CN101463370A - Method for preparing L-lactic acid by fermenting potato starch by Rhizopus oryzae - Google Patents
Method for preparing L-lactic acid by fermenting potato starch by Rhizopus oryzae Download PDFInfo
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- CN101463370A CN101463370A CNA2009100005324A CN200910000532A CN101463370A CN 101463370 A CN101463370 A CN 101463370A CN A2009100005324 A CNA2009100005324 A CN A2009100005324A CN 200910000532 A CN200910000532 A CN 200910000532A CN 101463370 A CN101463370 A CN 101463370A
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Abstract
The invention discloses a method for preparing L-lactic acid by fermenting potato starch with rhizopus oryzae. The method comprises the following steps: (1) the spore of the rhizopus oryzae is prepared; (2) the rhizopus oryzae spore is prepared into rhizopus oryzae spore emulsion suspension; (3) the rhizopus oryzae spore emulsion suspension is immobilized on an immobilized carrier to obtain an immobilized rhizopus oryzae seed; (4) the immobilized rhizopus oryzae seed is inoculated in a fermentation medium for immobilized fermentation. By the method of the invention, high-output rhizopus oryzae bacterial strain is cultured and immobilized on a cotton fabric carrier to obtain the immobilized rhizopus oryzae seed. The immobilized fermentation is carried out under the most suitable fermentation condition that is groped, the conversion rate of the potato starch is high, the biomass of fermented products is high, and the yield of the L-lactic acid is high. In addition to the advantage of high L-lactic acid yield, the invention also has the advantages of low cost, simple steps and easy control, etc.
Description
Technical field
The present invention relates to a kind of organic acid preparation method, relate in particular to a kind of method of utilizing the Rhizopus oryzae fermenting potato starch to prepare L-lactic acid, belong to the organic acid production field.
Background technology
Lactic acid is a kind of multiduty fine chemicals, and lactic acid, lactic acid salt and derivative thereof are widely used in fields such as food, medicine, chemical industry.Because only have the enzyme of metabolism L-lactic acid in the human body, D-lactic acid can not be absorbed by the body, and excessive use is poisonous to human body.Therefore, the World Health Organization advocates and uses L-lactic acid as foodstuff additive and medicine for oral administration, replaces the DL-lactic acid that generally uses at present.The appearance of poly(lactic acid) (PLA) plastics makes the development of L-lactic acid demonstrate huge market potential especially.
China's lactic acid-producing still has big gap with external advanced level, and the lactic acid-producing scale is less, and fermentor tank is 30-60 ton/platform only, and acid production rate is lower; Range of product accounts for 80% still based on DL-lactic acid, and product colourity is of low quality; In addition big gap is being arranged more aspect the extraction of back, production cost is higher generally.
At present, microbe fermentation method is adopted in the production of L-lactic acid substantially, i.e. head mold method and bacterium method.Lactic fermentation has obtained bigger progress aspect biological chemistry this century 50 to the sixties, just taking up now from genetic engineering aspect improvement lactic-acid-producing strain.The bacterial classification that produces L-lactic acid in the Rhizopus is a lot, and bacterial classification commonly used has Rhizopus oryzae, bread mould, zhizopchin, walking head mold, wheat starter head mold and beautiful head mold.
Wherein the ability of Rhizopus oryzae fermentation production of L-lactic acid is the strongest, but be subjected to also in actual production that fermentation period is long, sugared transformation efficiency is low, the restriction of shortcoming such as by product ethanol and fumaric acid content are more.Therefore, the lot of domestic and foreign scholar has done a large amount of research to Rhizopus oryzae at the aspects such as seed selection of the research of the improvement of zymotechnique, metabolic mechanism and metabolic flux analysis, superior strain.
Immobilized cell is cultivated (cell immobilization culture) and is meant and utilizes physics or chemical means that free cell or enzyme are combined with solid-state insoluble carrier, makes it keep active also repeatedly used a kind of technology.Compare with the free fermentation of traditional batch, utilize the fixation of microbe technology to have advantages such as the cell load is high, biocatalysis is efficient, cell can use repeatedly, and can obtain high target output and feed stock conversion, be easy to realize that cell separates with product, help realizing automatization, the serialization of technological process, reduce production costs, be in particular in: can obtain high cell concentration in (1) bio-reactor, thereby fermentation rate can improve greatly, and the production intensity of reactor apparatus is strengthened; (2) in the product separation and purification process thalline from fermented liquid, separate very easy; (3) immobilized cell can repeat or life-time service, can either simplify the operation that the process need of free cell is constantly cultivated thalline like this, has reduced the waste of nutritive substance again, has improved productivity.
Although immobilization technology has certain advantage, can make the cell highly dense, fast reaction speed shortens the work period, increases work efficiency.But yet have some shortcomings at present: as the cost of immobilized cell also than higher, in the immobilization process and the pollution control ratio of immobilized cell when using than difficulty etc.In addition, because cell fixation is in carrier, thereby increased the perforated space resistance of nutritive substance, oxygen and product, particularly the oxygen problem of transmission during aerobic fermentation is difficult to solve, so immobilization technology only is used for the fermentation of emiocytosis type product mostly.Therefore, select what cheap fixation support material, how to optimize immobilization technology and require further study.
The Rhizopus oryzae submerged fermentation is produced in the L-lactic acid process, and the thalli morphology complexity is how with form growths such as thread, sheet, spheroid, bulk, cotton-shaped, cakings.Rhizopus oryzae volume morphing intense influence performance such as mass transfer, dissolved oxygen in the fermenting process, and it is effectively controlled is one of successful key factor of fermentation.Up to now, do not see the report that utilizes Rhizopus oryzae solid state fermentation yam starch to produce L-lactic acid as yet.
Summary of the invention
The purpose of this invention is to provide a kind of method of utilizing the Rhizopus oryzae fermenting potato starch to prepare L-lactic acid, this method adopts the immobilization fermentation method and finds out optimum fermentation condition, have yam starch transformation efficiency height, L-lactic acid yield height, advantage such as with low cost, step is simple and direct.
The objective of the invention is to be achieved through the following technical solutions:
A kind of Rhizopus oryzae (Rhizopus oryzae) fermenting potato starch that utilizes prepares the method for L-lactic acid, may further comprise the steps:
(1) preparation Rhizopus oryzae spore; (2) the Rhizopus oryzae spore is prepared into Rhizopus oryzae spore emulsion suspension liquid; (3) Rhizopus oryzae spore emulsion suspension liquid is fixed to being fixed Rhizopus oryzae seed on the fixation support; (4) immobilization Rhizopus oryzae seed is inoculated into carries out immobilization fermentation in the fermention medium.
Preferably, the preparation method of described Rhizopus oryzae spore comprises: in potato glucose (PDA) slant medium, first day is 32 ℃ in temperature, to cultivate humidity relatively be to cultivate under 70% the condition with Rhizopus oryzae As3.3462 streak inoculation; Second day is 34 ℃ in temperature, to cultivate humidity relatively be to cultivate under 30% the condition; Inoculate 3-4 as stated above for activated spawn, treat that spore covers white hypha fully, promptly.The present invention is based on metabolic control fermentation and the metabolic control breeding theory of microorganism, is object with Rhizopus oryzae carbon metabolism key enzyme serum lactic dehydrogenase (LDH) with ethanol dehydrogenase (ADH), systematically studies separation and purification and the catalysis characteristics of LDH and ADH, Mn
2+And Zn
2+Rhizopus oryzae fermentation control pattern under ion and the different aeration condition, optimization the culture condition of the best of Rhizopus oryzae spore, thereby obtained the Rhizopus oryzae bacterial strain of high yield L-lactic acid;
Described Rhizopus oryzae spore emulsion suspension liquid can prepare with reference to following method: with sterilized water prepared Rhizopus oryzae spore is eluted, elutriant is inoculated into is cultured to concentration in the seed culture medium and reaches 10
6Individual/ml, promptly get Rhizopus oryzae spore emulsion suspension liquid; Wherein, consisting of of described seed culture medium: glucose 60g/L, urea 2.0g/L, KH
2PO
40.3g/L, MgSO
47H
2O 0.25g/L, ZnSO
47H
2O 0.08g/L;
Fixation support described in the step (3) is cotton preferably; Wherein, the cotton size is preferably 1cm * 1cm, and the carrier consumption is selected the 15mL/50mL substratum;
Described fixing means can carry out with reference to following method: cotton is cut into small pieces, puts into the bottle that seed culture medium is housed, autoclave sterilization cooling back inoculating spores suspension is cultivated after 24 hours for 30 ℃, and mycelium promptly is fixed on the cotton carrier; Wherein, consisting of of described seed culture medium: glucose 60g/L, urea 2.0g/L, KH
2PO
40.3g/L, MgSO
47H
2O 0.25g/L, ZnSO
47H
2O 0.08g/L.
Described inoculum size is preferably 5wt% (that is: immobilization Rhizopus oryzae seed by weight: fermention medium=5:95);
The fermentation condition of described immobilization fermentation is preferably: ferment in fermentor tank, every 50L canned 25L fermention medium that ferments, the pH value of fermentation is 5.0-5.5 (can adopt the mode of intermittently adding lime carbonate to control the pH value), and leavening temperature is 32-34 ℃, and fermentation period is 60 hours;
Consisting of of described fermention medium: potato 120g/L, KH
2PO
40.3g/L, MgSO
47H
2O 0.25g/L, ZnSO
47H
2O 0.08g/L, urea 2g/L, CaCO
360g/L.
The contriver is by further test discovery, to ventilate during the fermentation flow velocity and rotating speed controlled according to following condition, can improve ferment effect significantly, improve the output of L-lactic acid: the fermentation beginning was to the 20th hour certainly, the flow rate control of will ventilating is 0.3L/Lmin, and rotating speed is controlled to be 50-80rpm; From fermentation the 20th hour to the 40th hour, the flow rate control of will ventilating was 0.6L/Lmin, and rotating speed is controlled to be 450rpm; From fermentation the 40th hour to the 60th hour, the flow rate control of will ventilating was 0.6L/Lmin, and rotating speed is controlled to be 50rpm;
It is defoamer that the present invention selects tbp, the contriver is by further test discovery, defoamer addition and the interpolation time effect for fermentation also has certain influence, finally, the contriver finds: defoamer is 0.01wt% at the addition at fermentation initial stage, begins progressively to be added into 0.1wt% from the 30h that ferments; Adopt above-mentioned technique means can effectively improve ferment effect, improve biomass.
The inventive method has been cultivated the Rhizopus oryzae bacterial strain of high yield and has been fixed to being fixed Rhizopus oryzae seed on the cotton carrier, the optimum fermentation condition that passes through to be groped carries out immobilization fermentation, yam starch transformation efficiency height, tunning biomass height, L-lactic acid yield height; The inventive method also has with low cost, advantages such as step is simple and direct, easy control except that having L-lactic acid yield advantages of higher.
Description of drawings
Fig. 1 Rhizopus oryzae fermenting potato starch prepares the process flow sheet of L-lactic acid.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Experiment material
One, substratum
(1) PDA slant medium (g/L)
Potato 200, glucose 20, agar powder 18.
(2) seed culture medium (g/L)
Glucose 60, urea 2.0, KH
2PO
40.3, MgSO
47H
2O 0.25, ZnSO
47H
2O 0.08.
(3) fermention medium (g/L)
Potato 120, KH
2PO
40.3, MgSO
47H
2O 0.25, ZnSO
47H
2O 0.08, urea 2, CaCO
360.
Two, bacterial strain: Rhizopus oryzae (Rhizopus oryzae) As3.3462, available from BeiJing ZhongKe institute of microbiology of institute.
Embodiment 1
With the 50L ferment tank is example, and Rhizopus oryzae solid state fermentation yam starch production L-lactic acid technology is as follows:
1. the preparation of Rhizopus oryzae spore
The streak inoculation of Rhizopus oryzae Rhizopus oryzae fungi in potato glucose (PDA) slant medium, was provided with 32 ℃ of culture temperature in first day, and cultivating humidity relatively is 70%, is beneficial to mycelial growth; 34 ℃ of culture temperature were set in second day, and cultivating humidity relatively is 30%, be beneficial to spore and generate, thus method inoculate 3 generation activated spawn, treat can prepare to collect after the dense spore of black covers white hypha fully.
2. the preparation of Rhizopus oryzae spore emulsion suspension liquid
Cover with the surface of mycelia with aseptic water washing, be inoculated in the 5L fermentor tank, be cultured to concentration with seed culture medium and reach 10
6Individual/ml, get final product Rhizopus oryzae spore emulsion suspension liquid.
3. immobilization seed preparation
Cotton is cut into the fritter of about 1cm * 1cm, put into the 250mL triangular flask that the 50mL seed culture medium is housed, in 121 ℃ of steam sterilizings 20 minutes, cooling back inoculating spores suspension, behind 30 ℃ of cultivation 24h, mycelium is adsorbed on the carrier, discards former seed nutrient solution, wash repeatedly three times with sterilized water, being fixed mycelium pellet is an immobilization Rhizopus oryzae seed.
4. fermentor tank immobilization fermentation
The inoculum size inoculation immobilization Rhizopus oryzae seed of 5wt%, 25L fermented liquid/50L fermentor tank; Adopt the mode of intermittently adding lime carbonate that the pH value of fermented liquid is controlled 5.0-5.5, leavening temperature is 32-34 ℃, and fermentation period is 60 hours; To the 20th hour, the flow rate control of will ventilating was 0.3L/Lmin from the fermentation beginning, and rotating speed is controlled to be 50-80rpm; From fermentation the 20th hour to the 40th hour, the flow rate control of will ventilating was 0.6L/Lmin, and rotating speed is controlled to be 450rpm; From fermentation the 40th hour to the 60th hour, the flow rate control of will ventilating was 0.6L/Lmin, and rotating speed is controlled to be 500rpm; The fermentation initial stage that is added on of defoamer tbp is 0.01wt%, progressively is added into 0.1wt% since 30h.
Ferment and detected tunning in 60 hours, wherein L-lactic acid cumulative concentration can reach the 88.90g/L tunning, and the transformation efficiency of ative starch is reached 66.74%, and biomass reaches 7.2g/L.
Embodiment 2
With the 50L ferment tank is example, and Rhizopus oryzae solid state fermentation yam starch production L-lactic acid technology is as follows:
1. the preparation of Rhizopus oryzae spore
The streak inoculation of Rhizopus oryzae Rhizopus oryzae fungi in potato glucose (PDA) slant medium, was provided with 32 ℃ of culture temperature in first day, and cultivating humidity relatively is 70%, is beneficial to mycelial growth; 34 ℃ of culture temperature were set in second day, and cultivating humidity relatively is 30%, is beneficial to spore and generates, and method inoculation 3-4 treats can prepare to collect after the dense spore of black covers white hypha fully for activated spawn thus.
2. the preparation of Rhizopus oryzae spore emulsion suspension liquid
Cover with the surface of mycelia with aseptic water washing, be inoculated in the 5L fermentor tank, be cultured to concentration with seed culture medium and reach 106/ml, get final product Rhizopus oryzae spore emulsion suspension liquid.
3. immobilization seed preparation
Cotton is cut into the fritter of about 1cm * 1cm, put into the 250mL triangular flask that the 50mL seed culture medium is housed, in 121 ℃ of steam sterilizings 20 minutes, cooling back inoculating spores suspension, behind 30 ℃ of cultivation 24h, mycelium is adsorbed on the carrier, discards former seed nutrient solution, wash repeatedly three times with sterilized water, being fixed mycelium pellet is an immobilization Rhizopus oryzae seed.
4. fermentor tank immobilization fermentation
Adopt and regulate ventilation flow rate, parameters such as mixing speed, the inoculum size inoculation immobilization Rhizopus oryzae seed of 5wt%, 25L fermented liquid/50L fermentor tank; Adopt the mode of intermittently adding lime carbonate that the pH value of fermented liquid is controlled 5.0-5.5, leavening temperature is 32-34 ℃, and fermentation period is 60 hours, and the flow rate control of will ventilating is 0.3L/Lmin, and rotating speed is controlled to be 50rpm;
Ferment and detected tunning in 60 hours, wherein L-lactic acid cumulative concentration can reach the 84.69g/L tunning, and the transformation efficiency of ative starch is reached 62.63%, and biomass reaches 6.7g/L.
Embodiment 3
With the 50L ferment tank is example, and Rhizopus oryzae solid state fermentation yam starch production L-lactic acid technology is as follows:
1. the preparation of Rhizopus oryzae spore
The streak inoculation of Rhizopus oryzae Rhizopus oryzae fungi in potato glucose (PDA) slant medium, was provided with 32 ℃ of culture temperature in first day, and cultivating humidity relatively is 70%, is beneficial to mycelial growth; 34 ℃ of culture temperature were set in second day, and cultivating humidity relatively is 30%, is beneficial to spore and generates, and method inoculation 3-4 treats can prepare to collect after the dense spore of black covers white hypha fully for activated spawn thus.
2. the preparation of Rhizopus oryzae spore emulsion suspension liquid
Cover with the surface of mycelia with aseptic water washing, be inoculated in the 5L fermentor tank, be cultured to concentration with seed culture medium and reach 10
6Individual/ml, get final product Rhizopus oryzae spore emulsion suspension liquid.
3. immobilization seed preparation
Cotton is cut into the fritter of about 1cm * 1cm, put into the 250mL triangular flask that the 50mL seed culture medium is housed, in 121 ℃ of steam sterilizings 20 minutes, cooling back inoculating spores suspension, behind 30 ℃ of cultivation 24h, mycelium is adsorbed on the carrier, discards former seed nutrient solution, wash repeatedly three times with sterilized water, being fixed mycelium pellet is an immobilization Rhizopus oryzae seed.
4. fermentor tank immobilization fermentation
Adopt and regulate ventilation flow rate, parameters such as mixing speed, the inoculum size inoculation immobilization Rhizopus oryzae seed of 5wt%, 25L fermented liquid/50L fermentor tank; Adopt the mode of intermittently adding lime carbonate that the pH value of fermented liquid is controlled 5.0-5.5, leavening temperature is 32-34 ℃, and fermentation period is 60 hours, and the flow rate control of will ventilating is 0.6L/Lmin, and rotating speed is controlled to be 500rpm;
Ferment and detected tunning in 60 hours, wherein L-lactic acid cumulative concentration reaches the 81.76g/L tunning, is 61.98% to the transformation efficiency of ative starch, and biomass is 6.69g/L.
Claims (10)
1, a kind of Rhizopus oryzae (Rhizopus oryzae) fermenting potato starch that utilizes prepares the method for L-lactic acid, may further comprise the steps:
(1) preparation Rhizopus oryzae spore; (2) the Rhizopus oryzae spore is prepared into Rhizopus oryzae spore emulsion suspension liquid; (3) Rhizopus oryzae spore emulsion suspension liquid is fixed to being fixed Rhizopus oryzae seed on the fixation support; (4) immobilization Rhizopus oryzae seed is inoculated into carries out immobilization fermentation in the fermention medium.
2, in accordance with the method for claim 1, it is characterized in that, the preparation method of the Rhizopus oryzae spore described in the step (1) comprises: in the potato glucose slant medium, first day is 32 ℃ in temperature, to cultivate humidity relatively be to cultivate under 70% the condition with Rhizopus oryzae As3.3462 streak inoculation; Second day is 34 ℃ in temperature, to cultivate humidity relatively be to cultivate under 30% the condition; Inoculate 3-4 as stated above for activated spawn, treat that spore covers white hypha, promptly.
3, in accordance with the method for claim 1, it is characterized in that, the preparation method of the Rhizopus oryzae spore emulsion suspension liquid described in the step (2) comprises: with sterilized water prepared Rhizopus oryzae spore is eluted, elutriant is inoculated into is cultured to concentration in the seed culture medium and reaches 10
6Individual/ml, promptly.
4, in accordance with the method for claim 1, it is characterized in that the fixation support described in the step (3) is a cotton; Wherein, the size of described cotton is preferably 1cm * 1cm, and the consumption of cotton carrier is preferably the 15mL/50mL substratum.
5, in accordance with the method for claim 1, it is characterized in that: the fixing means described in the step (3) comprises: cotton is cut into small pieces, put into the bottle that seed culture medium is housed, autoclave sterilization cooling back inoculating spores suspension, cultivate after 24 hours for 30 ℃, mycelium promptly is fixed on the cotton carrier;
6, according to claim 3 or 5 described methods, it is characterized in that: the consisting of of described seed culture medium: glucose 60g/L, urea 2.0g/L, KH
2PO
40.3g/L, MgSO
47H
2O 0.25g/L, ZnSO
47H
2O0.08g/L.
7, in accordance with the method for claim 1, it is characterized in that the described inoculum size described in the step (4) is 5wt%; The fermentation condition of the immobilization fermentation described in the step (4) is: in fermentor tank, ferments, and every 50L canned 25L fermention medium that ferments, the pH value of fermentation is 5.0-5.5, and leavening temperature is 32-34 ℃, and fermentation period is 60 hours.
8, in accordance with the method for claim 1, it is characterized in that the consisting of of the fermention medium described in the step (4): potato 120g/L, KH
2PO
40.3g/L, MgSO
47H
2O 0.25g/L, ZnSO
47H
2O 0.08g/L, urea 2g/L, CaCO
360g/L.
9, according to the described method of claim 1, it is characterized in that the fermentation condition described in the step (4) comprises: the fermentation beginning was to the 20th hour certainly, and the flow rate control of will ventilating is 0.3L/Lmin, and rotating speed is controlled to be 50-80rpm; From fermentation the 20th hour to the 40th hour, the flow rate control of will ventilating was 0.6L/Lmin, and rotating speed is controlled to be 450rpm; From fermentation the 40th hour to the 60th hour, the flow rate control of will ventilating was 0.6L/Lmin, and rotating speed is controlled to be 50rpm.
10, according to the described method of claim 1, it is characterized in that the fermentation condition described in the step (4) comprises: add defoamer at the fermentation initial stage, the addition of defoamer is 0.01wt%, begins progressively to be added into 0.1wt% from the 30h that ferments.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776249A (en) * | 2012-07-21 | 2012-11-14 | 太仓市茂通化建有限公司 | Method for preparing L-lactic acid by fermenting sweet potato starch with rhizopus oryzae |
| CN109468261A (en) * | 2019-01-02 | 2019-03-15 | 河南科技学院 | A kind of method of the high vigor Rhizopus oryzae seed of fast culture |
| CN110499344A (en) * | 2019-09-03 | 2019-11-26 | 浙江一清环保工程有限公司 | A method of utilizing starch wastewater, soybean processing waste water fermenting lactic acid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI297360B (en) * | 2005-04-15 | 2008-06-01 | Tatung Co Ltd | A method for producing l-lactic acid |
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2009
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI297360B (en) * | 2005-04-15 | 2008-06-01 | Tatung Co Ltd | A method for producing l-lactic acid |
Non-Patent Citations (2)
| Title |
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| 付珊珊: "米根霉发酵L-乳酸研究", 《中国优秀博硕士学位论文全文数据库 (硕士) 工程科技Ⅰ辑》 * |
| 王国英 等: "马铃薯淀粉直接发酵制备L ( + ) —乳酸工艺过程研究", 《食品工业科技》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776249A (en) * | 2012-07-21 | 2012-11-14 | 太仓市茂通化建有限公司 | Method for preparing L-lactic acid by fermenting sweet potato starch with rhizopus oryzae |
| CN109468261A (en) * | 2019-01-02 | 2019-03-15 | 河南科技学院 | A kind of method of the high vigor Rhizopus oryzae seed of fast culture |
| CN110499344A (en) * | 2019-09-03 | 2019-11-26 | 浙江一清环保工程有限公司 | A method of utilizing starch wastewater, soybean processing waste water fermenting lactic acid |
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|---|---|
| CN101463370B (en) | 2014-04-16 |
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