus inulinus and method for preparing D-lactic acid by fermenting strains of sporolactobacillus inulinus |
| CN101974603B (en) * | 2010-09-30 | 2013-04-24 | 山东大学 | Method for producing D-alpha-hydroxybutyric acid |
| CN102643875B (en) * | 2012-04-24 | 2013-10-23 | 中国科学院微生物研究所 | Method for producing D-lactic acid by utilizing jerusalem artichoke hydrolysate |
| 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 |
| CN103555776B (en) * | 2013-10-31 | 2016-01-20 | 南京工业大学 | Method for producing D-lactic acid by repeated batch fermentation by utilizing surface immobilization technology |
| CN103756939B (en) * | 2014-01-17 | 2015-07-15 | 上海交通大学 | Sporolactobacillus terrae and application thereof |
| CN106884001B (en) * | 2015-12-16 | 2020-04-10 | 中国科学院微生物研究所 | Recombinant alkalophilic bacillus, preparation method and application thereof, and method for preparing D-lactic acid |
| CN105779353A (en) * | 2016-04-15 | 2016-07-20 | 深圳市计量质量检测研究院 | Lactobacillus and application thereof |
| WO2018084813A1 (en) * | 2016-11-01 | 2018-05-11 | Ptt Global Chemical Public Company Limited | Fermentation process for producing d-lactic acid or its salts |
| 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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