CN103627739A - Method for producing L-lactic acid by fermenting bagasse cellulose - Google Patents
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Abstract
The invention discloses a method for producing L-lactic acid by fermenting bagasse cellulose. The method is characterized by using bagasse as a raw material, carrying out raw material pretreatment and cellulose enzymolysis and producing L-lactic acid by using moulds as fermentation strains and enzymatic hydrolysate of bagasse cellulose as a carbon source through fermentation with free cells or immobilized cells. The method has the beneficial effects that the raw material is accessible and is low in cost; the amount of reducing sugar obtained through enzymolysis is higher than that of reducing sugar in other reports, the fermentation period is short, the fermentation liquor is easy to separate, and the conversion rate of the product is high; not only can free fermentation be adopted but also immobilized fermentation can be carried out, and the immobilized cells for immobilized fermentation can be reused and have stable acid yields and high lactic acid conversion rates. In short, the method is suitable for industrially producing L-lactic acid by efficiently converting bagasse cellulose, greatly reduces the production cost of lactic acid, solves the problems of difficulty in control of a lactobacillus fermentation process and difficulty in separation and purification caused by the free cells, and has good industrial application prospects.
Description
Technical field
The invention belongs to the technical field of microorganism fermentation production of organic acid, relate in particular to a kind of method of utilizing bagasse cellulose fermentation production of L-lactic acid.
Background technology
Pfansteihl (L-lactic acid) is the D-lactic acid that the katalysis by LDH converts pyruvic acid in fermentation process.Compare with D-ALPHA-Hydroxypropionic acid, because Pfansteihl can be directly absorbed by the body, and without any side effects, so be widely used in the fields such as food, medicine, chemical industry.
If microbial host milk-acid bacteria and the rhizopus of current L-lactic acid fermentation, milk-acid bacteria requires high to nutritive ingredient, and is amphimicrobian type microorganism, bad control in fermenting process, and the Pfansteihl optical purity of production is not high yet.Traditional zymotic adopts free cell fermentation mode more, in free fermentation, hypha,hyphae is more flourishing, often occur grumeleuse and clustering phenomena, the mycelium of suspension also can increase the viscosity of fermented liquid, has reduced intercellular oxygen transmission, reduced the output of lactic acid, and also influential to follow-up sepn process, separating difficulty is large, and the product obtaining is few.
Production cost is one of key factor of restriction Pfansteihl application.Suitability for industrialized production all adopts starchy material, and this has increased production cost greatly.And some cheap reproducible raw materials, as biomass material, contain abundant carbohydrate, can be used for the production of Pfansteihl.China is large agricultural country, produces every year the agriculture and forestry organic waste material of more than one hundred million tons, but only has only a few to be effectively utilized, and major part is used as waste and loses.This has not only wasted abundant renewable resources, and has polluted environment.Therefore, if can utilize these biomass celluloses to produce all kinds of high value added products such as Pfansteihl, walk the route of " turning waste into wealth ", application prospect is by boundless.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method of utilizing bagasse cellulose fermentation production of L-lactic acid that production cost is low, fermentation period is short, technique is simply controlled, product purity is high.
For solving the problems of the technologies described above, the present invention is by the following technical solutions: the method for utilizing bagasse cellulose fermentation production of L-lactic acid, take bagasse as raw material, by raw materials pretreatment, cellulase hydrolysis, take mould as fermented bacterium, the enzymolysis solution of bagasse cellulose of take is carbon source, and free cell fermentation or immobilized cell fermentation are produced Pfansteihl.
Raw materials pretreatment is undertaken by following operation: with pulverizer, pulverize bagasse, cross 20-40 mesh sieve; Accurately taking the bagasse 30g after sieving, is that to add mass concentration be the H of 0.6-0.8% to 1:30 by solid-liquid ratio
2o
2naOH mixing solutions with 3-5% stirs 3-5h at 85 ℃; Suction filtration, is washed with distilled water to filtrate for neutral, and residue is dried to constant weight in 105 ℃ of loft drier, then pulverizes with pulverizer, crosses 20-40 mesh sieve, collects standby.
Cellulase hydrolysis is undertaken by following operation: accurately take pretreated bagasse, by every g bagasse, adding concentration is the cellulase solution 4-8mL of 10mg/mL, concentration of substrate 28-66g/L, the citric acid solution 17-21mL of pH4.0-5.4, enzymolysis 36-60h in 45-55 ℃, the thermostat water bath of rotating speed 120r/min, then boils the enzyme that goes out and lives, centrifugation, recovery enzymolysis solution is standby, measures the reducing sugar content in enzymolysis solution.
The above-mentioned method of utilizing bagasse cellulose fermentation production of L-lactic acid, adopts free cell fermentation mode, by following operation, is undertaken:
<1> slant culture: by the test tube PDA substratum of bacterial classification Rhizopus oryzae (Gim3.129) access sterilizing, cultivate 4-6 days in 28 ℃, after in 4 ℃ of refrigerators, preserve;
<2> spore suspension: get cultured slant strains, by stroke-physiological saline solution, the spore on inclined-plane is washed down, in the triangular flask of sterilizing, add aseptic granulated glass sphere fully to break up, with the absorbent cotton of sterilizing, filter, use aseptic water washing filter residue, making filtrate volume is 30mL, is diluted to spore suspension containing 1 * 10
7individual spore/mL;
<3> seed culture: spore suspension is linked in the triangular flask that liquid amount is 20-70mL by 4% inoculum size, cultivates 12-24h under 28-32 ℃, 200r/min, make seed culture fluid;
<4> fermentation culture: seed culture fluid is linked in the triangular flask that liquid amount is 50mL by the inoculum size of 10-14%, and under 30 ℃, 180-200r/min, fermentation 36-72h, obtains the fermented liquid containing tunning Pfansteihl; After fermentation ends, fermented liquid is carried out to suction filtration, get clear liquid, by EDTA method, measure the concentration of Pfansteihl, calculate the transformation efficiency (transformation efficiency of Pfansteihl=(concentration of the concentration/reducing sugar of Pfansteihl) * 100%) of Pfansteihl.
Slant medium is prepared by following: potato 200g, adds water 1000mL, glucose 15-25g, agar 15-25g, heating for dissolving packing test tube, 121 ℃ of sterilizing 20min;
Seed culture medium is prepared by following: glucose is 50-70g/L, yeast extract paste (nitrogenous source) 5-7g/L, KH
2pO
40.3-0.5g/L, MgSO
4.7H
2o0.2-0.4g/L, ZnSO
4.7H
2o0.08g/L, CaCO
310g/L, 121 ℃ of sterilizing 20min, CaCO
3separately sterilizing.
Fermention medium is prepared by following: enzymolysis solution 30-38g/L, (NH
4)
2sO
43-7g/L, KH
2pO
40.2-0.4g/L, MgSO
4.7H
2o0.2-0.4g/L, ZnSO
4.7H
2o0.04-0.08g/L, CaCO
310-30g/L, 121 ℃ of sterilizing 20min, CaCO
3separately sterilizing.
The above-mentioned method of utilizing bagasse cellulose fermentation production of L-lactic acid, adopts immobilized cell fermentation mode, by following operation, is undertaken:
<1> slant culture: by the test tube PDA substratum of bacterial classification Rhizopus oryzae (Gim3.129) access sterilizing, cultivate 4-6 days in 28 ℃, after in 4 ℃ of refrigerators, preserve;
<2> spore suspension: get cultured slant strains, by stroke-physiological saline solution, the spore on inclined-plane is washed down, in the triangular flask of sterilizing, add aseptic granulated glass sphere fully to break up, with the absorbent cotton of sterilizing, filter, use aseptic water washing filter residue, making filtrate volume is 30mL, is diluted to spore suspension containing 1 * 10
7individual spore/mL;
<3>ACA microcapsule method immobilized cell preparation: the sodium alginate that is 1.5-2.5% by the mass concentration of sterilizing and the spore suspension preparing (by 5:1) are mixed together in sterilized triangular flask and fully shake up; With the syringe (internal diameter 1mm) of same model, mixed solution is dropwise joined to the CaCl of mass concentration 2.0-3.0%
2in solution, calcification 0.5-1.5h, the calcium alginate microsphere that formation diameter is 3-4mm, removes supernatant liquor, with sterilized water, washes away CaCl
2film forming 15min in the chitosan solution (being dissolved in 1% glacial acetic acid solution) of mass concentration 0.4-0.8% again, discard unnecessary chitosan, clean, the sodium alginate soln with 0.15% is processed surface of microcapsule 5min, then uses the sodium citrate solution CF 15-25min of 0.075mol/L, elimination treatment solution, with sterilized water, clean, by stroke-physiological saline solution, at 4 ℃, preserve (being no more than 3 days), standby.
<4> immobilized cell is cultivated: immobilized cell is linked in the triangular flask that liquid amount is 50mL, under 30 ℃, 200r/min, cultivates 12-24h, make immobilized cell nutrient solution;
<5> fermentation culture: immobilized cell nutrient solution is linked in the triangular flask that liquid amount is 50mL by the inoculum size of 10-14%, under 30 ℃, 180-200r/min, fermentation 36-50h, obtains the fermented liquid containing tunning Pfansteihl; After fermentation ends, fermented liquid is carried out to suction filtration, get clear liquid, by EDTA method, measure the concentration of Pfansteihl, calculate the transformation efficiency (transformation efficiency of Pfansteihl=(concentration of the concentration/reducing sugar of Pfansteihl) * 100% of Pfansteihl.
The immobilization fermentation time is 36h.
The recycling of immobilized cell batch is 7 batches.
For current Pfansteihl, produce the problem existing, contriver be take bagasse as raw material, set up first the method for utilizing bagasse cellulose fermentation production of L-lactic acid, by raw materials pretreatment, cellulase hydrolysis, take mould as fermented bacterium, the enzymolysis solution of bagasse cellulose of take is carbon source, and free cell fermentation or immobilized cell fermentation are produced Pfansteihl.This method raw material is easy to get, with low cost; The reducing sugar amount that enzymolysis the obtains height of comparing with other reports, fermentation period is short, and separation of fermentative broth is easy, and conversion rate of products is high; Both can adopt free fermentation, and also can being fixed ferment, the latter's fixed cell can reuse, and acid production rate is stable.In a word, the present invention is a kind of method that industrialized Efficient Conversion bagasse cellulose is produced Pfansteihl that is adapted to, greatly reduce the production cost of lactic acid, solved the difficulty problem that the difficult control of lactic acid bacteria fermentation process and free cell cause separating-purifying, there is good prospects for commercial application.
Embodiment
The enzymolysis of embodiment 1 bagasse cellulose
With pulverizer, pulverize bagasse, cross 20 mesh sieves; Accurately taking the bagasse 30g after sieving, is that to add mass concentration be 0.7% H to 1:30 by solid-liquid ratio
2o
2naOH mixing solutions with 4% stirs 4h at 85 ℃; Suction filtration, is washed with distilled water to filtrate for neutral, and residue is dried to constant weight in 105 ℃ of loft drier, then pulverizes with pulverizer, crosses 40 mesh sieves, collects standby.
Accurately take pretreated bagasse 1g, adding concentration is the cellulase solution 7mL of 10mg/mL, concentration of substrate 40g/L, the citric acid solution 18mL of pH5.0, enzymolysis 36h in 50 ℃, the thermostat water bath of rotating speed 120r/min, then boil the enzyme that goes out and live, centrifugation, reclaims enzymolysis solution standby.
Utilize DNS method to measure the concentration of the reducing sugar in enzymolysis solution, result demonstration, recording concentration of reduced sugar in enzymolysis solution is 34.36g/L.
Embodiment 2 free cell fermentations
The preparation of <1> slant medium: by peeling potatoes, take 200g, add water 1000mL, boil 30min, filter, elimination potato ball, mends filtrate to 1000mL, add glucose 20g, agar 20g, heating for dissolving packing test tube, 121 ℃ of sterilizing 20min, inclination is put, cooling and get final product;
The slant culture of <2> bacterial classification: by the test tube PDA substratum of bacterial classification Rhizopus oryzae (Gim3.129) access sterilizing, cultivate 5 days in 28 ℃, after in 4 ℃ of refrigerators, preserve;
The preparation of <3> spore suspension: get cultured slant strains, by stroke-physiological saline solution, the spore on inclined-plane is washed down, in the triangular flask of sterilizing, add aseptic granulated glass sphere fully to break up, with the absorbent cotton of sterilizing, filter, use aseptic water washing filter residue, making filtrate volume is 30mL, is diluted to spore suspension containing 1 * 10
7individual spore/mL;
By following formula, " glucose is 60g/L to <4>, yeast extract paste (nitrogenous source) 7g/L, KH
2pO
40.4g/L, MgSO
4.7H
2o0.3g/L, ZnSO
4.7H
2o0.08g/L, CaCO
310g/L " be made into the seed culture medium of 50mL, 121 ℃ of sterilizing 20min, CaCO
3separately sterilizing.
<5> seed culture: spore suspension is linked in the triangular flask that liquid amount is 50mL by 4% inoculum size, cultivates 18h under 30 ℃, 200r/min, make seed culture fluid;
<6> fermention medium is prepared by following: enzymolysis solution 36.49g/L, (NH
4)
2sO
45g/L, KH
2pO
40.4g/L, MgSO
4.7H
2o0.2g/L, ZnSO
4.7H
2o0.04g/L, CaCO
320g/L, 121 ℃ of sterilizing 20min, CaCO
3separately sterilizing.
<7> free cell fermentation: seed culture fluid is linked in the triangular flask that fermention medium liquid amount is 50mL by 10% inoculum size, under 30 ℃, 200r/min, fermentation 72h, obtains the fermented liquid containing tunning Pfansteihl.
After fermentation ends, fermented liquid is carried out to suction filtration, get clear liquid, by EDTA method, measure the concentration of Pfansteihl, the transformation efficiency (transformation efficiency of Pfansteihl=(concentration of the concentration/reducing sugar of Pfansteihl) * 100%) that calculates Pfansteihl, the transformation efficiency of the lactic acid recording is 28.45%.
Embodiment 3 immobilized cell fermentations
The preparation of <1> slant medium: by peeling potatoes, take 200g, add water 1000mL, boil 30min, filter, elimination potato ball, mends filtrate to 1000mL, add glucose 20g, agar 15g, heating for dissolving packing test tube, 121 ℃ of sterilizing 20min, inclination is put, cooling and get final product;
The slant culture of <2> bacterial classification: by the test tube PDA substratum of bacterial classification Rhizopus oryzae (Gim3.129) access sterilizing, cultivate 5 days in 28 ℃, after in 4 ℃ of refrigerators, preserve;
The preparation of <3> spore suspension: get cultured slant strains, by stroke-physiological saline solution, the spore on inclined-plane is washed down, in the triangular flask of sterilizing, add aseptic granulated glass sphere fully to break up, with the absorbent cotton of sterilizing, filter, use aseptic water washing filter residue, making filtrate volume is 30mL, is diluted to spore suspension containing 1 * 10
7individual spore/mL;
<4>ACA microcapsule method immobilized cell preparation: the sodium alginate that is 1.5% by the mass concentration of sterilizing and the spore suspension preparing (by 5:1) are mixed together in sterilized triangular flask and fully shake up; With the syringe (internal diameter 1mm) of same model, mixed solution is dropwise joined to the CaCl of mass concentration 2.5%
2in solution, calcification 1h, the calcium alginate microsphere that formation diameter is 3-4mm, removes supernatant liquor, with sterilized water, washes away CaCl
2film forming 15min in the chitosan solution (being dissolved in 1% glacial acetic acid solution) of mass concentration 0.6% again, discard unnecessary chitosan, clean, the sodium alginate soln with 0.15% is processed surface of microcapsule 5min, then uses the sodium citrate solution CF 15min of 0.075mol/L, elimination treatment solution, with sterilized water, clean, by stroke-physiological saline solution, at 4 ℃, preserve (being no more than 3 days), standby.
By following formula, " glucose is 50g/L to <5>, yeast extract paste (nitrogenous source) 7g/L, KH
2pO
40.4g/L, MgSO
4.7H
2o0.4g/L, ZnSO
4.7H
2o0.08g/L, CaCO
310g/L " be made into the seed culture medium of 50mL, 121 ℃ of sterilizing 20min, CaCO
3separately sterilizing.
<5> immobilized cell is cultivated: immobilized cell is linked in the triangular flask that liquid amount is 50mL, under 30 ℃, 200r/min, cultivates 24h, make immobilized cell nutrient solution;
<6> fermention medium is prepared by following: enzymolysis solution 36.49g/L, (NH
4)
2sO
45g/L, KH
2pO
40.4g/L, MgSO
4.7H
2o0.3g/L, ZnSO
4.7H
2o0.06g/L, CaCO
320g/L, 121 ℃ of sterilizing 20min, CaCO
3separately sterilizing.
<7> immobilized cell fermentation is cultivated: immobilized cell nutrient solution is linked in the triangular flask that liquid amount is 50mL by 14% inoculum size, under 30 ℃, 200r/min, fermentation 36h, obtains the fermented liquid containing tunning Pfansteihl.
After fermentation ends, fermented liquid is carried out to suction filtration, get clear liquid, measure the concentration of Pfansteihl by EDTA method, (transformation efficiency of Pfansteihl=(concentration of the concentration/reducing sugar of Pfansteihl) * 100%, the transformation efficiency that result records lactic acid is 31% to the transformation efficiency of calculating Pfansteihl.
The recycling fermentation of <8> immobilized cell: after first fermentation ends, under aseptic condition, use filtered through gauze fermented liquid, by stroke-physiological saline solution, clean immobilized spherule, be linked into fresh substratum, in 30 ℃, 200r/min bottom fermentation 36h, fermentation ends, repeat operation above, by EDTA method, measure the concentration of lactic acid in every batch fermentation liquid, found that, recycling batch be 7 batches, rotational rate of lactic acid is downward trend afterwards.
Claims (8)
1. a method of utilizing bagasse cellulose fermentation production of L-lactic acid, it is characterized in that take that bagasse is as raw material, by raw materials pretreatment, cellulase hydrolysis, take mould as fermented bacterium, the enzymolysis solution of bagasse cellulose of take is carbon source, and free cell fermentation or immobilized cell fermentation are produced Pfansteihl.
2. the method for utilizing bagasse cellulose fermentation production of L-lactic acid according to claim 1, is characterized in that raw materials pretreatment undertaken by following operation: with pulverizer, pulverize bagasse, cross 20-40 mesh sieve; Accurately taking the bagasse 30g after sieving, is that to add mass concentration be the H of 0.6-0.8% to 1:30 by solid-liquid ratio
2o
2naOH mixing solutions with 3-5% stirs 3-5h at 85 ℃; Suction filtration, is washed with distilled water to filtrate for neutral, and residue is dried to constant weight in 105 ℃ of loft drier, then pulverizes with pulverizer, crosses 20-40 mesh sieve, collects standby.
3. the method for utilizing bagasse cellulose fermentation production of L-lactic acid according to claim 2, it is characterized in that cellulase hydrolysis is undertaken by following operation: accurately take pretreated bagasse, by every g bagasse, adding concentration is the cellulase solution 4-8mL of 10mg/mL, concentration of substrate 28-66g/L, the citric acid solution 17-21mL of pH4.0-5.4, enzymolysis 36-60h in 45-55 ℃, the thermostat water bath of rotating speed 120r/min, then boils the enzyme that goes out and lives, centrifugation, reclaims enzymolysis solution standby.
4. the method for utilizing bagasse cellulose fermentation production of L-lactic acid according to claim 3, is characterized in that adopting free cell fermentation mode, by following operation, is undertaken:
<1> slant culture: by the test tube PDA substratum of bacterial classification Rhizopus oryzae (Gim3.129) access sterilizing, cultivate 4-6 days in 28 ℃, after in 4 ℃ of refrigerators, preserve;
<2> spore suspension: get cultured slant strains, by stroke-physiological saline solution, the spore on inclined-plane is washed down, in the triangular flask of sterilizing, add aseptic granulated glass sphere fully to break up, with the absorbent cotton of sterilizing, filter, use aseptic water washing filter residue, making filtrate volume is 30mL, is diluted to spore suspension containing 1 * 10
7individual spore/mL;
<3> seed culture: spore suspension is linked in the triangular flask that liquid amount is 20-70mL by 4% inoculum size, cultivates 12-24h under 28-32 ℃, 200r/min, make seed culture fluid;
<4> fermentation culture: seed culture fluid is linked in the triangular flask that liquid amount is 50mL by the inoculum size of 10-14%, and under 30 ℃, 180-200r/min, fermentation 36-72h, obtains the fermented liquid containing tunning Pfansteihl.
5. the method for utilizing bagasse cellulose fermentation production of L-lactic acid according to claim 4, is characterized in that:
Described slant medium is prepared by following: potato 200g, adds water 1000mL, glucose 15-25g, agar 15-25g, heating for dissolving packing test tube, 121 ℃ of sterilizing 20min;
Described seed culture medium is prepared by following: glucose is 50-70g/L, yeast extract paste 5-7g/L, KH
2pO
40.3-0.5g/L, MgSO
4.7H
2o0.2-0.4g/L, ZnSO
4.7H
2o0.08g/L, CaCO
310g/L, 121 ℃ of sterilizing 20min, CaCO
3separately sterilizing;
Described fermention medium is prepared by following: enzymolysis solution 30-38g/L, (NH
4)
2sO
43-7g/L, KH
2pO
40.2-0.4g/L, MgSO
4.7H
2o0.2-0.4g/L, ZnSO
4.7H
2o0.04-0.08g/L, CaCO
310-30g/L, 121 ℃ of sterilizing 20min, CaCO
3separately sterilizing.
6. the method for utilizing bagasse cellulose fermentation production of L-lactic acid according to claim 3, is characterized in that adopting immobilized cell fermentation mode, by following operation, is undertaken:
<1> slant culture: by the test tube PDA substratum of bacterial classification Rhizopus oryzae (Gim3.129) access sterilizing, cultivate 4-6 days in 28 ℃, after in 4 ℃ of refrigerators, preserve;
<2> spore suspension: get cultured slant strains, by stroke-physiological saline solution, the spore on inclined-plane is washed down, in the triangular flask of sterilizing, add aseptic granulated glass sphere fully to break up, with the absorbent cotton of sterilizing, filter, use aseptic water washing filter residue, making filtrate volume is 30mL, is diluted to spore suspension containing 1 * 10
7individual spore/mL;
<3>ACA microcapsule method immobilized cell preparation: the sodium alginate that is 1.5-2.5% by the mass concentration of sterilizing and the spore suspension preparing are mixed together in sterilized triangular flask and fully shake up by 5:1; With the syringe of the same model of internal diameter 1mm, mixed solution is dropwise joined to the CaCl of mass concentration 2.0-3.0%
2in solution, calcification 0.5-1.5h, the calcium alginate microsphere that formation diameter is 3-4mm, removes supernatant liquor, with sterilized water, washes away CaCl
2film forming 15min in the chitosan solution (being dissolved in 1% glacial acetic acid solution) of mass concentration 0.4-0.8% again, discard unnecessary chitosan, clean, the sodium alginate soln with 0.15% is processed surface of microcapsule 5min, then uses the sodium citrate solution CF 15-25min of 0.075mol/L, elimination treatment solution, with sterilized water, clean, by stroke-physiological saline solution, at 4 ℃, preserve, standby;
<4> immobilized cell is cultivated: immobilized cell is linked in the triangular flask that liquid amount is 50mL, under 30 ℃, 200r/min, cultivates 12-24h, make immobilized cell nutrient solution;
<5> fermentation culture: immobilized cell nutrient solution is linked in the triangular flask that liquid amount is 50mL by the inoculum size of 10-14%, under 30 ℃, 180-200r/min, fermentation 36-50h, obtains the fermented liquid containing tunning Pfansteihl.
7. the method for utilizing bagasse cellulose fermentation production of L-lactic acid according to claim 6, is characterized in that: the described immobilization fermentation time is 36h.
8. the method for utilizing bagasse cellulose fermentation production of L-lactic acid according to claim 7, is characterized in that: the recycling of immobilized cell batch is 7 batches.
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| CN105177067A (en) * | 2015-10-09 | 2015-12-23 | 河南科技大学 | Method for producing L-lactic acid through double-enzyme hydrolyzed crop leftover double-strain fermentation |
| CN108203693A (en) * | 2016-12-20 | 2018-06-26 | 重庆恒远晋通科技有限公司 | Utilize the method for tobacco waste production high concentration L-type lactic acid |
| CN111348753A (en) * | 2018-12-21 | 2020-06-30 | 中国石油化工股份有限公司 | Method for enhancing denitrification of denitrifying microorganisms |
| CN110964756A (en) * | 2019-11-11 | 2020-04-07 | 盐城工学院 | Method for preparing L-lactic acid by using jerusalem artichoke in full value |
| CN112646843A (en) * | 2020-12-31 | 2021-04-13 | 安徽丰原发酵技术工程研究有限公司 | Method for preparing lactic acid by using water hyacinth as raw material |
| CN112646843B (en) * | 2020-12-31 | 2022-03-08 | 安徽丰原发酵技术工程研究有限公司 | Method for preparing lactic acid by using water hyacinth as raw material |
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