CN114350718A - Method for producing malic acid by fermentation - Google Patents
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- CN114350718A CN114350718A CN202111671850.0A CN202111671850A CN114350718A CN 114350718 A CN114350718 A CN 114350718A CN 202111671850 A CN202111671850 A CN 202111671850A CN 114350718 A CN114350718 A CN 114350718A
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Images
Abstract
The invention belongs to the technical field of fermentation, and particularly discloses a method for producing malic acid by fermentation. The method of the invention can effectively prevent CO produced by fermentation by alternately performing aerobic fermentation and anaerobic fermentation in the fermentation process2Escape from the exhaust to make the part of CO2Complete access to the metabolic pathway, on the one hand enhancing CO2To avoid waste and on the other hand to reduce CO2Discharging and protecting the environment. Moreover, when fermentation enters an anaerobic fermentation stage, sterile air does not need to be introduced into the fermentation tank, so that the use amount of the sterile air is reduced, and energy is saved. Compared with the prior art, the method for producing malic acid by fermentation improves the malic acid yield and the total acid conversion rate to a certain extent, avoids the reduction of the thallus activity caused by long-term anaerobic fermentation, saves resources, and reduces the production costAnd (4) cost.
Description
Technical Field
The invention belongs to the technical field of fermentation, and particularly relates to a method for producing malic acid by fermentation.
Background
Malic acid is a dibasic acid, also known as 2-hydroxysuccinic acid, having a molecular formula of C4H6O5The apple is separated from immature apples by Scheele in 1785, and is named as L-malic acid, D-malic acid and DL-malic acid. At normal temperature and pressure, malic acid is a white crystalline powder, which is easily soluble in water and slightly soluble in ethanol. The L-malic acid has soft taste and wide application in the fields of food, medicine and the like. For example, as a third food acid agent (after citric acid and lactic acid) in the world in the food field, as an anticancer drug precursor in the medical field, and as a cosmetic additive in the chemical field, and the polymer thereof can also be used for synthesizing bioplastic.
At present, three methods are mainly used for producing malic acid: (1) chemical synthesis method: mainly uses petroleum as raw material, and the produced malic acid has high concentration, simple post-treatment operation and low price, thereby having great market competitiveness. However, the chemical synthesis method has the disadvantages of serious equipment loss, high temperature and high pressure requirement, no stereoselectivity of products and the like. (2) An enzymatic conversion method: the method has the advantages of simple process and high conversion rate, but has obvious disadvantages including higher production cost and difficult removal of residual fumaric acid, thereby limiting the large-scale application of the enzymatic conversion method. (3) And (3) a microbial fermentation method: the malic acid is produced by fermenting filamentous fungi such as myceliophthora thermophila, aspergillus oryzae, aspergillus niger, penicillium and ustilago. Compared with the two methods, the microbial fermentation method has the advantages of rich raw material sources, low cost, high edible safety and the like, and is widely concerned by researchers in recent years.
In microorganisms, there are five pathways for malic acid synthesis: (1) oxaloacetic acid is directly reduced to synthesize malic acid, and 1 molecule of CO is required to be fixed for synthesizing 1 molecule of L-malic acid by glucose through the way2The maximum theoretical yield is 2mol malic acid/mol glucose. (2) Synthesizing malic acid by TCA cyclic oxidation of citric acid, wherein 2 molecules of CO are required to be released in the method for synthesizing L-malic acid by 1 molecule of glucose2The maximum theoretical conversion is only 1mol malic acid/mol glucose. (3) Malic acid was synthesized from oxaloacetate via glyoxylate cycle with a maximum theoretical conversion of 1mol malic acid/mol glucose. (4) The malic acid is synthesized by acetyl coenzyme A and oxaloacetate through glyoxylate cycle, and the maximum theoretical conversion rate is 1.33mol of malic acid/mol of glucose. (5) Pyruvate is directly carboxylated to synthesize malic acid, and the maximum theoretical conversion rate is 2mol of malic acid/mol of glucose.
Although there are multiple pathways for malate synthesis, many are associated with CO2The fixation of (c) is closely related. Therefore, balancing the various metabolic pathways is crucial. According to the Liulong team of Jiangnan university, an Aspergillus oryzae system is taken as an initial strain, a reduction TCA pathway is strengthened by expressing pyruvate carboxylase and malate dehydrogenase, so that the malic acid yield reaches 42.3g/L, and then the glycolysis pathway and the malic acid transport capacity are further strengthened, so that the malic acid yield is increased to 165 g/L. Meanwhile, Chinese patent CN106148209B discloses a genetically modified malic acid synthesis engineering strain, which is constructed by taking myceliophthora thermophila or myceliophthora heterolepta as an initial strain and synthesizing a positive regulatory gene (expressing C4-dicarboxylic acid transporter) by introducing exogenous binary organic acid.
In summary, researchers have developed multiple malic acid fermentation strains through microbial metabolic engineering in order to improve the level of malic acid fermentation and reduce the fermentation cost. However, the prior art is rarely optimized from the point of view of the fermentation process. Therefore, there is a need to develop a new fermentation process to balance the metabolic pathways.
Disclosure of Invention
The invention mainly solves the technical problem of providing a method for producing malic acid by fermentation, which alternately carries out the steps ofOxygen fermentation and anaerobic fermentation to enhance CO2The fixation of the malic acid, the output of the malic acid and the total acid conversion rate are improved, the resources are saved, and the CO is reduced2Discharging and protecting the environment.
In order to solve the technical problems, the invention provides a method for producing malic acid by fermentation, which comprises the step of preparing a fermentation liquid containing malic acid by alternately performing aerobic fermentation and anaerobic fermentation in the fermentation process.
As a preferred embodiment of the present invention, the process starts with aerobic fermentation, and proceeds from aerobic fermentation (first) to anaerobic fermentation in the middle of fermentation, and then alternately starts with aerobic fermentation and anaerobic fermentation until fermentation is completed. Preferably, the first aerobic fermentation is switched to the anaerobic fermentation in the middle period of the fermentation, wherein the aerobic fermentation is carried out for 50-70h, more preferably 60-70h, and even more preferably 64 h.
As a preferred embodiment of the present invention, the conditions for the transition from anaerobic fermentation to aerobic fermentation in the middle and later stages of fermentation are: the sugar consumption of the fermentation liquor per hour is less than 1.155 g/L; preferably less than 1.145 g/L. The conditions for the subsequent shift from aerobic (not first) to anaerobic fermentation are: the sugar consumption of the fermentation liquor per hour is more than 1.175 g/L; preferably greater than 1.185 g/L. In order to adjust the aerobic/anaerobic fermentation stage more accurately, it is preferable to detect the change in glucose on line, for example, monitor the glucose content in the reaction system using an on-line biochemical analyzer throughout the fermentation or at the middle or late stage.
In a preferred embodiment of the present invention, the dissolved oxygen in the fermentation broth is controlled to be 10% to 30% during the aerobic fermentation. The dissolved oxygen control adopts the mode of controlling the ventilation ratio and the stirring rotating speed, wherein the ventilation ratio is 1: (0.1-0.3) V/V.m, and the stirring speed is 200-500 rpm. When aerobic fermentation is carried out, a certain amount of sterile air is introduced into the fermentation tank, and the ventilation ratio and the stirring rotation speed are controlled; after the anaerobic fermentation is switched to, the introduction of sterile air is stopped.
As a preferred embodiment of the invention, the fermented strain is a thermophilic bacterium, preferably myceliophthora thermophila. The myceliophthora thermophila is selected from a wild type strain or a genetic engineering strain. The genetic engineering strain can be a myceliophthora thermophila engineering strain introduced with an exogenous dibasic organic acid synthesis positive regulation gene (expressing C4-dicarboxylic acid transporter) disclosed in patent CN 106148209B.
As a preferred embodiment of the present invention, the fermentation temperature is controlled to be 40-50 ℃ during the fermentation. The fermentation temperature is maintained within the above range during the fermentation.
As a preferred embodiment of the invention, the pressure of the fermentation tank is controlled to be 0.02-0.05MPa in the fermentation process. The fermenter pressure was kept within the above range during the fermentation.
As a preferred embodiment of the present invention, the amount of inoculation of the fermentation is 2-10% (v/v).
As a preferred embodiment of the invention, the medium of the fermentation consists (per liter) of: glucose 180-2HPO4 0.1-0.3g，KH2PO4 0.1-0.3g，MgSO4·7H20.05-0.2g of O, 0.1-0.3mg of biotin, 0.01-0.1g of malic acid, 0.05-0.1g of NaCl, and ZnSO4·7H2O 0.05-0.2g，FeSO4·7H20.1-0.2g of O and water (such as tap water) as a solvent.
Preferably, the medium of the fermentation consists of (per liter): 220g of glucose, 180g of soybean meal and K2HPO4 0.15g，KH2PO4 0.15g，MgSO4·7H20.1g of O, 0.2mg of biotin, 0.05g of malic acid, 0.08g of NaCl and ZnSO4·7H2O 0.1g，FeSO4·7H20.15g of O and tap water as a solvent. And/or, no additional medium is required during the fermentation.
The invention provides a method for producing malic acid by fermentation, which can prevent CO produced by aerobic fermentation by setting a plurality of anaerobic fermentation stages2Escape from the exhaust to make the part of CO2Complete access to the metabolic pathway, on the one hand enhancing CO2To avoid waste and on the other hand to reduce CO2Discharging and protecting the environment. And, when fermented intoWhen the fermentation tank enters the anaerobic fermentation stage, sterile air does not need to be introduced into the fermentation tank, so that the use amount of the sterile air is reduced, and the energy is saved.
Compared with the prior art, the fermentation method provided by the invention improves the malic acid yield and the total acid conversion rate to a certain extent by alternately performing aerobic fermentation and anaerobic fermentation, avoids the reduction of thallus activity caused by long-term anaerobic fermentation, reduces the use amount of sterile air, and improves the metabolic byproduct CO2The utilization rate of the method saves resources and reduces production cost.
Drawings
FIG. 1 is a glucose concentration profile of the malic acid fermentation process of example 1;
FIG. 2 is a glucose concentration profile of the malic acid fermentation process of example 2;
FIG. 3 is a glucose concentration profile of the malic acid fermentation process of example 3;
FIG. 4 is a graph of glucose concentration change during malic acid fermentation of comparative example 1;
FIG. 5 is a graph showing the change in glucose concentration during malic acid fermentation in comparative example 2.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the specific embodiments. It will be understood by those skilled in the art that the embodiments described below are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. All other embodiments, such as modifications and substitutions, which can be obtained by one skilled in the art based on the embodiments of the present invention without any inventive step, belong to the protection scope of the present invention.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the raw materials, reagents, instruments and the like used are commercially available unless otherwise specified.
The seed culture and fermentation media were prepared as follows in the following examples:
(1) activating seeds and preparing a seed culture solution:
myceliophthora thermophila (purchased from Tianjin Industrial microbiology institute of Chinese academy of sciences) is inoculated on a solid slant culture medium under aseptic condition, cultured at constant temperature of 45 ℃ for 7 days, washed and counted by 0.8% NaCl and 0.1% Tween-80, and 2.5 × 107The spores were transferred into a 250mL triangular flask (containing 100mL of seed medium) and cultured at 45 ℃ and 150rpm for 24 hours to obtain a seed culture solution.
Wherein the slant culture medium (per liter) comprises the following components: 20g of cane sugar, 15g of agar, 2.5g of sodium citrate and KH2PO4 5g，NH4NO4 2g，MgSO4·7H20.1g of O, 0.1g of calcium chloride and 1mL of 0.1g/L biotin.
The shake flask culture medium (per liter) contains the following components: 30g of glucose, 9g of soybean meal and K2HPO4 0.15g，KH2PO4 0.15g，MgSO4·7H20.1g of O, 0.1g of calcium chloride and 1mL of 0.1g/L biotin.
(2) Preparing a fermentation medium:
the fermentation medium (per liter) consists of the following components: 220g of glucose, 180g of soybean meal and K2HPO4 0.15g，KH2PO4 0.15g，MgSO4·7H20.1g of O, 0.2mg of biotin, 0.05g of malic acid, 0.08g of NaCl and ZnSO4·7H2O 0.1g，FeSO4·7H20.15g of O, using tap water as a solvent to prepare a fermentation medium, and then sterilizing at high temperature and high pressure for later use.
Example 1
The embodiment provides a method for producing malic acid by fermentation, which comprises the following steps:
inoculating the seed culture solution into a fermentation culture medium by 6 percent of inoculation amount, fermenting at 45 ℃ and 0.03MPa, detecting the change condition of glucose in the fermentation process in real time by an online biochemical analyzer, introducing sterile air into the fermentation tank from the beginning of fermentation to within 64h of fermentation, wherein the ventilation ratio is 1: 0.3V/V.m, and the dissolved oxygen amount in the medium was controlled to 25% by controlling the stirring rotation speed (the stirring rotation speed was in the range of 200-500 rpm).
And when the fermentation is carried out for 64 hours, stopping introducing sterile air into the fermentation tank, allowing the fermentation to enter an anaerobic fermentation stage, and keeping the pressure of the fermentation tank at 0.03 MPa.
In the anaerobic fermentation stage, the change condition of glucose in the fermentation process is detected in real time by an online biochemical analyzer, when the sugar consumption of the fermentation liquor per hour is less than 1.15g/L, the fermentation is switched from anaerobic fermentation to aerobic fermentation, sterile air is introduced into the fermentation tank again, and the ventilation ratio is 1: 0.15V/V.m, and the dissolved oxygen amount in the medium was controlled to 25% by controlling the stirring rotation speed (the stirring rotation speed was in the range of 200-500 rpm).
In the aerobic fermentation stage, the change condition of glucose in the fermentation process is detected in real time by an online biochemical analyzer, when the sugar consumption of the fermentation liquor per hour is more than 1.18g/L, the fermentation is switched from aerobic fermentation to anaerobic fermentation, the introduction of sterile air into the fermentation tank is stopped, and the pressure of the fermentation tank is kept at 0.03 MPa.
Then according to the sugar consumption of the fermentation liquid per hour, aerobic fermentation and anaerobic fermentation are alternately carried out, and the fermentation is continued until the fermentation is finished (the glucose concentration change curve in the fermentation process is shown in detail in figure 1), so that the fermentation liquid containing malic acid is obtained.
The fermentation period of the example is 120h, the malic acid content is 179.2g/L, the succinic acid content is 29.3g/L, and the total acid conversion rate is 88.27%.
Example 2
The embodiment provides a method for producing malic acid by fermentation, which comprises the following steps:
inoculating 4% of seed culture solution into a fermentation culture medium, fermenting at 40 ℃ and 0.02MPa, detecting the change condition of glucose in the fermentation process in real time by an online biochemical analyzer, introducing sterile air into the fermentation tank from the beginning of fermentation to within 50h of fermentation, wherein the ventilation ratio is 1: 0.1V/V.m, and the dissolved oxygen amount in the medium was controlled to 10% by controlling the stirring rotation speed (the stirring rotation speed was in the range of 200-500 rpm).
And when the fermentation is carried out for 50 hours, stopping introducing sterile air into the fermentation tank, allowing the fermentation to enter an anaerobic fermentation stage, and keeping the pressure of the fermentation tank at 0.02 MPa.
In the anaerobic fermentation stage, the change condition of glucose in the fermentation process is detected in real time by an online biochemical analyzer, when the sugar consumption of the fermentation liquor per hour is less than 1.145g/L, the fermentation is switched from anaerobic fermentation to aerobic fermentation, sterile air is introduced into the fermentation tank again, and the ventilation ratio is 1: 0.1V/V.m, and the dissolved oxygen amount in the medium was controlled to 10% by controlling the stirring rotation speed (the stirring rotation speed was in the range of 200-500 rpm).
In the aerobic fermentation stage, the change condition of glucose in the fermentation process is detected in real time by an online biochemical analyzer, when the sugar consumption of the fermentation liquor per hour is more than 1.175g/L, the fermentation is switched from aerobic fermentation to anaerobic fermentation, the introduction of sterile air into the fermentation tank is stopped, and the pressure of the fermentation tank is kept at 0.02 MPa.
Then according to the sugar consumption of the fermentation liquid per hour, aerobic fermentation and anaerobic fermentation are alternately carried out, and the fermentation is continued until the fermentation is finished (the glucose concentration change curve in the fermentation process is shown in detail in figure 2), so as to obtain the fermentation liquid containing malic acid.
The fermentation period of the example is 123h, the malic acid content is 176.8g/L, the succinic acid content is 27.3g/L, and the total acid conversion rate is 86.41%.
Example 3
The embodiment provides a method for producing malic acid by fermentation, which comprises the following steps:
inoculating 8% of seed culture solution into a fermentation culture medium, fermenting at 50 ℃ and 0.05MPa, detecting the change condition of glucose in the fermentation process in real time by an online biochemical analyzer, introducing sterile air into the fermentation tank from the beginning of fermentation to within 70h of fermentation, wherein the ventilation ratio is 1: 0.3V/V.m, and the dissolved oxygen amount in the medium was controlled to 30% by controlling the stirring rotation speed (the stirring rotation speed was in the range of 200-500 rpm).
And when the fermentation is carried out for 70 hours, stopping introducing sterile air into the fermentation tank, allowing the fermentation to enter an anaerobic fermentation stage, and keeping the pressure of the fermentation tank at 0.05 MPa.
In the anaerobic fermentation stage, the change condition of glucose in the fermentation process is detected in real time by an online biochemical analyzer, when the sugar consumption of the fermentation liquor per hour is less than 1.155g/L, the fermentation is switched from anaerobic fermentation to aerobic fermentation, sterile air is introduced into the fermentation tank again, and the ventilation ratio is 1: 0.3V/V.m, and the dissolved oxygen amount in the medium was controlled to 30% by controlling the stirring rotation speed (the stirring rotation speed was in the range of 200-500 rpm).
In the aerobic fermentation stage, the change condition of glucose in the fermentation process is detected in real time by an online biochemical analyzer, when the sugar consumption of the fermentation liquor per hour is more than 1.185g/L, the fermentation is switched from aerobic fermentation to anaerobic fermentation, the introduction of sterile air into the fermentation tank is stopped, and the pressure of the fermentation tank is kept at 0.05 MPa.
Then according to the sugar consumption of the fermentation liquid per hour, aerobic fermentation and anaerobic fermentation are alternately carried out, and the fermentation is continued until the fermentation is finished (the glucose concentration change curve in the fermentation process is shown in detail in figure 3), so as to obtain the fermentation liquid containing malic acid.
The fermentation period of the embodiment is 121h, the malic acid content is 175.4g/L, the succinic acid content is 29.2g/L, and the total acid conversion rate is 86.62%.
Comparative example 1
The comparative example provides a conventional method for producing malic acid by fermentation, comprising the following steps:
inoculating the seed culture solution into a fermentation culture medium by 6 percent of inoculation amount, fermenting at 45 ℃ and 0.03MPa, detecting the change condition of glucose in the fermentation process in real time by an online biochemical analyzer, introducing sterile air into the fermentation tank from the beginning of fermentation to within 18 hours of fermentation, wherein the ventilation ratio is 1: 0.3V/V.m, and the dissolved oxygen amount in the medium was controlled to 25% by controlling the stirring rotation speed (the stirring rotation speed was in the range of 200-500 rpm).
After 18h of fermentation, the aeration ratio was controlled to be 1: 0.15V/V.m, changing the stirring speed (within the range of 200-500 rpm) to control the dissolved oxygen to be 10%, and continuing to ferment until the fermentation is finished (the glucose concentration change curve in the fermentation process is shown in detail in FIG. 4), thereby obtaining the fermentation liquid containing malic acid.
The fermentation period of the comparative example is 118h, the malic acid content is 173.2g/L, the succinic acid content is 28.4g/L, and the total acid conversion rate is 85.35%.
Comparative example 2
The comparative example provides a conventional method for producing malic acid by fermentation, comprising the following steps:
inoculating the seed culture solution into a fermentation culture medium by 6 percent of inoculation amount, fermenting at 45 ℃ and 0.03MPa, detecting the change condition of glucose in the fermentation process in real time by an online biochemical analyzer, introducing sterile air into the fermentation tank from the beginning of fermentation to within 64h of fermentation, wherein the ventilation ratio is 1: 0.3V/V.m, and the dissolved oxygen amount in the medium was controlled to 25% by controlling the stirring rotation speed (the stirring rotation speed was in the range of 200-500 rpm).
And when the fermentation is carried out for 64 hours, stopping introducing sterile air into the fermentation tank, allowing the fermentation to enter an anaerobic fermentation stage, keeping the pressure of the fermentation tank at 0.03MPa, and continuing fermenting until the fermentation is finished (the glucose concentration change curve in the fermentation process is shown in detail in figure 5), thereby obtaining the fermentation liquor containing malic acid.
The fermentation period of the comparative example is 172h, the malic acid content is 64.6g/L, the succinic acid content is 8.7g/L, and the total acid conversion rate is 30.08%.
The comparison results show that the method for producing malic acid by fermentation is simple and easy to implement, and the CO is enhanced by arranging a plurality of anaerobic fermentation stages (namely, aerobic fermentation and anaerobic fermentation are alternately carried out in the fermentation process)2The method improves the output of the malic acid and the total acid conversion rate to a certain extent, saves resources and is green and environment-friendly. Moreover, when fermentation enters an anaerobic fermentation stage, sterile air does not need to be introduced into the fermentation tank, so that the using amount of the sterile air is reduced, the energy consumption is saved, and the production cost is reduced.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for producing malic acid by fermentation, which is characterized in that the method prepares a fermentation liquor containing malic acid by alternately performing aerobic fermentation and anaerobic fermentation in the fermentation process.
2. The method for producing malic acid by fermentation according to claim 1, wherein the method comprises aerobic fermentation, and the aerobic fermentation is switched to anaerobic fermentation in the middle of fermentation, and then the aerobic fermentation and the anaerobic fermentation are alternately performed until the fermentation is finished; preferably, the middle stage of fermentation is aerobic fermentation for 50-70h, and more preferably aerobic fermentation for 64 h.
3. A method for the fermentative production of malic acid according to claim 1 or 2, wherein the conditions for the transition from anaerobic fermentation to aerobic fermentation in the middle and later stages of fermentation are: the sugar consumption of the fermentation liquor per hour is less than 1.155 g/L; the conditions for the transition from aerobic to anaerobic fermentation were then: the sugar consumption of the fermentation liquor per hour is more than 1.175 g/L.
4. The method for producing malic acid by fermentation according to claim 1, wherein during the aerobic fermentation, the dissolved oxygen in the fermentation liquid is controlled to 10% -30%; preferably, the dissolved oxygen control adopts a mode of controlling a ventilation ratio and a stirring rotating speed, wherein the ventilation ratio is 1: (0.1-0.3) V/V.m, and the stirring speed is 200-500 rpm.
5. Method for the fermentative production of malic acid according to claim 1, wherein the fermented strain is a thermophilic bacterium, preferably myceliophthora thermophila, selected from a wild-type strain or a genetically engineered strain.
6. The method for producing malic acid by fermentation according to claim 1 or 5, wherein the fermentation temperature is controlled to 40-50 ℃ during the fermentation.
7. The method for producing malic acid by fermentation according to claim 1 or 5, wherein the fermentation tank pressure is controlled to 0.02-0.05MPa during the fermentation.
8. The method for the fermentative production of malic acid according to claim 1 or 5, wherein the amount of inoculation for the fermentation is 2 to 10% (v/v).
9. A method for the fermentative production of malic acid according to claim 1 or 5, wherein the fermentation medium consists of the following constituents per liter: glucose 180-2HPO4 0.1-0.3g，KH2PO4 0.1-0.3g，MgSO4·7H20.05-0.2g of O, 0.1-0.3mg of biotin, 0.01-0.1g of malic acid, 0.05-0.1g of NaCl, and ZnSO4·7H2O 0.05-0.2g，FeSO4·7H20.1-0.2g of O and water as a solvent.
10. A method of fermentatively producing malic acid according to claim 9, wherein the culture medium of the fermentation consists of, per liter: 220g of glucose, 180g of soybean meal and K2HPO4 0.15g，KH2PO4 0.15g，MgSO4·7H20.1g of O, 0.2mg of biotin, 0.05g of malic acid, 0.08g of NaCl and ZnSO4·7H2O 0.1g，FeSO4·7H20.15g of O, and tap water as a solvent; and/or, no additional medium is required during the fermentation.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5869301A (en) * | 1995-11-02 | 1999-02-09 | Lockhead Martin Energy Research Corporation | Method for the production of dicarboxylic acids |
| WO2008143147A1 (en) * | 2007-05-11 | 2008-11-27 | Sumitomo Chemical Company, Limited | Method for producing malic acid |
| US20110045559A1 (en) * | 2007-05-18 | 2011-02-24 | Aaron Adriaan Winkler | Malic acid production in recombinant yeast |
| CN102352382A (en) * | 2011-10-13 | 2012-02-15 | 南京工业大学 | Method producing malic acid through double-phase fermentation |
| CN105543294A (en) * | 2016-03-07 | 2016-05-04 | 南京工业大学 | Method for conversion synthesis of malic acid by using low-activity succinic acid production strains |
| CN106337064A (en) * | 2016-04-07 | 2017-01-18 | 南京工业大学 | Production method of L-malic acid |
| CN110241147A (en) * | 2019-07-05 | 2019-09-17 | 安徽丰原发酵技术工程研究有限公司 | A kind of method of fermenting and producing L MALIC ACID coproduction succinic acid |
| CN112626134A (en) * | 2020-12-31 | 2021-04-09 | 安徽丰原发酵技术工程研究有限公司 | Method for preparing malic acid by using citric acid fermentation tail gas |
-
2021
- 2021-12-31 CN CN202111671850.0A patent/CN114350718A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5869301A (en) * | 1995-11-02 | 1999-02-09 | Lockhead Martin Energy Research Corporation | Method for the production of dicarboxylic acids |
| WO2008143147A1 (en) * | 2007-05-11 | 2008-11-27 | Sumitomo Chemical Company, Limited | Method for producing malic acid |
| US20110045559A1 (en) * | 2007-05-18 | 2011-02-24 | Aaron Adriaan Winkler | Malic acid production in recombinant yeast |
| CN102352382A (en) * | 2011-10-13 | 2012-02-15 | 南京工业大学 | Method producing malic acid through double-phase fermentation |
| CN105543294A (en) * | 2016-03-07 | 2016-05-04 | 南京工业大学 | Method for conversion synthesis of malic acid by using low-activity succinic acid production strains |
| CN106337064A (en) * | 2016-04-07 | 2017-01-18 | 南京工业大学 | Production method of L-malic acid |
| CN110241147A (en) * | 2019-07-05 | 2019-09-17 | 安徽丰原发酵技术工程研究有限公司 | A kind of method of fermenting and producing L MALIC ACID coproduction succinic acid |
| CN112626134A (en) * | 2020-12-31 | 2021-04-09 | 安徽丰原发酵技术工程研究有限公司 | Method for preparing malic acid by using citric acid fermentation tail gas |
Non-Patent Citations (1)
| Title |
|---|
| FABRIZIO FATICHENTI等: "Malic acid production and consumption by selected of Saccharomyces cerevisiae under anaerobic and aerobic conditions", 《APPL MICROBIOL BIOTECHNOL》, vol. 19, 31 December 1984 (1984-12-31), pages 427 - 429 * |
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