CN103789359B - A kind of method utilizing the fermentation of Testa Tritici hydrolysis sugar to produce oxysuccinic acid - Google Patents
A kind of method utilizing the fermentation of Testa Tritici hydrolysis sugar to produce oxysuccinic acid Download PDFInfo
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Abstract
The invention provides a kind of method utilizing the fermentation of Testa Tritici hydrolysis sugar to produce oxysuccinic acid, Testa Tritici hydrolysis sugar liquid is prepared by the method for acidolysis, then with gac, detoxification treatment is carried out to the fermentation inhibitor contained in hydrolysis sugar liquid, after selecting the Nutrious fermented material mixed fermentation that there is fine adaptive aspergillus (Aspergillusparasiticus) CICC40365 and coordinate optimization again, obtain malic acid fermentation liquid.Testa Tritici percent hydrolysis of the present invention is high, speed of response is fast, technique is simple, cost is low, and hydrolysis sugar liquid is through good detoxification treatment, fermenting process sugar loss low, adopt aspergillus to have adaptability, oxysuccinic acid transformation efficiency is high.In addition, production process of the present invention is nontoxic, environmental protection and energy saving, is applicable to commercial scale production, effectively can alleviates the energy dilemma in China and the world, remarkable in economical benefits.
Description
Technical field
The invention belongs to fermentation technical field, particularly relate to a kind of method utilizing the fermentation of Testa Tritici hydrolysis sugar to produce oxysuccinic acid.
Background technology
L MALIC ACID (L-malicacid), has another name called hydroxy-butanedioic acid, is a kind of saturated diprotic acid.Outward appearance is white crystals or crystalline powder, has stronger water absorbability, and soluble in water, ethanol has special tart flavour, and be extensively present in a kind of natural organic acids in vegetables and fruit, its content is only second to citric acid, and has special local flavor.In addition, L MALIC ACID is also the important intermediate in body metabolism, is easily absorbed by the body.Therefore, food, makeup, medicine and other fields is widely used in as excellent acidic flavoring agent and preservation agent.
Wheat is one of staple food crop of China, and its annual production is more than more than 100,000,000 tons.Testa Tritici is then the Main By product in wheat flour milling process, annual output nearly wheat processing amount 20%, this is a very large resource.Polysaccharide, protein, fat, VITAMIN, mineral matter and other components is rich in Testa Tritici.But, domesticly substantially Testa Tritici is used for fodder industry, utilization ratio is very low, rich cellulose and hemicellulose in Testa Tritici, adopt suitable hydrolysis process, be translated into the monose and then fermentation that can utilize for microorganism for organic acid, this is to the high-efficiency comprehensive utilization of wheat bran and improve its added value and have great importance.
Summary of the invention
The object of the present invention is to provide a kind of method utilizing the fermentation of Testa Tritici hydrolysis sugar to produce oxysuccinic acid, be intended to solve the low problem of existing Testa Tritici utilization ratio.
The present invention is achieved in that 1, a kind ofly utilize Testa Tritici hydrolysis sugar to ferment to produce the method for oxysuccinic acid, comprise the following steps:
(1) by Testa Tritici powder and mass concentration be 2% ~ 4% sulphuric acid soln be 1g:(7.5 ~ 12.5 by mass volume ratio) ml mixes, at 121 DEG C, react 1h, cooling, filters, and gets filtrate; In filtrate, add excess amount of Ca (OH) 2 regulate pH to 10 ~ 11, under 8000r/min condition, centrifugal 10min, gets supernatant liquor, in described supernatant liquor, acid liquid regulates pH to neutral, under 8000r/min condition, centrifugal 10min, gets supernatant liquor, obtains Testa Tritici hydrolysis sugar liquid;
(2) in described Testa Tritici hydrolysis sugar liquid, be incorporated as the gac of its quality 10%, detoxification 1h under 40 DEG C of bath temperatures, 120r/min hunting speed, by reacting liquid filtering, gets filtrate, obtaining the liquid glucose that ferments, is 100g/L by described fermentation liquid glucose evaporation concentration to total sugar concentration;
(3), after being mixed with nutritive substance by the fermentation liquid glucose that step (2) obtains, at 121 DEG C of temperature, sterilizing 20min, obtains fermentation culture; Wherein, after described fermentation liquid glucose mixes with nutritive substance, cumulative volume calculates by 1L, and the quality of described fermentation liquid glucose and each nutritive substance is respectively: the liquid glucose 100g ~ 110g that ferments, ammonium sulfate 1.9g ~ 2.2g, yeast soak powder 2.7g ~ 3.3g, potassium primary phosphate 0.1g ~ 0.15g, dipotassium hydrogen phosphate 0.2g ~ 0.3g, magnesium sulfate 0.1g ~ 0.2g, manganous sulfate 0.1g ~ 0.2g, ferrous sulfate 0.05g ~ 0.07g, calcium carbonate 70g ~ 100g;
(4) (1 ~ 10) × 10 are added toward the fermentation culture in step (3)
4cFU/mL aspergillus tubigensis seed culture fluid, 32 DEG C, air vent amount 2.5 ~ 30L/min, stirring velocity 160r/min condition bottom fermentation 5 days, collect fermented liquid, obtain the oxysuccinic acid existed with the form of calcium salt; Wherein, the volume ratio of described fermentation culture and aspergillus tubigensis seed culture fluid is 1:10.
Preferably, in step (1), described Testa Tritici powder is prepared by following steps: dried at 80 DEG C of temperature by Testa Tritici, is pulverized, crosses 60 mesh sieves by the Testa Tritici after drying, obtain Testa Tritici powder.
Preferably, in step (1), described interpolation Ca (OH) 2 is pressed powder, analytical pure, purity >=95%; Described acid solution is the sulfuric acid of mass concentration 3%.
Preferably, in step (4), described aspergillus tubigensis seed culture liquid and preparation method thereof is as follows: be that the aspergillus spore liquid of 1 × 106CFU/mL is linked into its volume ratio by concentration be in 10:1 ~ 15:1 seed culture medium, 30 DEG C ~ 32 DEG C, cultivate 16 ~ 18h under 180r/min ~ 200r/min condition; Wherein, described seed culture medium calculates by 1L and comprises following composition: glucose 30g, ammonium sulfate 1.9g ~ 2.2g, yeast leaching powder 2.7g ~ 3.3g, potassium primary phosphate 0.1g ~ 0.15g, dipotassium hydrogen phosphate 0.2g ~ 0.3g, magnesium sulfate 0.1g ~ 0.2g, manganous sulfate 0.1g ~ 0.2g, ferrous sulfate 0.05g ~ 0.07g.
Preferably, described aspergillus bacterial classification is aspergillus (Aspergillusparasiticus) CICC40365, is purchased from Chinese industrial Culture Collection.
Compare the shortcoming and defect with prior art, the present invention has following beneficial effect:
(1) the present invention adopts the method for acidolysis to prepare Testa Tritici hydrolysis sugar, and the method has the features such as percent hydrolysis is high, speed of response is fast, technique is simple, cost is low.
(2) the present invention has carried out gac detoxification treatment to hydrolyzed solution, and the fermentation inhibitory such as furfural, the acetic acid agent content in liquid glucose that obtains obviously reduces, and can be adapted to very well by aspergillus (Aspergillusparasiticus) CICC40365 adopted in the present invention; And the sugar that the method causes loss is also lower, makes the transformation efficiency of final wheat bran higher.Macroporous resin also has better detoxification characteristic, but its material itself has toxicity, only have by ethanol and NaOH solution repeatedly clean just can avoid toxicant remain, operation is loaded down with trivial details, and itself cost is also very high.Compare macroporous resin detoxification, gac does not have secondary pollution problem, and cheap.
(3) bacterial classification adopted in the present invention can utilize five simultaneously, hexose, compares other bacterial strains, utilizes the efficiency of Testa Tritici hydrolyzed solution higher.
(4) the present invention utilizes agricultural byproduct Testa Tritici to prepare L MALIC ACID for raw material, can break away from depending on unduly petroleum-based feedstock, alleviate the energy dilemma in China and the world further.And open a green, sustainable, potential production ways, production cost is low, has good economy.
(5) the present invention utilizes the method for fermentable to prepare oxysuccinic acid.Its advantage is in production process nontoxic, environmental protection and energy saving, is applicable to commercial scale production; Main product L MALIC ACID, is conducive to product separation and purification.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment 1
The preparation of A, Testa Tritici hydrolyzed solution
By Testa Tritici removal of impurities, be placed in hot air drier, dry a few hours in 80 DEG C.Testa Tritici after oven dry plant pulverizer is pulverized, and crosses 60 mesh sieves, is loaded in sealing bag for subsequent use by Testa Tritici powder.
Get Testa Tritici powder 200g, add the dilution heat of sulfuric acid 2.0L that mass concentration is 3%, stirring and evenly mixing, reacts 1h at 121 DEG C, and cooling, filtration, get filtrate, add excess amount of Ca (OH) in filtrate
2adjust pH to 10 ~ 11, under 8000r/min condition, centrifugal 10min, gets supernatant liquor, and in supernatant liquor, add the dilute sulphuric acid tune pH extremely neutrality that mass concentration is 3%, under 8000r/min condition, centrifugal 10min, gets supernatant liquor, namely obtains Testa Tritici hydrolysis sugar.
B, hydrolyzed solution detoxification treatment
200g gac is added in the Testa Tritici hydrolyzed solution that steps A obtains, is placed in shaking bath at 40 DEG C, detoxification 1h under 120r/min hunting speed condition, after reaction terminates, filters and to obtain fermentation liquid glucose; Be 100g/L by this fermentation liquid glucose Rotary Evaporators evaporation concentration to total sugar concentration.
C, medium preparing
Fermention medium is obtained after adding nutritive substance in fermentation liquid glucose after concentrating in stepb, calculate by 1L, the each concentration of component of fermention medium is: wheat bran hydrolyzed solution sugar 100g/L, ammonium sulfate 2g/L, yeast leaching powder 3g/L, potassium primary phosphate 0.1g/L, dipotassium hydrogen phosphate 0.2g/L, magnesium sulfate 0.1g/L, manganous sulfate 0.1g/L, ferrous sulfate 0.05g/L, calcium carbonate 70g/L.
5L fermention medium is pumped in the fermentor tank of 7L scale, direct 121 DEG C of sterilizing 20min in place.
Prepared by D, described aspergillus tubigensis seed culture fluid
Method is as follows: be that the aspergillus spore liquid of 1 × 106CFU/mL is linked into its volume ratio by concentration be in 10:1 ~ 15:1 seed culture medium, 30 DEG C ~ 32 DEG C, cultivate 16 ~ 18h under 180r/min ~ 200r/min condition, obtain concentration for (1 ~ 10) × 10
4the aspergillus tubigensis seed culture fluid of CFU/mL; Wherein, described seed culture medium calculates by 1L and comprises following composition: glucose 30g, ammonium sulfate 1.9g ~ 2.2g, yeast leaching powder 2.7g ~ 3.3g, potassium primary phosphate 0.1g ~ 0.15g, dipotassium hydrogen phosphate 0.2g ~ 0.3g, magnesium sulfate 0.1g ~ 0.2g, manganous sulfate 0.1g ~ 0.2g, ferrous sulfate 0.05g ~ 0.07g.
Wherein, fermented bacterium used is aspergillus (Aspergillusparasiticus) CICC40365, is purchased from Chinese industrial Culture Collection.
Oxysuccinic acid is produced in E, fermentation
By in 250mL aspergillus tubigensis seed culture fluid access fermentor tank, at temperature 32 DEG C, air vent amount 2.5L/min, mixing speed 160r/min condition bottom fermentation 5 days, in gained fermented liquid, product oxysuccinic acid exists with the form of calcium salt, malic acid concentration reaches 51.38g/L, prepares 256.90g oxysuccinic acid in 5L fermented liquid.
Embodiment 2
The preparation of A, Testa Tritici hydrolyzed solution
By Testa Tritici removal of impurities, be placed in hot air drier, dry a few hours in 80 DEG C.Testa Tritici after oven dry plant pulverizer is pulverized, and crosses 60 mesh sieves, is loaded in sealing bag for subsequent use by Testa Tritici powder.
Get Testa Tritici powder 15kg, add the dilution heat of sulfuric acid 150L that mass concentration is 3%, stirring and evenly mixing, reacts 1h at 121 DEG C, and cooling, filtration, get filtrate, add excess amount of Ca (OH) in filtrate
2adjust pH to 10 ~ 11, under 8000r/min condition, centrifugal 10min, gets supernatant liquor, and in supernatant liquor, add the dilute sulphuric acid tune pH extremely neutrality that mass concentration is 3%, under 8000r/min condition, centrifugal 10min, gets supernatant liquor, namely obtains Testa Tritici hydrolysis sugar.
B, hydrolyzed solution detoxification treatment
15kg gac is added in the Testa Tritici hydrolyzed solution that steps A obtains, is placed in shaking bath at 40 DEG C, detoxification 1h under 120r/min hunting speed condition, after reaction terminates, filters and to obtain fermentation liquid glucose; Be 100g/L by this fermentation liquid glucose Rotary Evaporators evaporation concentration to total sugar concentration.
C, medium preparing
Fermention medium is obtained after adding nutritive substance in fermentation liquid glucose after concentrating in stepb, calculate by 1L, the each concentration of component of fermention medium is: wheat bran hydrolyzed solution sugar 100g/L, ammonium sulfate 2g/L, yeast leaching powder 3g/L, potassium primary phosphate 0.1g/L, dipotassium hydrogen phosphate 0.2g/L, magnesium sulfate 0.1g/L, manganous sulfate 0.1g/L, ferrous sulfate 0.05g/L, calcium carbonate 70g/L.
450L fermention medium is pumped in the fermentor tank of 500L scale, direct 121 DEG C of sterilizing 20min in place.
Prepared by D, described aspergillus tubigensis seed culture fluid
Method is as follows: be that the aspergillus spore liquid of 1 × 106CFU/mL is linked into its volume ratio by concentration be in 10:1 ~ 15:1 seed culture medium, 30 DEG C ~ 32 DEG C, cultivate 16 ~ 18h under 180r/min ~ 200r/min condition, obtain concentration for (1 ~ 10) × 10
4the aspergillus tubigensis seed culture fluid of CFU/mL; Wherein, described seed culture medium calculates by 1L and comprises following composition: glucose 30g, ammonium sulfate 1.9g ~ 2.2g, yeast leaching powder 2.7g ~ 3.3g, potassium primary phosphate 0.1g ~ 0.15g, dipotassium hydrogen phosphate 0.2g ~ 0.3g, magnesium sulfate 0.1g ~ 0.2g, manganous sulfate 0.1g ~ 0.2g, ferrous sulfate 0.05g ~ 0.07g.
Wherein, fermented bacterium used is aspergillus (Aspergillusparasiticus) CICC40365, is purchased from Chinese industrial Culture Collection.
Oxysuccinic acid is produced in E, fermentation
By in 22.5L aspergillus tubigensis seed culture fluid access fermentor tank, at temperature 32 DEG C, air vent amount 30L/min, mixing speed 160r/min condition bottom fermentation 5 days, in gained fermented liquid, product oxysuccinic acid exists with the form of calcium salt, malic acid concentration reaches 49.22g/L, prepares 22.15kg oxysuccinic acid in 450L fermented liquid.
Embodiment 3
The preparation of A, Testa Tritici hydrolyzed solution
By Testa Tritici removal of impurities, be placed in hot air drier, dry a few hours in 80 DEG C.Testa Tritici after oven dry plant pulverizer is pulverized, and crosses 60 mesh sieves, is loaded in sealing bag for subsequent use by Testa Tritici powder.
Get Testa Tritici powder 100g, add the dilution heat of sulfuric acid 1.0L that mass concentration is 3%, stirring and evenly mixing, reacts 1h at 121 DEG C, and cooling, filtration, get filtrate, add excess amount of Ca (OH) in filtrate
2adjust pH to 10 ~ 11, under 8000r/min condition, centrifugal 10min, gets supernatant liquor, and in supernatant liquor, add the dilute sulphuric acid tune pH extremely neutrality that mass concentration is 3%, under 8000r/min condition, centrifugal 10min, gets supernatant liquor, namely obtains Testa Tritici hydrolysis sugar.
B, hydrolyzed solution detoxification treatment
100g gac is added in the Testa Tritici hydrolyzed solution that steps A obtains, is placed in shaking bath at 40 DEG C, detoxification 1h under 120r/min hunting speed condition, after reaction terminates, filters and to obtain fermentation liquid glucose; Be 100g/L by this fermentation liquid glucose Rotary Evaporators evaporation concentration to total sugar concentration.
C, medium preparing
Fermention medium is obtained after adding nutritive substance in fermentation liquid glucose after concentrating in stepb, calculate by 1L, the each concentration of component of fermention medium is: wheat bran hydrolyzed solution sugar 100g/L, ammonium sulfate 2g/L, yeast leaching powder 3g/L, potassium primary phosphate 0.1g/L, dipotassium hydrogen phosphate 0.2g/L, magnesium sulfate 0.1g/L, manganous sulfate 0.1g/L, ferrous sulfate 0.05g/L, calcium carbonate 70g/L.
1000mL fermention medium is added in 10 250mL triangular flasks, each triangular flask 100mL.Put into sterilizing 20min at Autoclave 121 DEG C.
Prepared by D, described aspergillus tubigensis seed culture fluid
Method is as follows: be 1 × 10 by concentration
6it is in 10:1 ~ 15:1 seed culture medium that the aspergillus spore liquid of CFU/mL is linked into its volume ratio, 30 DEG C ~ 32 DEG C, cultivate 16 ~ 18h under 180r/min ~ 200r/min condition, obtain concentration for (1 ~ 10) × 10
4the aspergillus tubigensis seed culture fluid of CFU/mL; Wherein, described seed culture medium calculates by 1L and comprises following composition: glucose 30g, ammonium sulfate 1.9g ~ 2.2g, yeast leaching powder 2.7g ~ 3.3g, potassium primary phosphate 0.1g ~ 0.15g, dipotassium hydrogen phosphate 0.2g ~ 0.3g, magnesium sulfate 0.1g ~ 0.2g, manganous sulfate 0.1g ~ 0.2g, ferrous sulfate 0.05g ~ 0.07g.
Wherein, fermented bacterium used is aspergillus (Aspergillusparasiticus) CICC40365, is purchased from Chinese industrial Culture Collection.
Oxysuccinic acid is produced in E, fermentation
After accessing 5mL aspergillus tubigensis seed culture fluid in each triangular flask, be placed in incubator, temperature 32 DEG C, rotating speed 200r/min condition bottom fermentation 5 days, in gained fermented liquid, product oxysuccinic acid exists with the form of calcium salt, and malic acid concentration reaches 52.14g/L.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. utilize Testa Tritici hydrolysis sugar to ferment and produce a method for oxysuccinic acid, it is characterized in that comprising the following steps:
(1) by Testa Tritici powder and mass concentration be 2% ~ 4% sulphuric acid soln be 1g by mass volume ratio: (7.5 ~ 12.5) ml mixes, at 121 DEG C, react 1h, cooling, filter, get filtrate; Excess amount of Ca (OH) is added in filtrate
2regulate pH to 10 ~ 11, under 8000r/min condition, centrifugal 10min, gets supernatant liquor, and in described supernatant liquor, acid liquid regulates pH to neutral, and under 8000r/min condition, centrifugal 10min, gets supernatant liquor, obtains Testa Tritici hydrolysis sugar liquid;
(2) in described Testa Tritici hydrolysis sugar liquid, be incorporated as the gac of its quality 10%, detoxification 1h under 40 DEG C of bath temperatures, 120r/min hunting speed, by reacting liquid filtering, gets filtrate, obtaining the liquid glucose that ferments, is 100g/L by described fermentation liquid glucose evaporation concentration to total sugar concentration;
(3) step (2) is obtained concentrated after fermentation liquid glucose mix with nutritive substance after, at 121 DEG C of temperature, sterilizing 20min, obtains fermentation culture; Wherein, after described fermentation liquid glucose mixes with nutritive substance, cumulative volume calculates by 1L, and the quality of described fermentation liquid glucose and each nutritive substance is respectively: the liquid glucose 100g ~ 110g that ferments, ammonium sulfate 1.9g ~ 2.2g, yeast soak powder 2.7g ~ 3.3g, potassium primary phosphate 0.1g ~ 0.15g, dipotassium hydrogen phosphate 0.2g ~ 0.3g, magnesium sulfate 0.1g ~ 0.2g, manganous sulfate 0.1g ~ 0.2g, ferrous sulfate 0.05g ~ 0.07g, calcium carbonate 70g ~ 100g;
(4) (1 ~ 10) × 10 are added toward the fermentation culture in step (3)
4cFU/mL aspergillus tubigensis seed culture fluid, 32 DEG C, air vent amount 2.5 ~ 30L/min, stirring velocity 160r/min condition bottom fermentation 5 days, collect fermented liquid, obtain the oxysuccinic acid existed with the form of calcium salt; Wherein, the volume ratio of described fermentation culture and aspergillus tubigensis seed culture fluid is 10: 1.
2. utilize Testa Tritici hydrolysis sugar to ferment as claimed in claim 1 and produce the method for oxysuccinic acid, it is characterized in that, in step (1), described Testa Tritici powder is prepared by following steps: dried at 80 DEG C of temperature by Testa Tritici, Testa Tritici after drying is pulverized, crossed 60 mesh sieves, obtains Testa Tritici powder.
3. utilize Testa Tritici hydrolysis sugar to ferment as claimed in claim 2 and produce the method for oxysuccinic acid, it is characterized in that, in step (1), described interpolation Ca (OH)
2for pressed powder, analytical pure, purity>=95%; Described acid solution is the sulfuric acid of mass concentration 3%.
4. utilize Testa Tritici hydrolysis sugar to ferment as claimed in claim 3 and produce the method for oxysuccinic acid, it is characterized in that, in step (4), described aspergillus tubigensis seed culture liquid and preparation method thereof is as follows: be 1 × 10 by concentration
6it is in 10: 1 ~ 15: 1 seed culture medium that the aspergillus spore liquid of CFU/mL is linked into its volume ratio, 30 DEG C ~ 32 DEG C, cultivate 16 ~ 18h under 180r/min ~ 200r/min condition; Wherein, described seed culture medium calculates by 1L and comprises following composition: glucose 30g, ammonium sulfate 1.9g ~ 2.2g, yeast leaching powder 2.7g ~ 3.3g, potassium primary phosphate 0.1g ~ 0.15g, dipotassium hydrogen phosphate 0.2g ~ 0.3g, magnesium sulfate 0.1g ~ 0.2g, manganous sulfate 0.1g ~ 0.2g, ferrous sulfate 0.05g ~ 0.07g.
5. utilize Testa Tritici hydrolysis sugar to ferment as claimed in claim 4 and produce the method for oxysuccinic acid, it is characterized in that, described aspergillus bacterial classification is aspergillus (Aspergillusparasiticus) CICC40365, is purchased from Chinese industrial Culture Collection.
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| CN108300740A (en) * | 2017-01-13 | 2018-07-20 | 山东阜丰发酵有限公司 | A method of preparing L MALIC ACID |
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| CN108300739B (en) * | 2017-01-13 | 2021-03-05 | 山东阜丰发酵有限公司 | Separation method for L-malic acid |
| CN107699537B (en) * | 2017-12-05 | 2020-08-18 | 杭州渔森农业技术开发有限公司 | Compound culture solution for culturing Platymonas subcordiformis |
| CN113913472A (en) * | 2021-09-30 | 2022-01-11 | 巢湖学院 | Method for efficiently preparing succinic acid by stimulating corynebacterium crenatum fermentation bran hydrolysate with weak current |
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| CN1059700C (en) * | 1994-05-20 | 2000-12-20 | 广东省微生物研究所 | High-yield saltant aspergillus N1-14' for L-malic acid and method for producing L-malic acid |
| CN106978454A (en) * | 2009-10-08 | 2017-07-25 | 帝斯曼知识产权资产管理有限公司 | The technique for preparing tunning from the material of lignocellulose-containing |
| CN102634545A (en) * | 2012-05-04 | 2012-08-15 | 苏州百趣食品有限公司 | Method for producing L-malic acid by fermenting sucrose molasses |
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| CN108300740B (en) * | 2017-01-13 | 2021-08-03 | 山东阜丰发酵有限公司 | Method for preparing L-malic acid |
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