CN100473720C - Method for producing L-lactic acid and coagulate bacillus cereus special for the same - Google Patents
Method for producing L-lactic acid and coagulate bacillus cereus special for the same Download PDFInfo
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Abstract
The invention discloses a production method of L-lactic acid and the special condensate bacillus CASH CGMCC No. 2184. L-lactic acid can be gotten by cultivating the special condensate bacillus CASH CGMCC No. 2184. Per litter of fermentation culture medium of the condensate bacillus comprises at least one of the following five nitrogen sources: yeast powder 5 to 15g, peptone 5 to 15g, soybean peptide 5 to 15g, soybean peptone 5 to 15g, and cottonseed protein 10 to 20g; in addition, the fermentation culture medium comprises glucose 80 to 170g (the content of initial glucose), calcium carbonate 50 to 100g and water; the pH of the fermentation culture medium is 5.5 to 7. With glucose as the substrate, under the condition of 45 to 60DEG C and with 98 to 99% of sugar acid conversion, the condensate bacillus CASH CGMCC No. 2184 efficiently ferments to produce L-lactic acid with optical purity of 99%; the concentration of the L-lactic acid maximum can attain 190 g/l and the volume production efficiency maximum can attain 5.7 g/l/h.
Description
Technical field
The present invention relates to a kind of method and coagulate bacillus cereus special 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, is widely used on food, cosmetic, pharmacy, weaving and the chemical industry.The poly(lactic acid) of being made by lactic acid has excellent biological compatibility and biodegradability, can be made into medical slow release material, Biodegradable fibers and biodegradable plastic etc., and is expected to replace in the near future petroleum-based plastics.In recent years, because the shortage day by day of petroleum resources and the enhancing gradually of people's environmental consciousness are also more and more around the research of poly(lactic acid), also more and more urgent as the demand of the monomeric lactic acid of poly(lactic acid).Lactic acid is a kind of chiral molecules, can be divided into L-lactic acid, D-lactic acid and DL-lactic acid.In the production process of poly(lactic acid), the physical properties of poly(lactic acid) and stability are influenced by the composition of L-lactic acid and D-lactic acid mainly, therefore need highly purified L-lactic acid and D-lactic acid in polyreaction.
The production method of lactic acid mainly contains three kinds of chemical synthesis, enzymic synthesis 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 because the used raw material of synthesis method is acetaldehyde and violent in toxicity prussic acid, synthesizing lactic acid is applied to food and can not be widely accepted, and its production cost is also higher in addition.Production by Enzymes can obtain to specificity optical purity lactic acid, but the production process more complicated is not widely used in 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 be that fermenting raw materials is produced lactic acid with renewable resourcess such as starch, Mierocrystalline celluloses, production cost is low, the Product Safety height is the main method of producing lactic acid.
Being successfully applied to lactic acid fermented microorganism at present mainly has two classes, and a class is the Rhizopus oryzae in the mould, and another kind of is Bacterium lacticum in the bacterium.It is aerobic fermentation that Rhizopus oryzae is produced lactic acid, and but Bacterium lacticum anaerobism or little aerobic fermentation are produced lactic acid, the common feature of this two classes bacterial strain is that the temperature of fermenting lactic acid is generally at 30～42 ℃, pollute DL-lactic-acid-producing strain (as plant lactobacillus) easily, therefore need sterilize to fermented liquid, cause the waste and the loss of nutritive components of energy.
Thermophilic genus bacillus is a kind of new microorganism that can be used for L-lactic fermentation production.Owing to have higher leavening temperature (45～60 ℃), significantly reduced the chance of pollution microbes in the fermenting process, improved the optical purity of L-lactic acid.Simultaneously, ferment with higher temperature, can reduce fermentation broth viscosity, help the operation of subsequent processing steps, can under the unsterilised condition of substratum, carry out fermentative production, reduced the production cost of energy consumption and L-lactic acid, and provided the foundation for producing lactic acid for the matrix simultaneous saccharification and fermentation with starch.
Once utilized thermophilic Bacillus coagulans (Bacillus coagulans DSM5196) to ferment in patent USP5079164, but the transformation efficiency of fermentation is lower, 19% first sugar only obtains 14% lactic acid.Patent USP7183088 B2 and CN 1498265 A mention and utilize an other bacillus coagulans (Bacillus coagulansSIM-7 DSM 14043) to carry out the L-lactic acid-producing, but the speed of producing acid is lower, fermented 98 hours, and produced acid 12.3%, transformation efficiency 95.5%.
Summary of the invention
An object of the present invention is to provide a strain and produce the Bacillus coagulans (Bacilluscoagulans) of L-lactic acid.
Bacillus coagulans provided by the present invention (Bacillus coagulans) bacterial strain CASH screens the soil near certain dairy factory of Mentougou District, Beijing and mutagenesis obtains, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 09 24th, 2007 and (be called for short CGMCC, the address is: the Datun Road, Chaoyang District, Beijing City), preservation registration number is CGMCC № 2184.
Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 Gram-positives, the elongated rod shape cell, cell can move, and forms the avette statospore of near-end.Its bacterium colony is circular, protruding, glossy, transparent, smooth surface, drying, diameter 2～3mm.It can utilize glucose, maltose, fructose, pectinose, dextrin and beta-cyclodextrin to produce not aerogenesis of acid, can not utilize wood sugar, lactose, raffinose, N.F,USP MANNITOL and sorbyl alcohol, can not utilize propionic salt, the catalase positive, can not gelatin hydrolysate, edwardsiella hoshinae can tolerate 0.02% trinitride, can not grow when the N,O-Diacetylmuramidase that has 0.01%, L-lactic acid is produced in the lactic fermentation of obligate homotype.Its 16S rDNA sequence (sequence 1 in the sequence table) reaches 99% with the 16S rDNA sequence alignment similarity of many bacillus coagulans.
Second purpose of the present invention provides a kind of method of utilizing Bacillus coagulans (Bacillus coagulans) CASHCGMCC № 2184 to produce L-lactic acid.
The method of production L-lactic acid provided by the present invention is that fermentation Bacillus coagulans (Bacilluscoagulans) CASH CGMCC № 2184 obtains L-lactic acid.
Can contain at least a in following five kinds of nitrogenous sources in every liter of the fermention medium of described Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184: yeast powder 5～15 grams, peptone 5～15 grams, soybean peptides 5～15 grams, soy peptone 5～15 grams, cottonseed protein 10～20 grams.Also contain in every liter of fermention medium in addition: glucose 80～170 restrains, and is used to regulate and control the neutralizing agent of fermented liquid pH, and surplus is a water; The pH of described fermention medium is 5.5～7.This fermention medium need not sterilized.
Wherein, described neutralizing agent is a lime carbonate, and every liter contains 50～100 grams in the described fermention medium.
The pH of described fermention medium specifically can be 5.5～6.5.
The fermentation condition of described Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 specifically can be 45 ℃～60 ℃ and cultivated 18～60 hours, cultivates 18～48 hours as 50～55 ℃.
In the described method, also Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 can be inserted earlier in the following seed culture medium, cultivate 6～8 hours, insert described fermention medium again at 45～60 ℃; Contain in every liter of the described seed culture medium: glucose 60～80 grams, yeast powder 10～15 grams, soy peptone 5～8 grams, lime carbonate 30～40 grams, surplus is a water; The pH of described seed culture medium is 5.5～6.
The training method of described fermentation specifically can be stir culture, and mixing speed is 200～300 rev/mins, and rotation radius is 33mm.
Bacillus coagulans of the present invention (Bacillus coagulans) CASH CGMCC № 2184 is a substrate with glucose, under 45～60 ℃ of conditions with the L-lactic acid of the efficient fermentative production optical purity of 98～99% glucose acid invert ratio more than 99%, wherein the L-lactic acid concn reaches as high as 190 grams per liters, volume production is most effective reach 5.7 grams per liters/hour.
The method of production of the present invention L-lactic acid is utilized the advantage of thermophilic fermentation process, and the probability of pollution microbes is little, can fermention medium not sterilized, and has reduced the consumption of energy and the loss of nutritive substance.
Description of drawings
Fig. 1 is the change curve of glucose and L-lactic acid in Bacillus coagulans (Bacillus coagulans) the CASH CGMCC № 2184 fermentation production of L-lactic acid processes.
Fig. 2 is Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 fermentation production of L-lactic acid gained fermented liquids and D-lactic acid and the color atlas of L-lactic acid standard substance under the liquid phase chiral column.A is L-lactic acid standard substance, and B is D-lactic acid standard substance, and C is a fermentation broth sample.
Embodiment
The sequence of steps that relates in Bacillus coagulans (Bacillus coagulans) the CASH CGMCC № 2184 fermentation production of L-lactic acid methods of utilizing of the present invention is as follows:
(1) slant culture: with Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 bacterial classification inoculations on the solid slant culture base of the agar that contains 1.5～2.0g/100ml, 45～60 ℃ of culture temperature, incubation time 8～12 hours;
(2) seed culture: the slant culture with step (1), under aseptic condition, be inoculated in the seed culture medium, 45～60 ℃ of culture temperature, training method is cultivated 120 rev/mins of rotating speeds, incubation time 6～8 hours for utilizing the Scroll-tupe shaking table;
(3) fermentation culture: the inoculum size of the volume ratio with 5～10%, insert fermention medium with seed culture fluid, 45～60 ℃ of culture temperature, training method is a stir culture, 200～300 rev/mins of mixing speed, incubation time 18～60 hours makes the fermentation culture that contains L-lactic acid;
(4) handle sample: get fermented liquid and be heated to 80～100 ℃ earlier, again 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor;
(5) sample detection: get the supernatant liquor dilution, detect glucose content and L-lactic acid, D-lactic acid content;
Wherein, the yeast culture temperature described in step (1), (2), (3) is preferably 50～55 ℃.
Wherein, preferably 9～10 hours yeast culture time described in the step (1).
Wherein, preferably 6～7 hours yeast culture time described in the step (2).
Wherein, preferably 18～48 hours yeast culture time described in the step (3).
Wherein, the adding of the yeast culture described in step (2), (3) lime carbonate control pH is 5.5～6.
Further illustrate the present invention below by embodiment.
Separation mutagenesis screening and the evaluation of embodiment 1, Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184
1, separates mutagenesis screening
Used substratum is composed as follows among this embodiment:
Nutrient broth: glucose 150 grams per liters, yeast powder 10 grams per liters, lime carbonate 50 grams per liters, pH are 6.
Nutrient agar: glucose 50 grams per liters, yeast powder 10 grams per liters, lime carbonate 20 grams per liters, agar powder 10 grams per liters, pH are 6.
Fermention medium: glucose 150 grams per liters, yeast powder 20 grams per liters, lime carbonate 90 grams per liters, pH are 6.
Nutrient broth medium: glucose 50 grams per liters, yeast powder 10 grams per liters, lime carbonate 10 grams per liters, pH are 6.
The specific operation process of this embodiment is as follows:
Obtain soil sample near certain dairy factory of Mentougou District, Beijing, per 2 gram soil samples are put into the 50ml Nutrient broth, 55 ℃ of enrichment culture 6～10 hours.Dilution is applied in the culture dish that contains nutrient agar then, cultivates 24 hours for 55 ℃.After waiting to grow single bacterium colony, choosing colony area and the big bacterium colony of transparent circle area are inoculated in the fermention medium, and 55 ℃ leave standstill cultivation 48 hours, measure the output of L-lactic acid, select the highest bacterial strain of output, are used for next step ion beam mutagenesis.
The bacterial strain of tentatively selecting is streak culture on the nutrient agar medium culture dish, choose single bacterium colony, be inoculated in the nutrient broth medium, 55 ℃ leave standstill and are cultured to mid-log phase, suspend with physiological saline washing back, make bacteria suspension.Draw 100 μ L 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, inserts respectively to contain in the Nutrient broth of 5% (5g/100ml) L-Sodium.alpha.-hydroxypropionate, and 55 ℃ leave standstill cultivation 24 hours, and having only implantation dosage is 10 * 10
14Ions/cm
2Nutrient solution occur muddyly, dilute this nutrient solution, be coated in the nutrient agar medium culture dish, after waiting to grow single bacterium colony, choosing colony area and the big bacterium colony of transparent circle area are inoculated in the fermention medium, 55 ℃ leave standstill cultivation 48 hours, measure the output of L-lactic acid, select the highest bacterial strain of output.Finally obtain the highest strain bacillus coagulans of output (Bacillus coagulans) CASH CGMCC № 2184.
2, the evaluation of bacterial strain
According to the authentication method that " Bai Jie system identification handbook (the 8th edition) " and " common bacteria system identification handbook " (Science Press) provides this bacterial strain is identified, qualification result shows that Bacillus coagulans (Bacilluscoagulans) CASH CGMCC № 2184 is Gram-positive, the elongated rod shape cell, cell can move, and forms the avette statospore of near-end.Its bacterium colony is circular, protruding, glossy, transparent, smooth surface, drying, diameter 2～3mm.It can utilize glucose, maltose, fructose, pectinose, dextrin and beta-cyclodextrin to produce not aerogenesis of acid, can not utilize wood sugar, lactose, raffinose, N.F,USP MANNITOL and sorbyl alcohol, can not utilize propionic salt, the catalase positive, can not gelatin hydrolysate, edwardsiella hoshinae can tolerate 0.02% trinitride, can not grow when the N,O-Diacetylmuramidase that has 0.01%, L-lactic acid is produced in the lactic fermentation of obligate homotype.Its 16S rDNA sequence (sequence 1 in the sequence table) reaches 99% with the 16S rDNA sequence alignment similarity of many bacillus coagulans.
Based on above feature, this strain L-lactic-acid-producing strain is accredited as the lactobacillus that condenses (Bacilluscoagulans), and it is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCC № 2184.
Embodiment 2, utilize Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 fermentation production of 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, 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 70 grams per liters, yeast powder 10 grams per liters, soy peptone 5 grams per liters, lime carbonate 30 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.
Fermention medium: glucose (the sugared company limited of Zibo rainbow space industry) 170 grams per liters, yeast powder (Beijing extensive and profound in meaning star biotechnology responsibility company limited, cat. no 01-013) 15 grams per liters, lime carbonate 80 grams per liters.The initial pH of this fermention medium is 6.5.Unsterilised.
The method of this fermentation production of L-lactic acid may further comprise the steps:
(1) slant culture: with Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 bacterial classification inoculations on slant medium, under 50 ℃ of conditions, static cultivation 10 hours;
(2) seed culture: with the bacterial strain of step (1) cultivation, encircle in the 100ml triangular flask that the 30ml seed culture medium is housed with inoculation articulating 1 under aseptic condition, under 50 ℃ of conditions, shaking table was cultivated 6 hours, 120 rev/mins of rotating speeds (13mm rotation radius) make seed liquor;
(3) fermentation culture: the seed that inserts 10ml step (2) in the 300ml triangular flask of 100ml fermention medium is housed is supported liquid, and under 50 ℃ of conditions, shaking table was cultivated 39 hours, finished fermentation, and the rotating speed of this shaking table is 120 rev/mins (13mm rotation radius).50 repetitions are established in experiment altogether.Got fermented liquid in per 3 hours, be heated to 80～100 ℃ earlier, again 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor and detect L-lactic acid concn, D-lactic acid concn, glucose concn in the fermented liquid, calculate glucose acid invert ratio, volume production efficient and L-lactic acid optical purity.
Wherein, the determination of glucose method is that fermented liquid dilution back is centrifugal, adopts bio-sensing analyser SBA-40C (Shandong Scientific Research Academy) to measure.
The measuring method of L-lactic acid and D-lactic acid content is to adopt Agilent 1100 liquid chromatographs, outfit chiral separation post (separation of optics allosome is used for Mitsubishi chemical company, MCI GEL-CRS10 W (3 μ) 4.6 ID * 50mm).The concrete operations condition is: 0.002mol/L copper sulfate is as moving phase, flow 0.5ml/min, and sample size 20 μ L, UV-detector detects wavelength 254nm, 25 ℃ of service temperatures.Utilize L-lactic acid and D-lactic acid standard substance to make typical curve, calculate the content of L-lactic acid and D-lactic acid in the fermented liquid again according to typical curve.
Among the present invention, be the product of German Sigma-Aldrich company as the D-lactic acid of standard substance, its article No. is L0625-25MG; As the L-lactic acid of standard substance is the product of German Sigma-Aldrich company, and its article No. is L1750-10G.Under as above chromatographic condition, D-lactic acid retention time is 10.150 minutes, and L-lactic acid retention time is 12.293 minutes.The color atlas of L-lactic acid standard substance, D-lactic acid standard substance and fermented liquid is seen accompanying drawing 2.
Optical purity (optical purity) be weigh in the opticity sample enantiomorph surpass another enantiomorph amount measure its available enantiomeric excess value (enantiomeric excess) expression.The optical purity of L-lactic acid is calculated as follows among the present invention: (L-lactic acid content-D-lactic acid content) ÷ (L-lactic acid content+D-lactic acid content) * 100%.
Glucose acid invert ratio is defined as: the consumption (grams per liter) * 100% of L-lactic acid production (grams per liter) ÷ glucose.
The volume production definitions of efficiency is: L-lactic acid production (grams per liter) ÷ fermentation time (hour).
After the fermentation ends, the concentration of glucose is 2.3 grams per liters ± 0.5 grams per liter (mean+SD), L-lactic acid concn 165.1 grams per liters ± 1.0 grams per liters (mean+SD), glucose acid invert ratio 98.5% ± 0.6% (mean+SD), volume production efficient 4.2 grams per liters/hour ± 0.3 grams per liter/hour (mean+SD), L-lactic acid optical purity 99.3% ± 0.3% (mean+SD).
The culture temperature of Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 is 55 ℃.Consisting of of fermention medium: glucose (the sugared company limited of Zibo rainbow space industry) 118 grams per liters, soy peptone (Beijing extensive and profound in meaning star biotechnology responsibility company limited, cat. no 01-004) 15 grams per liters, lime carbonate 80 grams per liters.The initial pH of this fermention medium is 5.5.Fermentation time is 24 hours.Other method is all identical with embodiment 2.
Fermentation ends, the concentration of glucose is 3.2 grams per liters ± 1.1 grams per liters (mean+SD), L-lactic acid concn 113.5 grams per liters ± 1.9 grams per liters (mean+SD), glucose acid invert ratio 98.9% ± 0.3% (mean+SD), volume production efficient 4.7 grams per liters/hour ± 0.2 grams per liter/hour (mean+SD), L-lactic acid optical purity 99.3% ± 0.2% (mean+SD).
Embodiment 4, utilize Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 fermentation production of L-lactic acid in triangular flask
The culture temperature of Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 is 60 ℃.Consisting of of fermention medium: glucose (the sugared company limited of Zibo rainbow space industry) 83 grams per liters, soybean peptides (Harbin Leneng Biological Engineering Co., Ltd., happy energy soybean protein peptide 3# powder) 15 grams per liters, lime carbonate 80 grams per liters.The initial pH of this fermention medium is 5.5.Fermentation time is 24 hours.Other method is all identical with embodiment 2.
Fermentation ends, the concentration of glucose is 2.1 grams per liters ± 0.5 grams per liter (mean+SD), L-lactic acid concn 79.0 grams per liters ± 1.5 grams per liters (mean+SD), glucose acid invert ratio 98.8% ± 0.3% (mean+SD), volume production efficient 3.3 grams per liters/hour ± 0.2 grams per liter/hour (mean+SD), L-lactic acid optical purity 99.1% ± 0.1% (mean+SD).
Embodiment 5, utilize Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 fermentation production of L-lactic acid in triangular flask
The culture temperature of Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 is 45 ℃.Consisting of of fermention medium: glucose (the sugared company limited of Zibo rainbow space industry) 120 grams per liters, cottonseed protein (Beijing Kang Mingwei substratum technology limited liability company) 20 grams per liters, lime carbonate 80 grams per liters.The initial pH of this fermention medium is 6.Fermentation time is 27 hours.Other method is all identical with embodiment 2.
Fermentation ends, the concentration of glucose is 2.5 grams per liters ± 0.6 grams per liter (mean+SD), L-lactic acid concn 116.0 grams per liters ± 1.5 grams per liters (mean+SD), glucose acid invert ratio 98.7% ± 0.4% (mean+SD), volume production efficient 4.3 grams per liters/hour ± 0.3 grams per liter/hour (mean+SD), L-lactic acid optical purity 99.2% ± 0.2% (mean+SD).
Consisting of of the fermention medium of Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184: glucose (the sugared company limited of Zibo rainbow space industry) 118 grams per liters, peptone (Beijing extensive and profound in meaning star biotechnology responsibility company limited, cat. no 01-001-2) 15 grams per liters, lime carbonate 80 grams per liters.The initial pH of this fermention medium is 7.Fermentation time is 28 hours.Other method is all identical with embodiment 2.
Fermentation ends, the concentration of glucose is 2.7 grams per liters ± 0.3 grams per liter (mean+SD), L-lactic acid concn 113.5 grams per liters ± 1.7 grams per liters (mean+SD), glucose acid invert ratio 98.5% ± 0.4% (mean+SD), volume production efficient 4.1 grams per liters/hour ± 0.2 grams per liter/hour (mean+SD), L-lactic acid optical purity 99.3% ± 0.2% (mean+SD).
Embodiment 7, utilize Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 to produce the L-lactic acid 5 liters of fermentation cylinder for fermentation
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, 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 70 grams per liters, yeast powder 10 grams per liters, soy peptone 5 grams per liters, lime carbonate 30 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.
Fermention medium: glucose (the sugared company limited of Zibo rainbow space industry) 125 grams per liters, yeast powder (Beijing extensive and profound in meaning star biotechnology responsibility company limited, cat. no 01-013) 9.8 grams per liters, soy peptone (Beijing extensive and profound in meaning star biotechnology responsibility company limited, cat. no 01-004) 5 grams per liters, lime carbonate 100 grams per liters.The initial pH of this fermention medium is 6.5.Unsterilised.
The method of this fermentation production of L-lactic acid may further comprise the steps:
(1) slant culture: with Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 bacterial classification inoculations on slant medium, under 50 ℃ of conditions, static cultivation 10 hours;
(2) seed culture: with the bacterial strain of step (1) cultivation, encircle in the 300ml triangular flask that the 100ml seed culture medium is housed with inoculation articulating 2 under aseptic condition, under 50 ℃ of conditions, shaking table was cultivated 6 hours, 120 rev/mins of rotating speeds (13mm rotation radius) make seed liquor;
(3) fermentation culture: (BIOSTAT B B.Braun) adds 3 liters of fermention mediums in 5 liters of fermentor tanks to German Bei Lang.In fermention medium, insert the 300ml seed culture fluid, 200 rev/mins of fermentor tank mixing speed (33mm rotation radius), 50 ℃ of condition bottom fermentations 21 hours.10 repetitions are established in experiment altogether.Got fermented liquid in per 3 hours according to the method for embodiment 2, be heated to 80～100 ℃ earlier, again 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor and detect L-lactic acid concn, D-lactic acid concn, glucose concn in the fermented liquid, calculate glucose acid invert ratio, volume production efficient and L-lactic acid optical purity.
After the fermentation ends, the concentration of glucose is 3.4 grams per liters ± 0.6 grams per liter (mean+SD), L-lactic acid concn 120 grams per liters ± 1.8 grams per liters (mean+SD), glucose acid invert ratio 99.0% ± 0.5% (mean+SD), volume production efficient 5.7 grams per liters/hour ± 0.4 grams per liter/hour (mean+SD), L-lactic acid optical purity 99.2% ± 0.1% (mean+SD).
Embodiment 8, utilize Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 to produce the L-lactic acid 5 liters of fermentation cylinder for fermentation
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, 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 70 grams per liters, yeast powder 10 grams per liters, soy peptone 5 grams per liters, lime carbonate 30 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.
Fermentation initial medium: glucose (the sugared company limited of Zibo rainbow space industry) 122 grams per liters, peptone (Beijing extensive and profound in meaning star biotechnology responsibility company limited, cat. no 01-001-2) 5 grams per liters, soybean peptides (Harbin Leneng Biological Engineering Co., Ltd., happy energy soybean protein peptide 3# powder) 12.8 grams per liters, lime carbonate 100 grams per liters.The initial pH of this fermention medium is 6.5.Unsterilised.
The method of this fermentation production of L-lactic acid may further comprise the steps:
(1) slant culture: with Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 bacterial classification inoculations on slant medium, under 55 ℃ of conditions, static cultivation 10 hours;
(2) seed culture: with the bacterial strain of step (1) cultivation, encircle in the 300ml triangular flask that the 100ml seed culture medium is housed with inoculation articulating 2 under aseptic condition, under 55 ℃ of conditions, shaking table was cultivated 6 hours, 120 rev/mins of rotating speeds (13mm rotation radius) make seed liquor;
(3) fermentation culture: (BIOSTAT B B.Braun) adds 3 liters of fermentation initial mediums in 5 liters of fermentor tanks to German Bei Lang.Insert the 300ml seed culture fluid in fermention medium, 200 rev/mins of fermentor tank mixing speed (33mm rotation radius) fermented 39 hours under 55 ℃ of conditions altogether.Wherein, ferment (after testing by 15 hours, the concentration of glucose is 30 grams per liters in the fermented liquid at this moment), add glucose according to the amounts of adding 42 gram glucose in every liter of fermented liquid, promptly add 126 gram glucose (the sugared company limited of Zibo rainbow space industry) in each fermentor tank again.10 repetitions are established in experiment altogether.Method according to embodiment 2 is got fermented liquid, be heated to 80～100 ℃ earlier, again 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor and detect L-lactic acid concn, D-lactic acid concn, glucose concn in the fermented liquid, calculate glucose acid invert ratio, volume production efficient and L-lactic acid optical purity.
After the result shows fermentation ends, the concentration of glucose is 4.6 grams per liters ± 0.7 grams per liter (mean+SD), L-lactic acid concn 160 grams per liters ± 3.2 grams per liters (mean+SD), glucose acid invert ratio 98.5% ± 0.4% (mean+SD), volume production efficient 4.1 grams per liters/hour ± 0.3 grams per liter/hour (mean+SD), L-lactic acid optical purity 99.2% ± 0.2% (mean+SD).
The culture temperature of Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 is 60 ℃, the prescription of fermention medium is: glucose (the sugared company limited of Zibo rainbow space industry) 83 grams per liters, yeast powder (Beijing extensive and profound in meaning star biotechnology responsibility company limited, cat. no 01-013) 10 grams per liters, soybean peptides (Harbin Leneng Biological Engineering Co., Ltd., happy energy soybean protein peptide 3# powder) 5 grams per liters, lime carbonate 80 grams per liters.The initial pH of this fermention medium is 7.Fermentation time is 18 hours.Other method is all identical with embodiment 7.
After the result shows fermentation ends, the concentration of glucose is 3.2 grams per liters ± 0.4 grams per liter (mean+SD), the L-lactic acid concn is 78.5 grams per liters ± 0.9 grams per liter (mean+SD), glucose acid invert ratio is 98.4% ± 0.3% (mean+SD), volume production efficient 4.4 grams per liters/hour ± 0.1 grams per liter/hour (mean+SD), L-lactic acid optical purity is 99.2% ± 0.1% (mean+SD).
Embodiment 10, utilize Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 to produce the L-lactic acid 5 liters of fermentation cylinder for fermentation
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, 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 70 grams per liters, yeast powder 10 grams per liters, soy peptone 5 grams per liters, lime carbonate 30 grams per liters.The initial pH of this substratum is 6.5.Sterilization is 20 minutes under 115 ℃ of conditions.
Fermentation initial medium: glucose (the sugared company limited of Zibo rainbow space industry) 122 grams per liters, yeast powder (Beijing extensive and profound in meaning star biotechnology responsibility company limited, cat. no 01-013) 5 grams per liters, cottonseed protein (Beijing Kang Mingwei substratum technology limited liability company) 20 grams per liters, lime carbonate 100 grams per liters.The initial pH of this fermention medium is 6.5.Unsterilised.
The method of this fermentation production of L-lactic acid may further comprise the steps:
(1) slant culture: with Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 bacterial classification inoculations on slant medium, under 55 ℃ of conditions, static cultivation 10 hours;
(2) seed culture: with the bacterial strain of step (1) cultivation, encircle in the 300ml triangular flask that the 100ml seed culture medium is housed with inoculation articulating 2 under aseptic condition, under 55 ℃ of conditions, shaking table was cultivated 6 hours, 120 rev/mins of rotating speeds (13mm rotation radius) make seed liquor;
(3) fermentation culture: (BIOSTAT B B.Braun) adds 3 liters of fermentation initial mediums in 5 liters of fermentor tanks to German Bei Lang.Insert the 300ml seed culture fluid in fermention medium, 300 rev/mins of fermentor tank mixing speed (33mm rotation radius) fermented 48 hours under 55 ℃ of conditions altogether.Wherein, ferment (after testing by 18 hours, the concentration of glucose is 16 grams per liters in the fermented liquid at this moment), add glucose, add yeast powder, add cottonseed protein and add lime carbonate according to the amounts of adding 76 gram glucose in every liter of fermented liquid according to the amounts of adding 30 gram lime carbonate in every liter of fermented liquid according to the amounts of adding 6 gram cottonseed proteins in every liter of fermented liquid according to the amounts of adding 3 gram yeast powders in every liter of fermented liquid; Be to add 228 gram glucose (the sugared company limited of Zibo rainbow space industry), 9 gram yeast powders (Beijing extensive and profound in meaning star biotechnology responsibility company limited, cat. no 01-013), 18 gram cottonseed proteins (Beijing Kang Mingwei substratum technology limited liability company) and 90 gram lime carbonate in each fermentor tank again.10 repetitions are established in experiment altogether.Method according to embodiment 2 is got fermented liquid, be heated to 80～100 ℃ earlier, again 6,000 rev/mins centrifugal 5 minutes, get supernatant liquor and detect L-lactic acid concn, D-lactic acid concn, glucose concn in the fermented liquid, calculate glucose acid invert ratio, volume production efficient and L-lactic acid optical purity.
The change curve that the result shows glucose and L-lactic acid in Bacillus coagulans (Bacillus coagulans) the CASH CGMCC № 2184 fermentation production of L-lactic acid processes shows the L-lactic acid that can be converted into high density by feed supplement glucose rapidly as shown in Figure 1.After the fermentation ends, the concentration of glucose is 5.0 grams per liters ± 0.8 grams per liter (mean+SD), L-lactic acid concn 190 grams per liters ± 5.2 grams per liters (mean+SD), glucose acid invert ratio 98.2% ± 0.3% (mean+SD), volume production efficient 4.0 grams per liters/hour ± 0.4 grams per liter/hour (mean+SD), L-lactic acid optical purity 99.5% ± 0.2% (mean+SD).
Sequence table
<160>1
<210>1
<211>612
<212>DNA
＜213〉Bacillus coagulans (Bacillus coagulans)
<400>1
Claims (10)
1, Bacillus coagulans (Bacillus coagulans) CASH, its preserving number is CGMCC № 2184.
2, a kind of method of producing L-lactic acid is that fermentation Bacillus coagulans (Bacillus coagulans) CASHCGMCC № 2184 obtains L-lactic acid.
3, method according to claim 2, it is characterized in that: contain at least a in following five kinds of nitrogenous sources in every liter of the fermention medium of described Bacillus coagulans (Bacilluscoagulans) CASH CGMCC № 2184: yeast powder 5～15 grams, peptone 5～15 grams, soybean peptides 5～15 grams, soy peptone 5～15 grams, cottonseed protein 10～20 grams; Also contain in every liter of the described fermention medium: glucose 80～170 grams, be used to regulate and control the neutralizing agent of fermented liquid pH, surplus is a water; The pH of described fermention medium is 5.5～7.
4, method according to claim 3 is characterized in that: described neutralizing agent is a lime carbonate, and every liter contains 50～100 grams in the described fermention medium.
5, according to claim 3 or 4 described methods, it is characterized in that: described fermention medium is unsterilised.
6, according to claim 3 or 4 described methods, it is characterized in that: the fermentation condition of described Bacillus coagulans (Bacilluscoagulans) CASH CGMCC № 2184 is 45 ℃～60 ℃ and cultivated 18～60 hours.
7, method according to claim 6 is characterized in that: described culture temperature is 50～55 ℃.
8, method according to claim 6 is characterized in that: described incubation time is 18～48 hours.
9, according to claim 3 or 4 described methods, it is characterized in that: in the described method, Bacillus coagulans (Bacillus coagulans) CASH CGMCC № 2184 is inserted earlier in the following seed culture medium, cultivated 6～8 hours, insert described fermention medium again at 45～60 ℃; Contain in every liter of the described seed culture medium: glucose 60～80 grams, yeast powder 10～15 grams, soy peptone 5～8 grams, lime carbonate 30～40 grams, surplus is a water; The pH of described seed culture medium is 5.5～6.
10, according to claim 3 or 4 described methods, it is characterized in that: the training method of described fermentation is a stir culture, and mixing speed is 200～300 rev/mins, and rotation radius is 33mm.
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| CN101503708B (en) * | 2009-03-05 | 2012-04-11 | 天津科技大学 | Cultivation fermentation method of Bacillus coagulans antimycotics active substance |
| CN101805757A (en) * | 2010-03-24 | 2010-08-18 | 天津工业生物技术研究所 | Method for producing optical pure L-lactic acid by open type whole-cell recovery cyclic fermentation |
| CN101792727B (en) * | 2010-04-02 | 2012-05-30 | 上海交通大学 | Bacillus coagulans and application thereof in L-sodium lactate preparation |
| CN101914465B (en) * | 2010-05-20 | 2012-10-03 | 上海交通大学 | Bacillus coagulans for preparing L-lactic acid and application method thereof |
| CN102174600B (en) * | 2010-12-31 | 2013-04-17 | 安徽丰原发酵技术工程研究有限公司 | Method for producing L-lactic acid through continuous fermentation |
| CN102382783B (en) * | 2011-10-17 | 2016-09-21 | 天津科技大学 | A kind of Bacillus coagulans and fermentation liquid thereof and fermentation process and fermentation liquid are as the application of biological pesticide |
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| 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 |
| CN102839140B (en) * | 2012-09-07 | 2013-07-17 | 吉林中粮生化科技有限公司 | L-lactic acid producing strain separated and screened out of corn soaking water |
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