CN100554405C - A kind of method and special-purpose lactobacillus rhamnosus thereof that produces L-lactic acid - Google Patents
A kind of method and special-purpose lactobacillus rhamnosus thereof that produces L-lactic acid Download PDFInfo
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Abstract
The invention discloses a kind of method and special-purpose lactobacillus rhamnosus thereof of the L-of production lactic acid.Lactobacillus rhamnosus provided by the present invention is lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183.Cultivate lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 and can obtain L-lactic acid.Contain carbon source, nitrogenous source in the fermention medium of this bacterial strain and be used to control the neutralizing agent of fermented liquid pH; Described carbon source is glucose 150-200 grams per liter (a fermentation starting point concentration); Described nitrogenous source is soybean meal hydrolysate, soybean meal hydrolysate+corn steep liquor or bean cake powder; When described nitrogenous source is bean cake powder, also can contain proteinase-10 .05-0.1 grams per liter; Described neutralizing agent is a lime carbonate 75-100 grams per liter, and surplus is a water, and the pH of this fermention medium is 5.5-7.Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183, with glucose is substrate, under 35 ℃ of-45 ℃ of conditions with the transformation efficiency of 94.5%-96.5%, the L-lactic acid of production optical purity 97.6%-98.7%, its concentration reaches as high as 235 grams per liters.
Description
Technical field
The present invention relates to a kind of method and special strain therefore thereof of the L-of production lactic acid, particularly relate to a kind of method and special-purpose lactobacillus rhamnosus thereof of the L-of production lactic acid.
Background technology
Lactic acid (Lactic Acid) has another name called dihydroxypropionic acid, is one of the world's three big organic acids.As a kind of traditional multi-usage fine chemicals, lactic acid can be used as acidic flavoring agent, perfume compound, sanitas, plant-growth regulator, biological degradable material, medicine and agricultural chemicals etc., is applied to food, pharmacy, brewages, in process hides, weaving, environmental protection and the agricultural.Lactic acid is a kind of chiral molecules, can be divided into L-lactic acid, D-lactic acid and DL-lactic acid.The poly(lactic acid) of being made as principal monomer by L-lactic acid has excellent biological compatibility and biodegradability, can be made into medical slow release material, Biodegradable fibers and biodegradable plastic etc.L-lactic acid is just becoming the research focus of domestic and international lactic acid industry.
The production method of lactic acid mainly contains three kinds of chemical synthesis, enzymic synthesis method and fermentation methods.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 because the used raw material of synthesis method is acetaldehyde and violent in toxicity prussic acid, so synthesizing lactic acid is applied to food and can not be widely accepted, its production cost is also higher in addition.Enzyme process catalysis can obtain to specificity optical purity lactic acid, but its reaction process research difficulty is very big, is not applied to suitability for industrialized production as yet.Production by Microorganism Fermentation lactic acid, can obtain D-lactic acid, L-lactic acid or both a certain proportion of mixtures by the selection of bacterial classification and culture condition, can decompose resulting glucose etc. with renewable resourcess such as starch, Mierocrystalline celluloses is that fermenting raw materials is produced lactic acid, and production cost is low, Product Safety is high, is the main method of scale operation lactic acid.
Application number is that 200480036931.1 Chinese patent discloses the method that a kind of Lactobacillus of employing paracasei produces lactic acid, is fermented substrate with glucose, yeast extract and peptone, produces the lactic acid of 181 grams per liters; With glucose, peptone and corn steeping powder is fermented substrate, produces the lactic acid of 179.1 grams per liters.Application number is the method that 200510119041.3 Chinese patent discloses a kind of employing mutant strain of lactobacillus casei (Lactobacillus caseisubsp.rhamnosus)-Lc-F34 fermentation production of L-lactic acid, this bacterial strain is a fermented substrate with glucose and yeast extract, shake bottle fed-batch fermentation and produce lactic acid, produce the lactic acid of 177.6 grams per liters; With corn steep liquor and glucose is fermented substrate, and 30 liters of fermentor tank fed-batch fermentations are produced lactic acid, produce the L-lactic acid of 154.6 grams per liters.
The production cost of L-lactic acid is one of restriction L-lactic acid key in application factor.Improve in the fermentative Production L-lactic acid process that the L-concentration of lactic acid helps reducing cost in the fermented liquid.In the process of fermentative Production L-lactic acid, nitrogenous source in the substratum is another important factor that influences L-lactic acid-producing cost, adopting wide material sources, the cheap nitrogen-containing material surrogate as the higher nitrogenous source of prices such as yeast powder and peptone, is another effective way of control L-lactic acid-producing cost.
Summary of the invention
An object of the present invention is to provide a strain and produce lactobacillus rhamnosus (Lactobacillusrhamnosus) CASL of L-lactic acid.
Lactobacillus rhamnosus provided by the present invention (Lactobacillus rhamnosus) CASL screens the soil to obtain near the dairy factory of Jinan, Shandong, is accredited as lactobacillus rhamnosus (Lactobacillus rhamnosus).Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 24th, 2007 and (is called for short CGMCC, the address is: the Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC № 2183.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 is a gram-positive microorganism, cell elongated rod shape, long 2.5-3.5 μ m, wide 0.7-0.8 μ m.This bacterium can utilize trehalose, Sorbitol Powder, glucose, lactose, ribose, cellobiose, maltose and fructose etc.
Second purpose of the present invention provides a kind of method of utilizing lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid.
The method of production L-lactic acid provided by the present invention is that fermentation lactobacillus rhamnosus (Lactobacillusrhamnosus) CASL CGMCC № 2183 obtains L-lactic acid.
Contain carbon source, nitrogenous source in the fermention medium of described lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 and be used to control the neutralizing agent of fermented liquid pH, described carbon source is cerelose (Zibo rainbow space industry sugared company limited) 150-200 grams per liter, and described nitrogenous source is selected from following 1) to 3) in any one:
1) soybean meal hydrolysate 100-300 grams per liter;
2) bean cake powder 20-30 grams per liter;
3) soybean meal hydrolysate 100-300 grams per liter, corn steep liquor 10-30 grams per liter;
The pH of described fermention medium is 5.5-7.
Described bean cake powder can also can carry out acidolysis or enzymolysis earlier directly as nitrogenous source, with the soybean meal hydrolysate that obtains as nitrogenous source.
Described soybean meal hydrolysate can take by weighing bean cake powder (Beijing Kang Mingwei substratum technology limited liability company) according to the preparation of following method, carries out abundant mixing with the ratio of 2% sulphuric acid soln of 1g bean cake powder: 5ml, 90 ℃ of water-baths 15 hours.
When described nitrogenous source is bean cake powder, also can contain the proteolytic enzyme of 0.05-0.1 grams per liter in the described fermention medium.
Described neutralizing agent specifically can be lime carbonate.
The fermentation condition of described lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 specifically can be 35-50 ℃ and cultivated 48-80 hour, cultivates 50-68 hour as 35-45 ℃.
In the described method, also lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMC C № 2183 can be inserted earlier in the following seed culture medium, cultivate 12-20 hour, insert described fermention medium again at 35-40 ℃; Contain in every liter of the described seed culture medium: glucose 20 grams, yeast powder 5 grams, peptone 5 grams, lime carbonate 10 grams, surplus is a water; The pH of described seed culture medium is 6.5.
The training method of described fermentation specifically can be intermittent control shaking and cultivates (as using the triangular flask fermentation culture) or stir culture at intermittence (as adopting fermentor cultivation), is 12 hours pitch time; The rotating speed of described stirring is 30-100 rev/min, and rotation radius is 33 or 42mm, and churning time is 20-40 minute; Described mixing speed is preferably 50 rev/mins, and churning time is preferably 30 minutes.
The present invention utilizes lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid, with the cerelose is substrate, with the L-lactic acid of the efficient fermentative production optical purity of the transformation efficiency of 94.5%-96.5% 97.6%-98.7%, wherein the L-lactic acid concn reaches as high as 235 grams per liters under 35 ℃ of-45 ℃ of conditions.The present invention utilizes lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid, when being nitrogenous source with the bean cake powder, simultaneously can add proteolytic enzyme, proteolysis and lactic fermentation are carried out simultaneously, improved the utilising efficiency of bean cake powder, simplify the raw material treating processes, also can control the production cost of L-lactic acid simultaneously.
Description of drawings
Fig. 1 is lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 fermentation production of L-lactic acid fermented liquids and L-lactic acid standard substance, D-lactic acid standard substance high-efficient liquid phase chromatogram.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.
Fig. 2 is that glucose and L-lactic acid is the change procedure curve in time in lactobacillus rhamnosus (Lactobacillus rhamnosus) the CASL CGMCC № 2183 fermentation production of L-lactic acid processes.
Embodiment
The step that relates in lactobacillus rhamnosus (Lactobacillus rhamnosus) the CASL CGMCC № 2183 fermentation production of L-lactic acid methods of utilizing of the present invention is as follows:
(1) slant culture: lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 bacterial classification inoculations on the solid slant culture base that contains 1.5g/100ml agar, under the 35-40 ℃ of condition, were cultivated 24-48 hour;
(2) seed culture: with the slant culture of step (1), be inoculated under aseptic condition in the 30ml seed culture medium, under the 35-40 ℃ of condition, static cultivation 10-24 hour adds neutralizing agent control fermented liquid pH, makes seed culture fluid;
(3) fermentation culture: with the inoculum size of 5-15% volume ratio, seed culture fluid is inoculated in the fermention medium, cultivated 48-80 hour under the 35-50 ℃ of condition, wherein, manually vibration is adopted in fermentation in the triangular flask, and every vibration in 12 hours once, fermentor tank adopts intermittently stir culture, stirred once every 12 hours, stirring velocity 30-100 rev/min, rotation radius is 33 or 42mm, churning time 20-40 minute, add neutralizing agent control fermented liquid pH, make the fermentation culture that contains L-lactic acid;
(4) handle sample: got 6,000 rev/mins of fermented liquids centrifugal 5 minutes, and got supernatant liquor;
(5) sample detection: get the supernatant liquor dilution, detect glucose content, L-lactic acid content and D-lactic acid content;
Wherein, the yeast culture temperature described in the step (1) specifically can be 37-40 ℃.
Wherein, the yeast culture temperature described in the step (2) specifically can be 37-40 ℃.
Wherein, the yeast culture temperature described in the step (3) specifically can be 35-45 ℃.
Wherein, the yeast culture time described in the step (1) specifically can be 24-36 hour.
Wherein, the yeast culture time described in the step (2) specifically can be 12-20 hour.
Wherein, the yeast culture time described in the step (3) specifically can be 50-68 hour.
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-CRS10W (3 μ) 4.6ID * 50mm, the optics allosome separates to be used), moving phase is 0.002mol/L copper sulfate, flow 0.5ml/min, sample size 20 μ L, UV-detector detects wavelength 254nm, 25 ℃ of service temperatures.L-lactic acid standard substance are Sigma-Aldrich company product, and article No. is L1750.D-lactic acid standard substance are Sigma-Aldrich company product, and article No. is L0625.
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, B is D-lactic acid standard substance high-efficient liquid phase chromatograms, and C is lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 fermentation production of L-lactic acid fermented liquid 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.
L-lactic acid optical purity method of calculation: L-lactic acid optical purity (%)=[L-lactic acid concn (grams per liter)-D-lactic acid concn (grams per liter)] ÷ [L-lactic acid concn (grams per liter)+D-lactic acid concn (grams per liter)] * 100%
Transformation efficiency method of calculation: transformation efficiency (%)=L-lactic acid concn (grams per liter) ÷ glucose consumption amount (grams per liter) * 100%
Further illustrate the present invention below by embodiment.
Separation, mutagenesis screening and the evaluation of embodiment 1, lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183
1, the separation of bacterial classification, mutagenesis screening
1) separation of bacterial classification
Near the dairy factory of Jinan, Shandong, obtain soil sample, take by weighing 5 gram soil samples in the 250ml triangular flask, add 100ml physiological saline, dilute 10 with sterilized water after fully being mixed, coat on the MRS solid medium for 000 times, 40 ℃ of cultivations, wait to grow single bacterium colony after, the single bacterium colony of part forms transparent circle owing to producing acid with the dissolution of calcium carbonate in the periphery of bacterial colonies substratum.Single bacterium colony that the picking transparent circle is bigger is inoculated in the 10ml fermention medium, and 40 ℃ leave standstill and cultivated 72 hours, measure L-lactic acid concn in the fermented liquid, the highest bacterial strain of picking one strain L-lactic acid production, and it is standby to be inoculated on the MRS medium slant refrigeration.
2) ion beam mutagenesis screening
In the MRS liquid nutrient medium, 40 ℃ leave standstill and are cultured to mid-log phase with the inoculation of above acquisition, and centrifugal back suspends with physiological saline, makes 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, be coated on after the dilution on the MRS solid medium, after waiting to grow single bacterium colony, select the bigger bacterium colony of transparent circle, be inoculated in the 10ml fermention medium, 40 ℃ leave standstill cultivation 72 hours, measure glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid.Through repeatedly screening, obtain a strain L-lactic acid production than the bacterial strain before the ion beam mutagenesis screening be significantly improved, L-lactic acid optical purity more than 98%, the bacterial strain of transformation efficiency more than 95%, be lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183, through the secondary fermentation test of repeatedly going down to posterity, L-lactic acid production, L-lactic acid optical purity and transformation efficiency do not have considerable change.
Used MRS solid medium is in lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 screenings:
Used MRS liquid nutrient medium is in lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 screenings:
Used fermention medium is in lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 screenings:
Cerelose (the sugared company limited of Zibo rainbow space industry) 180 grams per liters, soybean meal hydrolysate (soybean meal hydrolysate preparation method: bean cake powder (Beijing Kang Mingwei substratum technology limited liability company), ratio with 2% (volumn concentration) sulphuric acid soln of 1g bean cake powder: 5ml is carried out abundant mixing, 90 ℃ of water-baths 15 hours, obtain soybean meal hydrolysate) 100 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 10 grams per liters, lime carbonate 90 grams per liters.The pH value of substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.
2, the evaluation of bacterial strain
According to " the outstanding Bacteria Identification handbook of uncle " (the 8th edition) and " common bacteria system identification handbook " (eastern elegant pearl, Cai Miaoying etc. write, Beijing: Science Press, 2001.2) method of describing in is carried out morphological specificity observation and physio-biochemical characteristics evaluation to this bacterial strain;
Qualification result shows that lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 is gram-positive microorganism, cell elongated rod shape, long 2.5-3.5 μ m, wide 0.7-0.8 μ m.This bacterium can utilize trehalose, Sorbitol Powder, glucose, lactose, ribose, cellobiose, maltose, fructose, N.F,USP MANNITOL, melizitose, rhamnosyl, semi-lactosi, Vitamin C2, seminose, can not utilize melibiose, amygdaloside, pectinose, inositol, raffinose.Oxidase negative.The hemolytic test feminine gender.Can be 15 ℃ and 45 ℃ of growths.
Based on above feature, this strain L-lactic-acid-producing strain is accredited as lactobacillus rhamnosus (Lactobacillusrhamnosus) CASL, and 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 location is: the Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC № 2183.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 and " the lactic-acid-bacterium classification is identified and experimental technique " (Ling Daiwen chief editor; Ling Daiwen, eastern elegant pearl is write Beijing: China Light Industry Press, 1999.3) described in lactobacillus rhamnosus (Lactobacillus rhamnosus) physiological and biochemical property relatively see Table 1.
Table 1
| Physiological and biochemical property | Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 | Lactobacillus rhamnosus (Lactobacillus rhamnosus) described in " the lactic-acid-bacterium classification is identified and experimental technique " |
| Amygdaloside | - | + |
| Pectinose | - | d |
| Cellobiose | + | + |
| Polychrom (Vitamin C2) | + | + |
| Fructose | + | + |
| Semi-lactosi | + | + |
| Glucose | + | + |
| Lactose | + | + |
| Maltose | + | + |
| N.F,USP MANNITOL | + | + |
| Seminose | + | + |
| Melizitose | + | + |
| Melibiose | - | - |
| Raffinose | - | - |
| Rhamnosyl | + | + |
| Ribose | + | + |
| Trehalose | + | + |
| 15 ℃ of growths | + | + |
In the table 1 ,+represent the positive or growth of experiment ,-expression is tested negative or is not grown, and d represents the 11%-89% bacterial strain positive.
Embodiment 2, utilize lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 fermentative Production L-lactic acid in triangular flask
Used substratum is composed as follows among this 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.The initial pH of this substratum is 6.5.Sterilized 20 minutes for 115 ℃.
Seed culture medium: glucose 20 grams per liters, yeast powder 5 grams per liters, peptone 5 grams per liters, lime carbonate 10 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 150 grams per liters, soybean meal hydrolysate (preparation method is with embodiment 1) 100 grams per liters, lime carbonate 75 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.
The method of fermentative Production L-lactic acid of the present invention may further comprise the steps:
(1) slant culture: lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 bacterial classification inoculations on the solid slant culture base, were cultivated 36 hours for 37 ℃;
(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 inoculation articulating 2,37 ℃ leave standstill cultivation 20 hours, make seed culture fluid;
(3) fermentation culture: the seed that inserts 10ml step (2) in the 300ml triangular flask of 90ml fermention medium is housed is supported liquid, and 40 ℃ leave standstill cultivation 72 hours, finish fermentation.Wherein, stir once every vibration in 12 hours.3 repetitions are established in experiment altogether.
During fermentation ends, get fermented liquid, 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor, detect glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid according to aforesaid method, calculate L-lactic acid optical purity and transformation efficiency.The result shows that the concentration of glucose is 10 grams per liters ± 0.5 grams per liter, L-lactic acid concn 132 grams per liters ± 3 grams per liters, transformation efficiency 94.5% ± 0.3%, L-lactic acid optical purity 98.5% ± 0.4%.
Embodiment 3, utilize lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 fermentative Production L-lactic acid in triangular flask
Used substratum is composed as follows among this 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 (preparation method is with embodiment 1) 300 grams per liters, lime carbonate 75 grams per liters.The initial pH of this substratum is 7.Sterilization is 20 minutes under 115 ℃ of conditions.
The method of fermentative Production L-lactic acid of the present invention may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 2;
(3) fermentation culture: the seed that inserts 15ml step (2) in the 300ml triangular flask of 85ml fermention medium is housed is supported liquid, and 37 ℃ leave standstill cultivation 65 hours, finish fermentation.Wherein, stir once every vibration in 12 hours.3 repetitions are established in experiment altogether.
During fermentation ends, get fermented liquid, 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor, according to above-mentioned detection method, detect glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid, calculate L-lactic acid optical purity and transformation efficiency.The result shows that the concentration of glucose is 2 grams per liters ± 0.3 grams per liter, L-lactic acid concn 140 grams per liters ± 4 grams per liters, transformation efficiency 95.2% ± 0.5%, L-lactic acid optical purity 98.0% ± 0.2%.
Embodiment 4, utilize lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 fermentative Production L-lactic acid in triangular flask
Used substratum is composed as follows among this 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 (preparation method is with embodiment 1) 220 grams per liters, lime carbonate 75 grams per liters.The initial pH of this substratum is 5.5.Sterilization is 20 minutes under 115 ℃ of conditions.
The method of fermentative Production L-lactic acid of the present invention may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 2;
(3) fermentation culture: the seed that inserts 5ml step (2) in the 300ml triangular flask of 95ml fermention medium is housed is supported liquid, and 40 ℃ leave standstill cultivation 50 hours, finish fermentation.Wherein, stir once every vibration in 12 hours.3 repetitions are established in experiment altogether.
During fermentation ends, get fermented liquid, 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor, according to above-mentioned detection method, detect glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid, calculate L-lactic acid optical purity and transformation efficiency.The result shows that the concentration of glucose is 1 grams per liter ± 0.3 grams per liter, L-lactic acid concn 142 grams per liters ± 2 grams per liters, transformation efficiency 95.3% ± 0.3%, L-lactic acid optical purity 98.5% ± 0.5%.
Embodiment 5, utilize lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 fermentative Production L-lactic acid in triangular flask
Used substratum is composed as follows among this 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 (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 90 grams per liters.The initial pH of this substratum is 6.Sterilization is 20 minutes under 115 ℃ of conditions.
The method of fermentative Production L-lactic acid of the present invention may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 2;
(3) fermentation culture: the seed that inserts 10ml step (2) in the 300ml triangular flask of 90ml fermention medium is housed is supported liquid, and 37 ℃ leave standstill cultivation 60 hours, finish fermentation.Wherein, stir once every vibration in 12 hours.3 repetitions are established in experiment altogether.
During fermentation ends, get fermented liquid, 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor, according to above-mentioned detection method, detect glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid, calculate L-lactic acid optical purity and transformation efficiency.The result shows that the concentration of glucose is 8 grams per liters ± 1.0 grams per liters, L-lactic acid concn 165 grams per liters ± 5 grams per liters, transformation efficiency 96.0% ± 0.5%, L-lactic acid optical purity 98.2% ± 0.3%.
Embodiment 6, utilize lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 fermentative Production L-lactic acid in triangular flask
Used substratum is composed as follows among this embodiment:
Slant medium and seed culture medium are with embodiment 2.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 200 grams per liters, soybean meal hydrolysate (preparation method is with embodiment 1) 300 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 10 grams per liters, lime carbonate 100 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.
The method of fermentative Production L-lactic acid of the present invention may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 2;
(3) fermentation culture: the seed that inserts 15ml step (2) in the 300ml triangular flask of 85ml fermention medium is housed is supported liquid, and 35 ℃ leave standstill cultivation 48 hours, finish fermentation.Wherein, stir once every vibration in 12 hours.3 repetitions are established in experiment altogether.
During fermentation ends, get fermented liquid, 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor, according to above-mentioned detection method, detect glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid, calculate L-lactic acid optical purity and transformation efficiency.The result shows that the concentration of glucose is 1 grams per liter ± 0.5 grams per liter, L-lactic acid concn 192 grams per liters ± 2 grams per liters, transformation efficiency 96.5% ± 0.4%, L-lactic acid optical purity 97.6% ± 0.2%.
Embodiment 7, utilize lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 fermentative Production L-lactic acid in triangular flask
Used substratum is composed as follows among this 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 (fermentation starting point concentration), soybean meal hydrolysate (preparation method is with embodiment 1) 250 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 20 grams per liters, lime carbonate 75 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.
The method of fermentative Production L-lactic acid of the present invention may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 2;
(3) fermentation culture: the seed that inserts 10ml step (2) in the 300ml triangular flask of 90ml fermention medium is housed is supported liquid, and 40 ℃ leave standstill cultivation 50 hours, stirs once every vibration in 12 hours.Wherein, added 8 gram cereloses (the sugared company limited of Zibo rainbow space industry) and 4 gram lime carbonate at 30 hours.3 repetitions are established in experiment altogether.
During fermentation ends, get fermented liquid, 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor, according to above-mentioned detection method, detect glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid, calculate L-lactic acid optical purity and transformation efficiency.The result shows that the concentration of glucose is 2 grams per liters ± 0.3 grams per liter, L-lactic acid concn 220 grams per liters ± 3 grams per liters, transformation efficiency 96.3% ± 0.4%, L-lactic acid optical purity 98.0% ± 0.2%.
Embodiment 8, utilize lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 fermentative Production L-lactic acid in triangular flask
Used substratum is composed as follows among this 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, bean cake powder (Beijing Kang Mingwei substratum technology limited liability company) 30 grams per liters, lime carbonate 75 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.
The method of fermentative Production L-lactic acid of the present invention may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 2;
(3) fermentation culture: the seed that inserts 10ml step (2) in the 300ml triangular flask of 90ml fermention medium is housed is supported liquid, and 50 ℃ leave standstill cultivation 65 hours, finish fermentation.Wherein, stir once every vibration in 12 hours.3 repetitions are established in experiment altogether.
During fermentation ends, get fermented liquid, 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor, according to above-mentioned detection method, detect glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid, calculate L-lactic acid optical purity and transformation efficiency.The result shows that the concentration of glucose is 28 grams per liters ± 0.8 grams per liter, L-lactic acid concn 115 grams per liters ± 2 grams per liters, transformation efficiency 95.2% ± 0.5%, L-lactic acid optical purity 98.0% ± 0.3%.
Embodiment 9, utilize lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 fermentative Production L-lactic acid in triangular flask
Used substratum is composed as follows among this 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, bean cake powder (Beijing Kang Mingwei substratum technology limited liability company) 30 grams per liters, proteolytic enzyme (Novi's trade wind flavor proteolytic enzyme, Flavourzyme 500MG) 0.05 grams per liter, lime carbonate 90 grams per liters.The initial pH of this substratum is 7.Sterilization is 20 minutes under 115 ℃ of conditions.Wherein, proteolytic enzyme adds after sterilization.
The method of fermentative Production L-lactic acid of the present invention may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 2;
(3) fermentation culture: the seed that inserts 10ml step (2) in the 300ml triangular flask of 90ml fermention medium is housed is supported liquid, and 42 ℃ leave standstill cultivation 72 hours, finish fermentation.Wherein, stir once every vibration in 12 hours.3 repetitions are established in experiment altogether.
During fermentation ends, get fermented liquid, 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor, according to above-mentioned detection method, detect glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid, calculate L-lactic acid optical purity and transformation efficiency.The result shows that the concentration of glucose is 28 grams per liters ± 0.7 grams per liter, L-lactic acid concn 145 grams per liters ± 3 grams per liters, transformation efficiency 95.5% ± 0.3%, L-lactic acid optical purity 98.5% ± 0.2%.
Used substratum is composed as follows among this 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, bean cake powder (Beijing Kang Mingwei substratum technology limited liability company) 20 grams per liters, proteolytic enzyme (Novi's trade wind flavor proteolytic enzyme, Flavourzyme 500MG) 0.1 grams per liter, lime carbonate 90 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.Wherein, proteolytic enzyme adds after sterilization.
The method of fermentative Production L-lactic acid of the present invention may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 2;
(3) fermentation culture: the seed that inserts 10ml step (2) in the 300ml triangular flask of 90ml fermention medium is housed is supported liquid, and 42 ℃ leave standstill cultivation 80 hours, finish fermentation.Wherein, stir once every vibration in 12 hours.3 repetitions are established in experiment altogether.
During fermentation ends, get fermented liquid, 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor, according to above-mentioned detection method, detect glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid, calculate L-lactic acid optical purity and transformation efficiency.The result shows that the concentration of glucose is 17 grams per liters ± 0.5 grams per liter, L-lactic acid concn 156 grams per liters ± 5 grams per liters, transformation efficiency 96.0% ± 0.3%, L-lactic acid optical purity 98.2% ± 0.7%.
Embodiment 11, utilize lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce the L-lactic acid 5 liters of fermentation cylinder for fermentation methods
Used substratum is composed as follows among this 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, bean cake powder (Beijing Kang Mingwei substratum technology limited liability company) 28 grams per liters, proteolytic enzyme (Novi's trade wind flavor proteolytic enzyme, Flavourzyme 500MG) 0.08 grams per liter, lime carbonate 90 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.Wherein, proteolytic enzyme adds after sterilization.
The method of fermentative Production L-lactic acid of the present invention may further comprise the steps:
(1) slant culture: with embodiment 2;
(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 inoculation articulating 2,37 ℃ leave standstill cultivation 20 hours; Add the 400ml seed culture medium in 1 liter of triangular flask, the 30ml seed culture fluid is inserted the 400ml seed culture medium, 37 ℃ leave standstill cultivation 20 hours, make seed culture fluid;
(3) fermentation culture: Shanghai is protected and is added 3.6 liters of fermention mediums in 5 liters of fermentor tanks of emerging BIOTECH, inserts the 400ml seed culture fluid in fermention medium, and 45 ℃ leave standstill cultivation 72 hours, finish fermentation.Wherein, stirred once every 12 hours.30 rev/mins of mixing speed, rotation radius are 33mm, churning time 40 minutes.3 repetitions are established in experiment altogether.
During fermentation ends, get fermented liquid, 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor, according to above-mentioned detection method, detect glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid, calculate L-lactic acid optical purity and transformation efficiency.The result shows that the concentration of glucose is 10 grams per liters ± 0.6 grams per liter, L-lactic acid concn 160 grams per liters ± 4 grams per liters, transformation efficiency 94.8% ± 0.4%, L-lactic acid optical purity 98.7% ± 0.3%.
Embodiment 12, utilize lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce the L-lactic acid 5 liters of fermentation cylinder for fermentation methods
Used substratum is composed as follows among this embodiment:
Slant medium and seed culture medium are with embodiment 2.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 200 grams per liters (fermentation starting point concentration), soybean meal hydrolysate (preparation method is with embodiment 1) 230 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 22 grams per liters, lime carbonate 100 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.
The method of fermentative Production L-lactic acid of the present invention may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 11;
(3) fermentation culture: Shanghai is protected and is added 3.6 liters of fermention mediums in 5 liters of fermentor tanks of emerging BIOTECH, inserts the 400ml seed culture fluid in fermention medium, and 40 ℃ leave standstill cultivation 60 hours.Wherein, stirred once every 12 hours.50 rev/mins of mixing speed, rotation radius are 33mm, churning time 30 minutes.Wherein, added 200 gram cereloses (the sugared company limited of Zibo rainbow space industry) and 100 gram lime carbonate at 40 hours.3 repetitions are established in experiment altogether.
During fermentation ends, get fermented liquid, 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor, according to above-mentioned detection method, detect glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid, calculate L-lactic acid optical purity and transformation efficiency.The result shows that the concentration of glucose is 3 grams per liters ± 0.4 grams per liter, L-lactic acid concn 235 grams per liters ± 3 grams per liters, transformation efficiency 95.2% ± 0.3%, L-lactic acid optical purity 97.9% ± 0.5%.
Fermented liquid high performance liquid chromatography detected result is seen accompanying drawing 1.Wherein C is lactobacillus rhamnosus (Lactobacillusrhamnosus) CASL CGMCC № 2183 fermentation production of L-lactic acid fermented liquid high-efficient liquid phase chromatograms.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 fermenting process curves are seen accompanying drawing 2.
Embodiment 13, utilize lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce the L-lactic acid 30 liters of fermentation cylinder for fermentation methods
Used substratum is composed as follows among this embodiment:
Slant medium and seed culture medium are with embodiment 2.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 200 grams per liters, soybean meal hydrolysate (preparation method is with embodiment 1) 200 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 30 grams per liters, lime carbonate 100 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.
The method of fermentative Production L-lactic acid of the present invention may further comprise the steps:
(1) slant culture: with embodiment 2;
(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 inoculation articulating 2,37 ℃ leave standstill cultivation 20 hours; Add the 400ml seed culture medium in 1 liter of triangular flask, the 30ml seed culture fluid is inserted the 400ml seed culture medium, 37 ℃ of static cultivations 20 hours; Add 2 liters of seed culture mediums in 5 liters of triangular flasks, the 400ml seed culture fluid is inserted 2 liters of seed culture mediums, 37 ℃ leave standstill cultivation 20 hours, make seed culture fluid;
(3) fermentation culture: Shanghai is protected and is added 18 liters of fermention mediums in 30 liters of fermentor tanks of emerging BIOTECH, in fermention medium, insert 2 liters of seed culture fluids, 42 ℃ leave standstill cultivation 48 hours, wherein, stirred once every 12 hours, 50 rev/mins of mixing speed, rotation radius are 42mm, churning time 30 minutes.3 repetitions are established in experiment altogether.
During fermentation ends, get fermented liquid, 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor, according to above-mentioned detection method, detect glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid, calculate L-lactic acid optical purity and transformation efficiency.The result shows that the concentration of glucose is 9 grams per liters ± 0.3 grams per liter, L-lactic acid concn 182 grams per liters ± 4 grams per liters, transformation efficiency 95.3% ± 0.4%, L-lactic acid optical purity 98.3% ± 0.3%.
Embodiment 14, utilize lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce the L-lactic acid 30 liters of fermentation cylinder for fermentation methods
Used substratum is composed as follows among this embodiment:
Slant medium and seed culture medium are with embodiment 2.
Fermention medium: cerelose (the sugared company limited of Zibo rainbow space industry) 200 grams per liters (fermentation starting point concentration), soybean meal hydrolysate (preparation method is with embodiment 1) 230 grams per liters, corn steep liquor (west, Shanghai king's Dian Fentang company limited) 25 grams per liters, lime carbonate 100 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.
The method of fermentative Production L-lactic acid of the present invention may further comprise the steps:
(1) slant culture: with embodiment 2;
(2) seed culture: with embodiment 13;
(3) fermentation culture: Shanghai is protected and is added 18 liters of fermention mediums in 30 liters of fermentor tanks of emerging BIOTECH, in fermention medium, insert 2 liters of seed culture fluids, 37 ℃ leave standstill cultivation 68 hours, wherein, stirred once every 12 hours, 100 rev/mins of mixing speed, rotation radius are 42mm, churning time 20 minutes.Wherein, added 1000 gram cereloses (the sugared company limited of Zibo rainbow space industry) and 500 gram lime carbonate at 48 hours.3 repetitions are established in experiment altogether.
During fermentation ends, get fermented liquid, 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor, according to above-mentioned detection method, detect glucose concn, L-lactic acid concn and D-lactic acid concn in the fermented liquid, calculate L-lactic acid optical purity and transformation efficiency.The result shows that the concentration of glucose is 15 grams per liters ± 0.6 grams per liter, L-lactic acid concn 226 grams per liters ± 3 grams per liters, transformation efficiency 96.3% ± 0.5%, L-lactic acid optical purity 98.0% ± 0.3%.
Claims (12)
1, lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL, its preserving number is CGMCC № 2183.
2, a kind of method of producing L-lactic acid is that fermentation lactobacillus rhamnosus (Lactobacillus rhamnosus) CASLCGMCC № 2183 obtains L-lactic acid.
3, method according to claim 2 is characterized in that: contain carbon source, nitrogenous source in the fermention medium of described lactobacillus rhamnosus (Lactobacillusrhamnosus) CASL CGMCC № 2183 and be used to control the neutralizing agent of fermented liquid pH; Carbon source in the described fermention medium is a glucose 150-200 grams per liter; Nitrogenous source in the described fermention medium is following 1) to 3) in any one:
1) soybean meal hydrolysate 100-300 grams per liter;
2) bean cake powder 20-30 grams per liter;
3) soybean meal hydrolysate 100-300 grams per liter, corn steep liquor 10-30 grams per liter.
4, method according to claim 3 is characterized in that: when the nitrogenous source in the described fermention medium was soybean meal hydrolysate, its consumption was 220 grams per liters.
5, method according to claim 3 is characterized in that: when the nitrogenous source in the described fermention medium is bean cake powder, also contain the proteolytic enzyme of 0.05-0.1 grams per liter in the described fermention medium.
6, method according to claim 3 is characterized in that: the neutralizing agent in the described fermention medium is a lime carbonate, and the concentration of described lime carbonate is the 75-100 grams per liter.
7, method according to claim 6 is characterized in that: the concentration of described lime carbonate is 90 grams per liters.
8, according to claim 2 or 3 described methods, it is characterized in that: the pH of described fermention medium is 5.5-7.
9, according to claim 2 or 3 described methods, it is characterized in that: described fermentation condition is 35-50 ℃ and cultivated 48-80 hour.
10, method according to claim 9 is characterized in that: described fermentation condition is 35-45 ℃ and cultivated 50-68 hour.
11, method according to claim 2, it is characterized in that: the training method of described fermentation adopts intermittent control shaking to cultivate in triangular flask, adopt intermittently stir culture in the fermentor tank, be 12 hours pitch time, the rotating speed of described stirring is 30-100 rev/min, rotation radius is 33 or 42mm, and churning time is 20-40 minute.
12, method according to claim 11 is characterized in that: described mixing speed is 50 rev/mins; Churning time is 30 minutes.
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| CN101805759B (en) * | 2010-04-21 | 2012-07-04 | 山东大学 | Method for producing L-lactic acid by taking cassava powder as material |
| 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 |
| CN101880696B (en) * | 2010-07-14 | 2013-04-17 | 华中科技大学 | Method for producing L-lactic acid by fermentation and bacterial strain using same |
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| CN102747112A (en) * | 2012-07-21 | 2012-10-24 | 太仓市茂通化建有限公司 | Method for producing L-lactic acid by fermentation of lactobacillus rhamnosus |
| CN102864186A (en) * | 2012-08-29 | 2013-01-09 | 太仓市茂通化建有限公司 | Method for fermenting L-lactic acid by utilizing lactic streptococci |
| CN104894171A (en) * | 2014-03-06 | 2015-09-09 | 广西科学院 | Method for producing (R,R)-2,3-butylene glycol by fermenting raw material cassava starch |
| CN104894170A (en) * | 2014-03-06 | 2015-09-09 | 广西科学院 | Method for preparing (R,R)-2,3-butylene glycol by using synchronous enzymatic hydrolysis and fermentation of soybean cake powder |
| CN103898028A (en) * | 2014-04-12 | 2014-07-02 | 东北农业大学 | High-density liquid-state cultivation method of feeding lactobacillus plantarum |
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Assignee: Yancheng Haijianuo Biological Engineering Co., Ltd. Assignor: Institute of Microbiology, Chinese Academy of Sciences Contract record no.: 2011320000489 Denomination of invention: Method for producing L-lactic acid and isoduicitol lactobacillus special for the same Granted publication date: 20091028 License type: Exclusive License Open date: 20080507 Record date: 20110401 |