CN1908184A - Method of fermenting ligno-cellulose to prepare high optical purity L-lactic acid - Google Patents
Method of fermenting ligno-cellulose to prepare high optical purity L-lactic acid Download PDFInfo
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- CN1908184A CN1908184A CN 200610041291 CN200610041291A CN1908184A CN 1908184 A CN1908184 A CN 1908184A CN 200610041291 CN200610041291 CN 200610041291 CN 200610041291 A CN200610041291 A CN 200610041291A CN 1908184 A CN1908184 A CN 1908184A
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- fermentation
- aspergillus niger
- lignocellulose
- rhizopus oryzae
- lactic acid
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Abstract
the invention discloses a preparing method of high-optical purity L-lactic acid based on wood cellulose as raw material, which comprises the following steps: fermenting 64-72 h for inoculated aspergillus niger for 64-72 h; fermenting for 12-36 h for filtered inoculated enzyme.
Description
Technical field
The present invention relates to a kind of lignocellulose material and produce the method for high-optical-purity L-lactic acid and high-quality feed through the microorganism two-step fermentation.
Background technology
Lactic acid, English name Lactic acid, formal name used at school alpha-hydroxypropionic acid (α-hydroxypropanoic acid), structural formula is CH3 CHOHCOOH, molecular weight is 90.08, it is a kind of naturally occurring organic acid, extensively be present in human body, animal, plant and the microorganism, have two kinds of configurations of D-type and L-type, lactic acid has been widely used in fields such as food, medicine, chemical industry, environmental protection, because human body only has L one serum lactic dehydrogenase of metabolism L one lactic acid, therefore have only L one lactic acid can be by the complete metabolism utilization of human body.D-lactic acid and the excessive absorption of DL one lactic acid then might cause metabolism disorder even cause poisoning.Therefore, consider that the World Health Organization stipulates that clearly the amount that per day for adults is taken in D one lactic acid must not surpass the 100mg/kg body weight, for not adding D one lactic acid in the infant food below three months, does not then add restriction for L one lactic acid from health perspectives.The production of L-lactic acid and polymkeric substance thereof are deep day by day as the research of degradable plastics and medical material, and therefore, the L-lactic acid of high-optical-purity all has broad application prospects.
At present, the production of lactic acid mainly is to utilize the generation of fermenting of glucose or wood sugar by microorganism, a problem that very big problem is a production cost that exists, and have very big lignocellulose physical resources in China, agricultural crop straw for example, plant wastes such as molasses, wood chip, waste paper etc., it is reported that only agricultural crop straw and agricultural-food husk etc. just have more than 700,000,000 ton every year in China, the words quantity of adding industrial fiber wastes is just huger, on-site incineration is not only wasted resource, also cause serious environmental to be polluted, but owing to do not have good bacterial classification and effective means limiting the application of these resources always.
Publication number be CN1594584 a patent disclosure a kind of by the fermenting crops straws method of obtain lactic acid and feed simultaneously, anteroposterior diameter has been crossed solid state fermentation → liquid state fermentation → three fermentations of solid state fermentation just can produce lactic acid and feed, raw material sources are agricultural crop straw, in the pre-treatment of stalk, utilized the alkali lye processing, environment has been caused secondary pollution again.
Summary of the invention
The object of the present invention is to provide a kind of is raw material with the plain class material of natural wooden fiber, produce the technology of high-optical-purity L-lactic acid and high-quality feed by the microorganism two-step fermentation, not only preferably resolve the problem of utilizing of lignocellulose material, and greatly reduce cost for the production of L-lactic acid.
Technical scheme of the present invention is:
A kind ofly produce the method for high-optical-purity L-lactic acid, it is characterized in that carrying out successively following steps with the lignocellulose fermentation of materials:
(1), the pre-treatment of lignocellulose-containing class material:, standby with the pulverizing of lignocellulose-containing class material;
(2), preparation aspergillus niger substratum: take by weighing a certain amount of lignocellulose material, add the needed nutrition saline solution of aspergillus niger solid fermentation, sterilization;
(3), carry out the aspergillus niger inoculation: at the aspergillus niger inoculation of medium;
(4), fermentation of Aspergillus niger: fermentation culture obtains zytase and cellulase;
(5), add water, reaction: in fermented liquid, add sterilized water, zytase and Mierocrystalline cellulose enzyme-to-substrate are fully reacted, produce recuding sugars, as the carbon source of next step liquid fermenting;
(6), filter: with filtering fermentation liquor, the residue of lignocellulose material is a livestock feed;
(7), formulated rice head mold substratum: in filtrate, add Rhizopus oryzae fermentation desired nutritional material, sterilization;
(8), carry out the Rhizopus oryzae inoculation: at the Rhizopus oryzae inoculation of medium;
(9), Rhizopus oryzae fermentation: fermentation culture obtains L-lactic acid.
The needed nutrition saline solution of aspergillus niger solid fermentation composition is (g/l): KH
2PO
43.0; NaNO
35.0; Corn starch 5.0; MgSO
40.25 initial PH is 5.0, after the lignocellulose material joined the nutrition saline solution, its water content was 65%.
Fermentation of Aspergillus niger adopts 32 ℃ of-28 ℃ of temperature-variable fermentations, cultivates 64-72 hour.
Add sterilized water in fermented liquid after, pH value is about 5.0, and temperature of reaction is 50 ℃, and zytase and Mierocrystalline cellulose enzyme-to-substrate are fully reacted.
The Rhizopus oryzae that adds in the described filtrate needed nutritive substance that ferments, its composition is: (NH
4)
2SO
43g/l; ZnSO
47H
2O 0.15g/l; KH
2PO
40.3g/l; MgSO
47H
2O 0.75g/l; CaCO
310g/l (sterilization separately), inoculum size is 5-10%.
The meegan enzyme fermentation condition is: 37 ℃, shaking speed are 200rpm, finish through secondary fermentation in 12-36 hour.
Described aspergillus niger and Rhizopus oryzae, it chooses N respectively
+The pulsed radiation treatment, wherein the energy of aspergillus niger processing is 10～15keV, dosage is 20 * 2.6 * 10
13～120 * 2.6 * 10
13Ions/cm
2, Rhizopus oryzae is handled to such an extent that energy is 10～15keV, and dosage is 40 * 2.6 * 10
13～60 * 2.6 * 10
13Ions/cm
2
The material of described lignocellulose-containing class is meant the waste of some industrial or agricultural, agricultural wastes such as straw powder, corn cob, bagasse etc., industrial waste such as waste wood etc.
The present invention only need can produce L-lactic acid and feed through two-step fermentation, the lignocellulose material does not need to handle through alkali lye, the selected lactic acid that this strain fermentation produced of the present invention simultaneously optical purity after measured is a kind of bacterial classification of very good production high-optical-purity L-lactic acid greater than 98%.Raw materials used is all lignocellulose materials, and it is more extensive to originate.
The present invention has the following advantages: technical process is simple, the aspergillus niger that utilizes ion implantation seed selection produces the enzyme bacterium that sets out as solid state fermentation, the yield of enzyme height, reached international most advanced level, second step utilized the Rhizopus oryzae of ion implantation seed selection to carry out fermentation and acid, experimental results show that it not only can utilize hexose, utilize the level of five-carbon sugar also very high, thus the utilization ratio of hemicellulose increased greatly, and the L-lactic acid optical purity height that produces, the economic worth height, step is simple, and fermentation time is short, less investment, produced simultaneously waste residue can also have bigger economic benefit as high-quality feed.
Description of drawings
Fig. 1 produces the process flow sheet of high-optical-purity L-lactic acid and high-quality feed for lignocellulose material two-step fermentation.
Concrete embodiment
Two plant heights produce the preparation of bacterial strain:
One strain aspergillus niger mutant strain (Aaspergillus niger mutant P602) is to be 10～15keV by mutant strain Aaspergillus niger mutant N218 in the middle of the aspergillus niger through energy, and dosage is 20 * 2.6 * 10
13～120 * 2.6 * 10
13Ions/cm
2Carry out N
+The injection processing obtains.
One Rhizopus oryzae mutant strain (Rhizopus oryzae 6041) be by Rhizopus oryzae set out bacterium through energy be 10～15keV, dosage is 40 * 2.6 * 10
13～60 * 2.6 * 10
13Ions/cm
2Carry out N
+Inject and handle the high yield bacterium that obtains.
Ion implantationly carry out on Hefei Inst. of Plasma Physics, Chinese Academy of Sciences's ion implanter, it is made up of essential parts such as ion source, focusing system, mass analysis, accelerating system, vacuum system, target chamber and TT﹠C system.Principle of work is: produce plasma body in ion source, its intermediate ion is drawn through extraction system, after mass analyzer is selected, obtains energy in accelerating system, just can inject sample.
Product enzyme and the acid yield of these two kinds of bacterium all are improved largely after ion implantation mutagenesis is handled.
Example 1
Seed culture:
Aspergillus niger: get the 10ml sterilized water spore on the cultured fresh product spore inclined-plane is washed, it is standby to make spore suspension.
Rhizopus oryzae: 37 ℃, 200r/min cultivates 12h.Liquid amount: the 250mL triangular flask 50mL liquid nutrient medium of packing into.
(1), the pre-treatment of lignocellulose material: lignocellulose materials such as corn cob, straw powder are crushed to the 40-60 order, standby.
(2), utilize aspergillus niger to carry out solid fermentation and produce enzyme: take by weighing a certain amount of dry-matter, add the needed nutrition saline solution of aspergillus niger solid fermentation, composition is (g/l): KH
2PO
43.0; NaNO
35.0; Corn starch 5.0; MgSO
40.25, initial PH is 5.0, initial moisture content is 65%, after preparing, substratum packs in the Erlenmeyer flask of 250ml, sterilized 20 minutes for 121 ℃, by the time substratum has gone out and has inoculated after the cooling, getting the 10ml sterilized water washes the spore on the cultured fresh product spore inclined-plane, make spore suspension, the 10-15% that draws substrate weight is in the fermention medium for preparing and sterilize, carry out temperature-variable fermentation [32 ℃-28 ℃], cultivated 64-72 hour, produced zytase and cellulase a large amount of this moment after measured.
Aspergillus niger can ferment the lignocellulose material that utilizes very extensively, but want to make it to produce enzyme and reach highest level, will rationally adjust the proportioning of all kinds of materials, in order to make it produce the enzyme height, also to add suitable nutrition saline solution, the following condition of the preferential recommendation of the present invention: the nutrition saline solution that needs, composition is (g/l): KH
2PO
43.0; NaNO
35.0; Corn starch 5.0; MgSO
40.25 initial PH is 5.0, initial moisture content is 65%, carries out temperature-variable fermentation [32 ℃-28 ℃] in the Erlenmeyer flask of the 200ml that packs into after substratum prepares, and shaking speed 200rpm cultivated 64-72 hour.
(3), after solid fermentation finishes, in fermentation flask, add the 80-100ml sterilized water, place under 50 ℃ of conditions, and it is shaken gently, the enzyme that can digest lignin class material and the lignin class material of generation fully reacted (solid state fermentation finish after, its pH value is about 5.0, is the optimal pH of enzyme reaction), produce recuding sugars, as the carbon source of next step liquid fermenting.
(4), with filtering fermentation liquor, the lignocellulose material residue after the filtration is then owing to passed through the digestion of zytase and eliminate " antinutritional factor " piperylene and can be used as livestock feed.Get a certain amount of filtrate, add the Rhizopus oryzae needed nutritive element that ferments, Rhizopus oryzae fermentation and acid substratum consists of (g/l):: (NH
4)
2SO
43, ZnSO
47H
2O 0.15, KH
2PO
40.3, MgSO
47H
2O 0.75.In order to control the pH value in the fermenting process, in fermented liquid, added a certain amount of CaCO simultaneously
3Temperature is that 37 ℃, shaking speed are 200rpm, and inoculum size is 5-10%, and concrete seeded process is identical with aspergillus niger, and secondary fermentation in 12-36 hour finishes after measured, produces L-lactic acid.
Example 2
Corn cob, crushed stalk is to the 40-60 order, standby.
Utilize aspergillus niger to carry out solid fermentation and produce enzyme: corn cob, straw powder are mixed by a certain percentage, and add wheat bran as nutritional factor, final corn cob: wheat bran: straw powder=4: 2.5: 1, take by weighing 7.5 gram dry-matteies, add the needed nutrition saline solution of aspergillus niger solid fermentation, composition is (g/l): KH
2PO
43.0; NaNO
35.0; Corn starch 5.0; MgSO
40.25, initial PH is 5.0, and initial moisture content is 65%, in the Erlenmeyer flask of the 250ml that packs into after substratum prepares, sterilized 20 minutes for 121 ℃, by the time substratum has gone out and has inoculated after the cooling, gets the 10ml sterilized water spore on the cultured fresh product spore inclined-plane is washed, and makes spore suspension, draw 1ml in the fermention medium for preparing and sterilize, carry out temperature-variable fermentation [32 ℃-28 ℃], cultivated 64 hours, produced zytase and cellulase a large amount of this moment after measured.
After measured, the enzyme of the zytase of generation is lived and is 6320IU/g, and cellulose enzyme activity is 28IU/g.
After solid fermentation finishes, in fermentation flask, add the 100ml sterilized water, place under 50 ℃ of conditions, and it is shaken gently, the enzyme that can digest the lignocellulose material and the substrate of generation are fully reacted, produce recuding sugars, as the carbon source of next step liquid fermenting.
With filtering fermentation liquor, the lignocellulose material residue after the filtration then can be used as livestock feed owing to passed through digestion elimination " antinutritional factor " piperylene of zytase.Get filtrate 50ml, add the Rhizopus oryzae needed nutrition saline solution that ferments, Rhizopus oryzae fermentation and acid substratum consists of (g/l): (NH
4)
2SO
43, ZnSO
47H
2O 0.15, KH
2PO
40.3, MgSO
47H
2O 0.75.In order to control the pH value in the fermenting process, we have added a certain amount of CaCO in fermented liquid simultaneously
3Culture condition is that liquid amount is 50ml when carrying out the meegan enzyme fermentation, temperature is that 37 ℃, shaking speed are 200rpm, inoculum size is 5%, concrete seeded process is identical with aspergillus niger, and secondary fermentation in 12-36 hour finishes after measured, produces L-lactic acid, the amount that produces is the 7-8mg/g dry-matter, that is to say that every 100g dry-matter can produce the lactic acid about 10g through after the fermenting twice, its transformation efficiency to the lignocellulose material reaches about 10%.
Claims (8)
1, a kind ofly produce the method for high-optical-purity L-lactic acid, it is characterized in that carrying out successively following steps with the lignocellulose fermentation of materials:
(1), the pre-treatment of lignocellulose-containing class material:, standby with the pulverizing of lignocellulose-containing class material;
(2), preparation aspergillus niger substratum: take by weighing a certain amount of lignocellulose material, add the needed nutrition saline solution of aspergillus niger solid fermentation, sterilization;
(3), carry out the aspergillus niger inoculation: at the aspergillus niger inoculation of medium;
(4), fermentation of Aspergillus niger: fermentation culture obtains zytase and cellulase;
(5), add water, reaction: in fermented liquid, add sterilized water, zytase and Mierocrystalline cellulose enzyme-to-substrate are fully reacted, produce recuding sugars, as the carbon source of next step liquid fermenting;
(6), filter: with filtering fermentation liquor, the residue of lignocellulose material is a livestock feed;
(7), formulated rice head mold substratum: in filtrate, add Rhizopus oryzae fermentation desired nutritional material, sterilization;
(8), carry out the Rhizopus oryzae inoculation: at the Rhizopus oryzae inoculation of medium;
(9), Rhizopus oryzae fermentation: fermentation culture obtains L-lactic acid.
2, method according to claim 1 is characterized in that the needed nutrition saline solution of aspergillus niger solid fermentation composition is (g/l): KH
2PO
43.0; NaNO
35.0; Corn starch 5.0; MgSO
40.25 initial PH is 5.0, after the lignocellulose material joined the nutrition saline solution, its water content was 65%.
3, method according to claim 1 is characterized in that fermentation of Aspergillus niger adopts 32 ℃ of-28 ℃ of temperature-variable fermentations, cultivates 64-72 hour.
4, method according to claim 1, after it is characterized in that adding sterilized water in fermented liquid, pH value is about 5.0, temperature of reaction is 50 ℃, and zytase and Mierocrystalline cellulose enzyme-to-substrate are fully reacted.
5, method according to claim 1 is characterized in that the Rhizopus oryzae that adds in the filtrate needed nutritive substance that ferments, and its composition is: (NH
4)
2SO
43g/l; ZnSO
47H
2O 0.15g/l; KH
2PO
40.3g/l; MgSO
47H
2O 0.75g/l; CaCO
310g/l (sterilization separately), inoculum size is 5-10%.
6, method according to claim 1 is characterized in that the meegan enzyme fermentation condition is: 37 ℃, shaking speed are 200rpm, finish through secondary fermentation in 12-36 hour.
7, method according to claim 1 is characterized in that described aspergillus niger and Rhizopus oryzae, and it chooses N pulsed radiation treatment respectively, and wherein the energy of aspergillus niger processing is 10～15keV, and dosage is 20 * 2.6 * 10
13～120 * 2.6 * 10
13Ions/cm
2, Rhizopus oryzae is handled to such an extent that energy is 10～15keV, and dosage is 40 * 2.6 * 10
13～60 * 2.6 * 10
13Ions/cm
2
8, method according to claim 1, the material that it is characterized in that described lignocellulose-containing class is meant the waste of some industrial or agricultural, agricultural wastes such as straw powder, corn cob, bagasse etc., industrial waste such as waste wood etc.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174602A (en) * | 2011-03-07 | 2011-09-07 | 南京林业大学 | Method for producing L-lactic acid through biomass fermentation |
| CN102300994A (en) * | 2009-02-11 | 2011-12-28 | 希乐克公司 | Processing biomass |
| CN101724663B (en) * | 2008-10-24 | 2012-08-22 | 中国农业大学 | Method for producing L-lactic acid by utilizing corn cob and special rhizopus oryzae thereof |
| CN102690845A (en) * | 2012-06-26 | 2012-09-26 | 太仓市周氏化学品有限公司 | Method for producing lactic acid by fermenting fiber residue zymolyte |
| CN103710407A (en) * | 2012-09-29 | 2014-04-09 | 江苏省农业科学院 | Efficient preparation of glucose from corn starch by rhizopus oryzae liquid fermentation under corncob biological catalysis |
-
2006
- 2006-08-05 CN CN 200610041291 patent/CN1908184A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101724663B (en) * | 2008-10-24 | 2012-08-22 | 中国农业大学 | Method for producing L-lactic acid by utilizing corn cob and special rhizopus oryzae thereof |
| CN102300994A (en) * | 2009-02-11 | 2011-12-28 | 希乐克公司 | Processing biomass |
| CN102174602A (en) * | 2011-03-07 | 2011-09-07 | 南京林业大学 | Method for producing L-lactic acid through biomass fermentation |
| CN102690845A (en) * | 2012-06-26 | 2012-09-26 | 太仓市周氏化学品有限公司 | Method for producing lactic acid by fermenting fiber residue zymolyte |
| CN103710407A (en) * | 2012-09-29 | 2014-04-09 | 江苏省农业科学院 | Efficient preparation of glucose from corn starch by rhizopus oryzae liquid fermentation under corncob biological catalysis |
| CN103710407B (en) * | 2012-09-29 | 2016-12-21 | 江苏省农业科学院 | Corn cob living things catalysis Rhizopus oryzae liquid fermentation corn starch Efficient preparation of glucose |
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