CN104673691A - Novel lactobacillus plantarum for high-yield production of lactic acid by efficiently utilizing biomass material - Google Patents
Novel lactobacillus plantarum for high-yield production of lactic acid by efficiently utilizing biomass material Download PDFInfo
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- CN104673691A CN104673691A CN201310625005.9A CN201310625005A CN104673691A CN 104673691 A CN104673691 A CN 104673691A CN 201310625005 A CN201310625005 A CN 201310625005A CN 104673691 A CN104673691 A CN 104673691A
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- lactic acid
- biomass material
- lactobacillus plantarum
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- efficiency utilization
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
Abstract
The invention discloses novel lactobacillus plantarum (Lactobacillus plantarum) Li-2013-01 for high-yield production of lactic acid by efficiently utilizing a biomass material. The preservation number of the novel lactobacillus plantarum is CGMCC No.7928. A culture medium of a fermentation tank of the novel lactobacillus plantarum comprises the following components: 12g of a beef extract, 60g of peptone, 30g of a yeast extract, 120g of a carbon source, 30g of sodium acetate, 12g of ammonium citrate, 12g of dipotassium phosphate, 1.2g of magnesium sulfate heptahydrate and 0.3g of manganese sulfate heptahydrate, wherein the components are dissolved one by one and are added with tap water to regulate the volume to 6L, and then the pH is regulated to 7.0 to 7.2. The strain can be used for efficiently utilizing various biomass materials such as common starch raw materials, monosaccharide, disaccharide and hexose as well as pentose, and is rapid in metabolism, high in generated lactic acid concentration, high in transformation rate, few in impurity acids and high in hereditary stability.
Description
Technical field:
The invention belongs to microorganism field, relate to milk-acid bacteria.
Background technology:
Along with the shortage of fossil energy and the day by day serious of white pollution, bio-based macromolecular material more and more draws attention.Poly(lactic acid) be a kind of can the important degradable biological based high molecular material of Some substitute fossil level plastics.It take lactic acid as the high molecular polymer of monomer synthesize, have boundless application prospect, international and domestic at present had numerous scientific research institution and company to utilize poly(lactic acid) to develop the multiple products such as degradable plastic film, cutlery box, cup, operating sutures, CD, computer shell.
But because the production cost of lactic acid remains high, the extensive application of poly(lactic acid) is severely limited.In lactic acid-producing cost, most is its carbon source cost, the carbon source of current production lactic acid mainly adopts the grain raw material such as W-Gum, tapioca (flour), even if the starchy material in employing agricultural wastes, its production cost still can not be equal to fossil base plastics mutually.
The cellulose base wastes such as maize straw, straw and wood chip Comparatively speaking its price then can reduce greatly.But current Main Bottleneck is that most milk-acid bacteria can only utilize the hexose in these raw materials, and five-carbon sugar almost can not utilize completely.The residual waste both having caused resource of a large amount of five-carbon sugar in fermented liquid, brings certain pressure also to the process of fermentation wastes.Therefore, the excavation of five-carbon sugar metabolism bacterium has great importance for the large-scale application of poly(lactic acid).
Summary of the invention:
Technical problem to be solved by this invention is to provide a strain can the novel plant lactobacterium strain of efficiency utilization biomass material high-yield lactic acid.
Bacterium lacticum provided by the present invention is plant lactobacillus (Lactobacillusplantarum) Li-2013-01, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.7928, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.Preservation date on July 15th, 2013.This bacterial strain feature is as follows: examine under a microscope, and this bacterial strain is rod-short, and gramstaining is positive, and atrichia does not produce gemma; On solid medium, this bacterium bacterium colony is white, and smooth surface is fine and close, and form is circular, and edge is more neat.Physicochemical characteristics is: catalase (-), gelatine liquefication (-), indoles experiment (+), mobility (-), fermentation gas (-), nitrate reductase (-), and fermentation gas (-) produces hydrogen sulfide (-), grows (+) in pH4.5MRS substratum.
Plant lactobacillus of the present invention adopts following flow process to carry out seed selection:
The original bacterial classification that sets out → test tube activation → ethyl sulfate (DES) mutagenesis → dull and stereotyped primary dcreening operation → nitrosoguanidine (NTG) mutagenesis → dull and stereotyped primary dcreening operation → shaking flask sieves → mitotic stability test again.
The original bacterial classification that sets out is CICC20242, is purchased from Chinese industrial Microbiological Culture Collection administrative center.
Original strain of the present invention is in xylan substratum, and the output of lactic acid is 12.5g/L.In order to improve its lactic acid production, DES and NTG is adopted to carry out mutagenesis to this bacterial classification successively, mutagenesis adopts MRS calcium carbonate flat board to carry out primary dcreening operation, then 500mL shake flask fermentation is adopted, biosensor analysis instrument carries out multiple sieve to Producing Strain, the lactobacterium plantarum strain that seed selection is excellent, then does passage assays, evaluates its genetic stability.
Bacterial strain CGMCCNo.7928 genetic stability result shows: through continuous passage ten times, property indices is all more stable, and heredity is better, and proterties is not replied, therefore using the object bacterial strain that bacterial strain CGMCCNo.7928 obtains as seed selection.
Object bacterial strain CGMCCNo.7928 is done the experiment of 10L fermentor tank, result shows: after fermentation 72h, take xylan as carbon source, the lactic acid concn of plant lactobacillus CGMCCNo.7928 can reach 57g/L, improves 356% compared with starting strain.
Object bacterial strain CGMCCNo.7928 is done the experiment of 10L fermentor tank, result shows: after fermentation 72h, take glucose as carbon source, the lactic acid concn of plant lactobacillus CGMCCNo.7928 can reach 68g/L.
Described fermentation tank culture medium consists of: extractum carnis 12g, peptone 60g, yeast extract paste 30g, carbon source 120g, sodium acetate 30g, ammonium citrate 12g, dipotassium hydrogen phosphate 12g, magnesium sulfate heptahydrate 1.2g, seven water manganous sulfate 0.3g, after dissolving one by one, tap water is settled to 6L, regulates pH7.0-7.2.
Detailed process is as follows:
Substratum:
Liquid MRS xylan substratum (extractum carnis 2g, peptone 10g, yeast extract paste 5g, xylan 20g, sodium acetate 5g, ammonium citrate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.2g, seven water manganous sulfate 0.05g, after dissolving one by one, tap water constant volume 1000mL, regulates pH7.0-7.2); MRS xylan screening solid medium (extractum carnis 2g, peptone 10g, yeast extract paste 5g, xylan 90g, sodium acetate 5g, ammonium citrate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.2g, seven water manganous sulfate 0.05g, after dissolving one by one, tap water constant volume 1000mL, regulate pH7.0-7.2, add 20g agar);
1. ethyl sulfate (DES) mutagenic and breeding
1) on super clean bench, get plant lactobacillus one ring on test tube slant, access is equipped with in the 250mL triangular flask of 50mL liquid MRS xylan substratum, 200rpm, cultivates about 12h for 40 DEG C, makes thalline be in logarithmic growth in earlier stage.
2) get 5mL bacterium liquid, the centrifugal 10min of 5000rpm collects thalline, with brine 2 times.
3) 107/mL bacteria suspension is diluted to pH7.0 phosphoric acid buffer.
4) get the potassium phosphate buffer of 32mLpH7.0,8mL bacteria suspension, 150mL triangular flask that 0.4mLDES to put into rotor in advance fully mix, make DES ultimate density be 1%(v/v).
5) in 30 DEG C of shaking tables, 150rpm reacts 30min, gets 1mL mixed solution, adds 0.5mL25%Na2S2O3 solution stopped reaction.
6) dilution spread screens in solid medium plate in the MRS xylan containing 90g/L xylan.The bacterial strain that after cultivating 2 ~ 3 days at 40 DEG C, picking transparent circle/colony diameter is maximum, label is DES bacterium.
2. nitrosoguanidine mutagenesis
1) on super clean bench, get plant lactobacillus DES mono-ring on test tube slant, access is equipped with in the 250mL triangular flask of 50mL liquid MRS xylan substratum, 200rpm, cultivates about 12h for 40 DEG C, makes thalline be in logarithmic growth in earlier stage.
2) get the centrifugal 10min of 5mL bacterium liquid 5000rpm and collect thalline, with brine 2 times.
3) 107/mL bacteria suspension is diluted to pH6.0 phosphoric acid buffer.
4) get 10mL bacteria suspension to be transferred in 100mL triangular flask, add the NTG of 10mg, be mixed with the NTG solution that final concentration is 10mg/mL, and add 4-5 and drip acetone, be beneficial to NTG and dissolve.
5) at 30 DEG C, the centrifugal 10min of 200rpm oscillatory reaction 30min, 5000rpm collects thalline, with stroke-physiological saline solution washing several, and stopped reaction.
6) suitably dilute, get last dilution bacterium liquid 0.2mL, coat in the MRS xylan screening solid medium plate containing 90g/L xylan.The bacterial strain 150 that after cultivating 2 ~ 3 days at 40 DEG C, picking transparent circle/colony diameter is larger.
3. shaking flask is sieved again
1) on super clean bench, get plant lactobacillus one ring on each test tube slant respectively, access is equipped with in the 250mL triangular flask of 50mL liquid MRS xylan substratum, 200rpm, cultivates 3-4 days, detects xylan concentration and Pfansteihl change in concentration every day for 40 DEG C.After fermentation ends, compare the xylan wear rate of 150 strain bacterial classifications and lactic acid and produce speed, the transformation efficiency of lactic acid and heteroacid content.
2) selection xylan metabolic rate is fast, lactic acid concn is high, transformation efficiency is high and the poor bacterial classification of heteroacid is final bacterial classification, called after Li bacterium.
4. genetic stability test
Li-2013-01 bacterial strain is gone down to posterity for continuous ten times on inclined-plane, and detects the fermentation situation after at every turn going down to posterity by the method that shaking flask is sieved again.Experiment finds, inclined-plane goes down to posterity for continuous ten times, and this bacterial classification proterties does not have considerable change, and property indices is all normal, illustrates that the genetic stability of this bacterial classification is stronger.
Beneficial effect:
This bacterial strain can the various biomass material of efficiency utilization, not only can utilize common starchy material, monosaccharide and disaccharide, hexose etc., and can utilize five-carbon sugar, and metabolic rate is fast, and lactic acid producing concentration is high, and transformation efficiency is high, and heteroacid content is few.Take xylan as carbon source, after fermentation 72h, lactic acid concn can reach 57g/L, improves 356% compared with starting strain.Bacterial strain goes down to posterity for continuous ten times on inclined-plane, and detects the fermentation situation after at every turn going down to posterity by the method that shaking flask is sieved again.Found that, inclined-plane goes down to posterity for continuous ten times, and this bacterial classification proterties does not have considerable change, and property indices is all normal, illustrates that the genetic stability of this bacterial classification is stronger.
Embodiment:
Embodiment 1
This bacterial strain CGMCCNo.7928 is done the experiment of 10L fermentor tank, be carbon source with sucrose, result shows: after fermentation 72h, lactic acid concn can reach 60g/L.
Fermentation tank culture medium is: extractum carnis 12g, peptone 60g, yeast extract paste 30g, sucrose 120g, sodium acetate 30g, ammonium citrate 12g, dipotassium hydrogen phosphate 12g, magnesium sulfate heptahydrate 1.2g, seven water manganous sulfate 0.3g, after dissolving one by one, tap water is settled to 6L, regulates pH7.0-7.2.
Embodiment 2
This bacterial strain CGMCCNo.7928 is done the experiment of 10L fermentor tank, be carbon source with lyxose, result shows: after fermentation 72h, lactic acid concn can reach 54g/L.
Fermentation tank culture medium is: extractum carnis 12g, peptone 60g, yeast extract paste 30g, lyxose 120g, sodium acetate 30g, ammonium citrate 12g, dipotassium hydrogen phosphate 12g, magnesium sulfate heptahydrate 1.2g, seven water manganous sulfate 0.3g, after dissolving one by one, tap water is settled to 6L, regulates pH7.0-7.2.
Embodiment 3
This bacterial strain CGMCCNo.7928 is done the experiment of 10L fermentor tank, be carbon source with pectinose, result shows: after fermentation 72h, lactic acid concn can reach 52g/L.
Fermentation tank culture medium is: extractum carnis 12g, peptone 60g, yeast extract paste 30g, pectinose 120g, sodium acetate 30g, ammonium citrate 12g, dipotassium hydrogen phosphate 12g, magnesium sulfate heptahydrate 1.2g, seven water manganous sulfate 0.3g, after dissolving one by one, tap water is settled to 6L, regulates pH7.0-7.2.
Claims (6)
1. the novel plant Bacterium lacticum of a strain efficiency utilization biomass material high-yield lactic acid, described plant lactobacillus (Lactobacillus plantarum) Li-2013-01, deposit number is CGMCCNo.7928.
2. the novel plant Bacterium lacticum of efficiency utilization biomass material high-yield lactic acid according to claim 1, it is characterized in that, described plant lactobacillus can ferment and utilize five-carbon sugar.
3. the novel plant Bacterium lacticum of efficiency utilization biomass material high-yield lactic acid according to claim 1, it is characterized in that, take xylan as carbon source, and after fermentation 72h, lactic acid concn can reach 57g/L.
4. the novel plant Bacterium lacticum of efficiency utilization biomass material high-yield lactic acid according to claim 1, it is characterized in that, take glucose as carbon source, and after fermentation 72h, lactic acid concn can reach 68g/L.
5. the novel plant Bacterium lacticum of efficiency utilization biomass material high-yield lactic acid according to the arbitrary claim of claim 2-4, it is characterized in that, fermentation tank culture medium consists of: extractum carnis 12g, peptone 60g, yeast extract paste 30g, carbon source 120g, sodium acetate 30g, ammonium citrate 12g, dipotassium hydrogen phosphate 12g, magnesium sulfate heptahydrate 1.2g, seven water manganous sulfate 0.3g, after dissolving one by one, tap water is settled to 6L, regulates pH7.0-7.2.
6. the application of novel plant Bacterium lacticum in fermenting lactic acid of efficiency utilization biomass material high-yield lactic acid according to claim 1.
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| CN201310625005.9A CN104673691A (en) | 2013-11-29 | 2013-11-29 | Novel lactobacillus plantarum for high-yield production of lactic acid by efficiently utilizing biomass material |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350465A (en) * | 2016-08-29 | 2017-01-25 | 江南大学 | Lactobacillus plantarum and its application in the high-acid yellow wine production for acid modulation |
| WO2021014460A1 (en) * | 2019-07-22 | 2021-01-28 | Venkata Satya Sarveswara Sairam Kuchimanchi | Production of high purity organic lactic acid and its salts and various applications thereof |
Citations (2)
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| CN102492735A (en) * | 2011-12-15 | 2012-06-13 | 天津工业大学 | Application of strain of high temperature and glucose resistant lactobacillus in lactate production |
| CN102533591A (en) * | 2011-12-15 | 2012-07-04 | 天津工业大学 | High temperature resisting and high-glucose resisting lactic acid bacteria |
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2013
- 2013-11-29 CN CN201310625005.9A patent/CN104673691A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102492735A (en) * | 2011-12-15 | 2012-06-13 | 天津工业大学 | Application of strain of high temperature and glucose resistant lactobacillus in lactate production |
| CN102533591A (en) * | 2011-12-15 | 2012-07-04 | 天津工业大学 | High temperature resisting and high-glucose resisting lactic acid bacteria |
Non-Patent Citations (1)
| Title |
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| 李倩倩等: "高效转化木聚糖为乳酸植物乳杆菌L3的诱变选育及发酵条件的优化", 《中国酿造》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350465A (en) * | 2016-08-29 | 2017-01-25 | 江南大学 | Lactobacillus plantarum and its application in the high-acid yellow wine production for acid modulation |
| CN106350465B (en) * | 2016-08-29 | 2019-10-29 | 江南大学 | One lactobacillus plantarum and the application in tune acid highly acidity rice wine production |
| WO2021014460A1 (en) * | 2019-07-22 | 2021-01-28 | Venkata Satya Sarveswara Sairam Kuchimanchi | Production of high purity organic lactic acid and its salts and various applications thereof |
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