CN1059700C - High-yield saltant aspergillus N1-14' for L-malic acid and method for producing L-malic acid - Google Patents

High-yield saltant aspergillus N1-14' for L-malic acid and method for producing L-malic acid Download PDF

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CN1059700C
CN1059700C CN94105633A CN94105633A CN1059700C CN 1059700 C CN1059700 C CN 1059700C CN 94105633 A CN94105633 A CN 94105633A CN 94105633 A CN94105633 A CN 94105633A CN 1059700 C CN1059700 C CN 1059700C
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malic acid
acid
described production
spore
substratum
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CN1112160A (en
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吴清平
周小燕
陈素云
钟瑜
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Guangdong Institute of Microbiology
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Guangdong Institute of Microbiology
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Abstract

The present invention discloses high-yield mutant aspergillus (Aspergillus sp.) N1-14' for L-malic acid and a method for producing the L-malic acid by the strains, which relates to industrial microorganisms and utilization thereof. The aspergillus N1-14'CCTCCM94103 provided by the present invention can be used for the direct fermentation of saccharine materials so as to generate the L-malic acid. The acid producing strains ferment the saccharine materials for 100 to 120 hours on a 20m<3> of pot so as to produce 75 to 85 g/l of acid, acid producing velocity is in a scope ranging from 0.65 to 0.71 g/l. h, and conversion rate for sugar is in a scope ranging from 65 to 68%. The present invention also provides a set of feasible industrialization process flows comprising a fermentation process and an extraction process for the L-malic acid, and has the outstanding characteristics of reasonable and simple process flows and very low fumaric acid contents. L-malic acid contents in products are larger than or equal to 99*100<-2>, and the fumaric acid contents are smaller than 0.2*10<-2>.

Description

L MALIC ACID high productive mutant aspergillus N1-14' and the method for producing L MALIC ACID
But the present invention relates to a kind of direct fermentation saccharine material produces the microorganism strains of L MALIC ACID and utilizes this bacterial strain to produce the method for L MALIC ACID.
The L MALIC ACID formal name used at school is L-hydroxy-butanedioic acid (L-hydroxysuccinic acid), is a very promising product after citric acid, can be widely used in all respects such as food, medicine, printing and dyeing, chemical industry, plating and material of construction.
Up to now, the approach of manufacturing L MALIC ACID roughly has 5: 1, extract from plant stem-leaf and fruit; 2, split the DL-oxysuccinic acid of chemosynthesis; 3, utilize the fumarase conversion fumaric acid of microorganisms to be L MALIC ACID (abbreviation enzyme process); 4, utilize the microbial fermentation saccharine material to produce L MALIC ACID; 5, utilize the non-saccharine material of microbial fermentation to produce L MALIC ACID.At present, the L MALIC ACID product of domestic and international market mainly adopts Production by Enzymes, yet because the high purity fumaric acid price of chemosynthesis is higher, add the more high factor of fumaric acid content in the product, the method for direct fermentation saccharine material production L MALIC ACID is being inquired into always and developed in a lot of countries.Nineteen ninety Israelis Battat etc., adopt flavus (Aspergillus flavus) about 192 hours of 16L fermentor tank top fermentation glucose, produce sour 113g/L, rate of producing acid 0.59g/L.h, but this bacterial classification produces flavacin, therefore can not be applied to produce (Biotechnology and Bioengineering 37:1108～1116).Day in 1991 disclosure special permission communique (flat 3-180187) was announced employing barnyard grass wart ustilago shake flask fermentation glucose such as (Ustilago crus-galli) 168 hours, produced sour 52g/L, the result of rate of producing acid 0.31g/L.h.The flavus shake flask fermentation rice hydrolysis sugar that (Food science 2:12-19) such as Chinese Wuxi Light Industry College in 1988 its honor of gold reported to adopt the aspergillus flavus not producing toxin 140 hours, produce sour 60-70g/L, rate of producing acid 0.43-0.50g/L.h, the result that sour 44g/L is on average produced in the pilot scale of 500L scale.Above-mentioned L MALIC ACID produces acid yield of bacterium and the level that the zymotechnique technology does not all reach suitability for industrialized production thereof.
The purpose of this invention is to provide a kind of energy direct fermentation saccharine material and produce L MALIC ACID, rate of producing acid reaches sugared transformation efficiency height, can carry out the microorganism strains of suitability for industrialized production L MALIC ACID and utilize this microorganism strains to produce the feasible industrialized preparing process of L MALIC ACID.
Microorganism strains provided by the present invention is L MALIC ACID high productive mutant aspergillus (AspergillusSP.) N1-14 ' through seed selection, the sample of this aspergillus N1-14 ' is preserved in Chinese typical culture collection center (CCTCC) on March 22nd, 1994, is numbered: M94013.This bacterial strain is designated hereinafter simply as: N1-14 ' CCTCC M94013.
N1-14 ' CCTCC M94013 cultivated the about 1-2cm of its colony diameter 5-7 days down for 28-32 ℃ on Cha Shi (Czapek) substratum.At early growth period, the bacterium colony ivory buff is the light brown yellow after old, and is fine and close, do not have special odor, and reverse side is faint yellow, and tool is gauffer significantly.Mycelia short wool shape, white, have every; Conidiophore vertically grows from the podocyte that wall thickness expands, nothing every, top is thicker, and ultimate swelling becomes subglobose vesicle, bears stigma radially from all surfaces of vesicle, stigma individual layer or bilayer, conidium is born in the stigma top, is initially oval or foreign pyriform, and ripe back is spherical or subsphaeroidal, band tawny and coarse spinule, and have not really significantly double wall.The growth suitable condition of N1-14 ' CCTCC M94013 is: 1, cultivate material: 20 * 10 -2Potato juice 500-1000ml/L, glucose 10-30g/L, KH 2PO 42-5g/L, MgSO 47H 2O 1-3g/L, agar 15-20g/L; 2, culture temperature: 25-35 ℃; 3, cultivate pH value: 5.5-7.5.This mutant strain does not produce aflatoxin after measured, the nontoxic level in its true border of whole beer acute poisoning test, and chronic accumulation test is accumulated a little less than belonging to.
Utilize N1-14 ' CCTCC M94013 direct fermentation saccharine material can produce L MALIC ACID, the present invention on the basis of shake flask test, by 1.2L, 20L, 240L, 1.7m 3And 20m 3The progressively expanding test of fermentor tank has formed the suitable technological condition for fermentation that utilizes N1-14 ' CCTCC M94013 direct fermentation saccharine material to produce L MALIC ACID, and has set up the feasible method that extracts L MALIC ACID from fermented liquid.
The method of production L MALIC ACID provided by the present invention, its principal feature are to utilize N1-14 ' CCTCC M94013 that saccharine material is carried out direct fermentation.Said saccharine material can be glucose, amylum hydrolysate of the sugar, and sucrose and starch etc. are raw material as adopting starch, then need add α-Dian Fenmei and carry out liquefaction processing.
The technological process of utilizing N1-14 ' CCTCC M94013 direct fermentation saccharine material to produce L MALIC ACID comprises:
1, fermentation technology process:
1.1,25-35 ℃ of temperature range and to add its major ingredient be glucose and KH 2PO 4The condition of product spore promotor under, make N1-14 '-CCTCC M94013 a large amount of spore, i.e. strain preparation of producing in the triangular flask bran mass;
1.2, sophisticated spore inserted the substratum that contains lime carbonate in the seeding tank carry out seed culture;
1.3, sophisticated seed moved in the substratum that contains lime carbonate in the breeding jar carries out the production of deep-layer liquid spore;
1.4, the seed of suitable cell age and sophisticated spore moved in the fermentor tank contain in the substratum of lime carbonate, saccharine material is carried out direct fermentation obtains karusen;
2, L MALIC ACID extraction process process:
2.1, directly the vacuum filtration karusen is received to such an extent that contain the L MALIC ACID calcium of thalline;
2.2, contain the L MALIC ACID calcium of thalline with no pozzuolite acid acidolysis, and acid hydrolysis solution is carried out removal of impurities and decolouring;
2.3, the acid hydrolysis solution vacuum filtration after the above-mentioned removal of impurities decolouring is removed thalline and calcium sulfate, obtain the thick solution of L MALIC ACID;
2.4, in the thick solution of L MALIC ACID, add the smart filter in flocculation agent clarification back, smart filtrate is purified by anion-cation exchange resin;
2.5, the L MALIC ACID solution after will purifying concentrates, crystallization, at last with centrifugal receive wet crystal carry out drying.
The present invention adopts in the strain preparation stage and produces spore promotor, can greatly improve spore output, generally can make bran mass produce spore by 1.0 * 10 3/ gram substratum (dry weight) brings up to 4.0 * 10 3/ gram substratum (dry weight), the major ingredient that produces spore promotor is glucose and KH 2PO 4, both ratios are glucose: KH 2PO 4=1: 0.005-0.05 (weight part).Employed bran mass composition (contained grammes per square metre g/kg calculates by the per kilogram substratum) is: fresh wheat bran 400-600, surplus is a water.The consumption that adds the product spore promotor in the substratum is (1-10) * 10 of bran mass -2, and with (3-6) * 10 -2For good.The suitable product spore temperature range of N1-14 ' CCTCC M94013 is 25-35 ℃, and producing the spore temperature range preferably is 28-32 ℃.
Deep-layer liquid spore production stage (step 1.3) purpose that the present invention adopts is the spore output problem that further solves fungi fermentation, can make liquid miospore output reach 5.0 * 10 3Individual/milliliter.The used substratum composition (calculating by every liter of contained grammes per square metre g/L of substratum) of suitable seed culture and deep-layer liquid spore production stage is: reducing sugar 30-70, (NH 4) 2SO 41.0-5.0, KH 2PO 40.1-1.0, MgSO 47H 2O 0.2-1.2, MnSO 4H 2O0.2-1.2, FeSO 47H 2O 0.3-1.5, CaCO 3, 20-60.Nature pH, wherein (NH 4) SO 4Separately sterilization.
In step 1.4, the substratum composition of suitable fermentation and acid (calculating by every liter of contained grammes per square metre g/L of substratum) is: reducing sugar 115-135, (NH 4) 2SO 41.0-5.0, KH 2PO 40.1-1.0, MgSO 47H 2O 0.2-1.2, MnSO 4H 2O 0.2-1.2, FeSO 47H 2O 0.3-1.5, CaCO 370-120.Nature pH, wherein (NH 4) 2SO 4Separately sterilization.
The present invention mainly adopts polyacrylamide in order to the flocculation agent (step 2.4) of the thick solution of clarification L MALIC ACID, and its consumption is 1-30mg/L (the thick solution of L MALIC ACID), and optimum amount is 5-10mg/L (the thick solution of L MALIC ACID).
F-1 citric acid anionite-exchange resin can be adopted in order to the anionite-exchange resin (step 2.4) that purifies L MALIC ACID solution, also A561 anionite-exchange resin can be adopted.Cationic exchange fat can adopt 732 Zeo-karbs or C26 Zeo-karb.
The specific embodiment of utilizing N1-14 ' CCTCC M94103 direct fermentation saccharine material to produce L MALIC ACID is:
1, fermentation technology process:
1.1, strain preparation: after the potato dextrose agar that will newly prepare (PDA) the test tube slant inoculation, putting 28-32 ℃ cultivated 5-7 days down, after treating the spore maturation, add sterilized water, the spore suspension that makes is inserted in the triangular flask bran mass, be added with in the bran mass and produce spore promotor, put 28-32 ℃ and cultivate after 5-7 days down, be placed in the 4-6 ℃ of refrigerator, preserve standby;
1.2, seed culture: sophisticated wheat bran is inserted in the seeding tank, keeps 30-37 ℃ of temperature, stirring velocity 200-300r/m, air flow 0.4-0.7vvm, tank pressure 0.01-0.1MPa cultivated 20-30 hour;
1.3, the deep-layer liquid spore produces: sophisticated seed is moved in the breeding jar, keeps 33-38 ℃ of temperature, stirring velocity 150-250r/m, air flow 0.3-0.6vvm, tank pressure 0.01-0.1MPa cultivated 18-23 hour;
1.4, ferment tank: seed that contains suitable cell age and sophisticated spore are moved in the fermentor tank saccharine material direct fermentation, keep 32-37 ℃ of temperature, stirring velocity 50-150r/m, air flow 0.05-0.3vvm, tank pressure 0.01-0.1MPa cultivated 100-120 hour, treated that residual sugar drops to 5g/L, stop fermentation, obtain karusen.
2, L MALIC ACID extraction process process:
2.1, the direct sophisticated karusen of vacuum filtration, and the residual sugar in the filter cake (the L MALIC ACID calcium that contains thalline) is clean with a small amount of no salt solution;
2.2, with the diluted acid water of 1-3 times of filter cake weight with filter cake furnishing pulpous state, add (20-60) * 10 -2No pozzuolite acid, keep stirring velocity 50-100r/m, temperature 30-50 ℃ mild conditions carries out acidolysis to L MALIC ACID calcium, adopts double-tube method to check the acidolysis terminal point, and acid hydrolysis solution is carried out removal of impurities and decolouring;
2.3, vacuum filtration removes thalline and calcium sulfate, and the L MALIC ACID in the filter cake is eluted with a small amount of no salt solution;
2.4, after the thick solution of L MALIC ACID adds the flocculation agent clarification, carry out the essence filter, then smart filtrate is delivered to header tank, introduce 732 cation exchange resin columns or C26 cation exchange resin column earlier, introduce special-purpose anion-exchange resin column of F-1 citric acid or A561 anion-exchange resin column then, positive post effluent liquid is mainly checked and is had or not Fe 3+And Ca 2+Spill, cloudy post effluent liquid is mainly checked and is had or not SO 4 2-And Cl -Spill.
2.5, the L MALIC ACID solution after purifying is introduced in the upgrading tower, under 50-70 ℃, be concentrated into 1000-1300g/L, again concentrated solution is introduced in the crystallizer, be cooled to 5-20 ℃, add an amount of crystal seed, crystallization under 1-10r/m slowly stirs, with whizzer crystal is separated from mother liquor then, and it is clean with a small amount of salt-free water washing, crystal after will washing is again put in the boiling-bed drying, dry under inlet temperature 30-80 ℃, at last dried L MALIC ACID packing is promptly finished whole technological processs.
The present invention utilizes good L MALIC ACID high productive mutant N1-14 ' CCTCC M94103, at 20m 3Jar top fermentation saccharine material 100-120 hour produces sour 75-85g/L, and rate of producing acid 0.65-0.71g/Lh to sugared transformation efficiency 65-68%, is better than the acid yield that domestic and international existing L MALIC ACID produces bacterium greatly.
The Technology of production L MALIC ACID provided by the present invention, technological process is simplified, and the L MALIC ACID rate of recovery reaches 50-60%, the content of L MALIC ACID 〉=99 * 10 in the product -2, its outstanding characteristics are that fumaric acid content is extremely low, less than 0.2 * 10 -2
Embodiment one
Produce L MALIC ACID with sucrose for raw material direct fermentation.
With the N1-14 ' slant strains of preserving in 4 ℃ of refrigerators, be transferred on the potato dextrose agar test tube slant of new preparation, put 28-32 ℃ and cultivate after 6 days down, obtain fresh test tube slant bacterial classification.Potato dextrose agar composition (calculating): 20 * 10 by every liter of contained amount of substratum -2Potato juice 800ml, glucose 20g, KH 2PO 43g, MgSO 47H 2O 2g, pH6.5, agar 18g.Then,, add sterilized water, make spore suspension, insert in the triangular flask bran mass fresh sophisticated test tube slant bacterial classification.The composition of bran mass (pressing the contained grammes per square metre g/kg of per kilogram substratum calculates): fresh wheat bran 500, water 500.Add product spore promotor wherein composition be glucose: KH 2PO 4=1: 0.01, its consumption is 3 * 10 of a bran mass -2Postvaccinal substratum is put 28-32 ℃ and is cultivated after 6 days down, and its spore output is 4.5 * 10 3Individual/gram substratum (dry weight).
The seeding tank volume is 600L, and dress liquid 450L adopts seed culture medium.The composition of seed culture medium (calculating): sucrose 50, (NH by every liter of contained grammes per square metre g/L of substratum 4) 2SO 42.0, KH 2PO 40.3, MgSO 47H 2O0.5, MnSO 4H 2O 0.5, FeSO 47H 2O 0.5, CaCO 340; Nature pH, wherein (NH 4) 2SO 4Separately sterilization.Seed culture medium adopts 121 ℃ of sterilizations 25 minutes, inserts the wheat bran kind after the cooling, keeps 33-35 ℃ of temperature, stirring velocity 230-240r/m, and air flow 0.5vvm, tank pressure 0.03-0.05MPa cultivated 23 hours, and residual sugar 10g/L moves in the breeding jar and cultivates.
The breeding tank volume is 3m 3, dress liquid 2.4m 3Adopt the deep layer spore to produce substratum (composition is identical with seed culture medium), sterilized 20 minutes for 121 ℃, the cooling back moves into the mature seed nutrient solution, keeps 35-36 ℃ of temperature, stirring velocity 200-220r/m, air flow 0.4vvm, tank pressure 0.03-0.05MPa cultivated 20 hours, and spore output is 6.7 * 10 5Individual/milliliter, residual sugar 8g/L moves into fermentor tank.
The fermentor tank volume is 20m 3, dress liquid 16m 3, adopt the fermentation and acid substratum.Fermentation and acid substratum composition (calculating): sucrose 115, (NH by every liter of contained grammes per square metre g/L of substratum 4) 2SO 42.0, KH 2PO 40.3, MgSO 47H 2O 0.5, MnSO 4H 2O 0.5, FeSO 47H 2O 0.5, CaCO 390; Nature pH, wherein (NH 4) 2SO 4Separately sterilization.121 ℃ of sterilizations of fermentation and acid substratum 10 minutes, the cooling back moves into the seed and the sophisticated spore nutrient solution of suitable cell age, keep 34-35 ℃ of temperature, stirring velocity 80-100r/m, air flow 0.1vvm, tank pressure 0.03-0.05MPa, cultivated 107 hours, producing acid is 76.1g/L, and residual sugar is 5g/L, stop fermentation, carry out L MALIC ACID and extract.
Put directly vacuum filtration karusen of jar back, obtain to contain the L MALIC ACID calcium filter cake of thalline; Behind filter cake usefulness diluted acid water (no pozzuolite acid) furnishing pulpous state,, carry out acidolysis, removal of impurities and decolouring under the mild conditions of stirring velocity 70-80r/m 40 ℃ of temperature; After removing by filter gypsum tailings and thalline etc., add polyacrylamide flocculant and clarify, the consumption of flocculation agent is 10mg/L; The thick solution of L MALIC ACID carries out the essence filter and by behind F-1 citric acid anion-exchange resin column and 732 cation exchange resin columns, under 60 ℃, adopts two-stage to concentrate, when being concentrated into 1200g/L, introduce in the crystallizer, under 5-6r/m stirs, be cooled to 10 ℃, add an amount of crystal seed and carry out crystallization; After the crystallization, with whizzer crystal separation is come out, mother liquor and wash water carry out reconcentration or purifying treatment, and crystal is then packed behind inlet temperature 50-60 ℃ of following fluidized drying immediately.
Embodiment two
Produce L MALIC ACID with starch for raw material direct fermentation.
With the N1-14 ' slant strains of preserving in 4 ℃ of refrigerators, be transferred on the potato dextrose agar test tube slant of new preparation, put 25-27 ℃ and cultivated 7 days down, obtain fresh test tube slant bacterial classification.Potato dextrose agar composition (calculating): 20 * 10 by every liter of contained amount of substratum -2Potato juice 500ml, glucose 10g, KH 2PO 42g, MgSO 47H 2O 1g, pH5.0, agar 15g.Then,, add sterilized water, make spore suspension, insert in the triangular flask bran mass fresh sophisticated test tube slant bacterial classification.The composition of bran mass (pressing the contained grammes per square metre g/kg of per kilogram substratum calculates): fresh wheat bran 600, water 400.Add the composition glucose of product spore promotor wherein: KH 2PO 4=1: 0.005, its consumption is 5 * 10 of a bran mass -2Postvaccinal substratum is put 25-27 ℃ and is cultivated after 7 days down, and its spore output is 4.3 * 10 3Individual/gram substratum (dry weight).
Starch is wanted the starch slurry of first furnishing 300-350g/L concentration before directly using, when being warming up to 50-55 ℃, add the α-Dian Fenmei of 5-10 units/gram starch and the CaCl of 1g/L 2, and then be warming up to and carry out liquefaction processing under 85-90 ℃.
The seeding tank volume is 600L, and dress liquid 450L adopts seed culture medium.The composition of seed culture medium (calculating): starch 70, (NH by every liter of contained grammes per square metre g/L of substratum 4) 2SO 41.0, KH 2PO 40.1, MgSO 47H 2O 0.2, MnSO 4H 2O 0.2, FeSO 47H 2O 0.3, CaCO 320; Nature pH, wherein (NH 4) 2SO 4Separately sterilization.Seed culture medium adopts 121 ℃ of sterilizations 25 minutes, inserts the wheat bran kind after the cooling, keeps 30-32 ℃ of temperature, stirring velocity 200-220r/m, and air flow 0.4vvm, tank pressure 0.08-0.1MPa cultivated 30 hours, and residual sugar 10g/L moves in the breeding jar and cultivates.
The breeding tank volume is 3m 3, dress liquid 2.4m 3Adopt the deep layer spore to produce substratum (composition is identical with seed culture medium), sterilized 20 minutes for 121 ℃, the cooling back moves into the mature seed nutrient solution, keeps 33-34 ℃ of temperature, stirring velocity 150-180r/m, air flow 0.3vvm, tank pressure 0.08-0.1MPa cultivated 23 hours, and spore output is 5.5 * 10 5Individual milliliter, residual sugar 10g/L moves into fermentor tank.
The fermentor tank volume is 20m 3, dress liquid 16m 3, adopt the fermentation and acid substratum.Fermentation and acid substratum composition (calculating): starch 135, (NH by every liter of contained grammes per square metre g/L of substratum 4) 2SO 41.0, KH 2PO 40.1, MgSO 47H 2O0.2, MnSO 4H 2O 0.2, FeSO 47H 2O 0.3, CaCO 370; Nature pH, wherein (NH 4) 2SO 4Separately sterilization.121 ℃ of sterilizations of fermentation and acid substratum 10 minutes, the cooling back moves into the seed and the sophisticated spore nutrient solution of suitable cell age, keep 32-34 ℃ of temperature, stirring velocity 50-70r/m, air flow 0.05vvm, tank pressure 0.08-0.1MPa, cultivated 115 hours, and produced sour 75.0g/L, residual sugar 5g/L, stop fermentation, carry out L MALIC ACID and extract.
Put directly vacuum filtration karusen of jar back, obtain to contain the L MALIC ACID calcium filter cake of thalline; Behind filter cake usefulness diluted acid water (no pozzuolite acid) furnishing pulpous state,, carry out acidolysis, removal of impurities and decolouring under the mild conditions of stirring velocity 50-60r/m 30 ℃ of temperature; After removing by filter gypsum tailings and thalline etc., add polyacrylamide flocculant and clarify, the consumption of flocculation agent is 30mg/L; The thick solution of L MALIC ACID carries out the essence filter and by behind F-1 citric acid anion-exchange resin column and the C26 cation exchange resin column, under 50 ℃, adopts two-stage to concentrate, when being concentrated into 1000g/L, introduce in the crystallizer, under 1-3r/m stirs, be cooled to 5 ℃, add an amount of crystal seed and carry out crystallization; After the crystallization, with whizzer crystal separation is come out, mother liquor and wash water carry out reconcentration or purifying treatment, and crystal is then packed behind inlet temperature 30-50 ℃ of following fluidized drying immediately.
Embodiment three
Produce L MALIC ACID with glucose for raw material direct fermentation.
With the N1-14 ' slant strains of preserving in 4 ℃ of refrigerators, be transferred on the potato dextrose agar test tube slant of new preparation, put 32-35 ℃ and cultivated 5 days down, obtain fresh test tube slant bacterial classification.Potato dextrose agar composition (calculating): 20 * 10 by every liter of contained amount of substratum -2Potato juice 1000ml, glucose 30g, KH 2PO 45g, MgSO 47H 2O 3g, pH7.5, agar 20g.Then, make spore suspension, insert in the triangular flask bran mass.The composition of bran mass (pressing the contained grammes per square metre g/L of per kilogram substratum calculates): fresh wheat bran 400, water 600.Add product spore promotor wherein composition be glucose: KH 2PO 4=1: 0.05, its consumption is 10 * 10 of a bran mass -2Postvaccinal substratum is put 32-35 ℃ and is cultivated after 5 days down, and its spore output is 4.0 * 10 3Individual/gram substratum (dry weight).
The seeding tank volume is 600L, and dress liquid 450L adopts seed culture medium.The composition of seed culture medium (calculating): glucose 30, (NH by every liter of contained grammes per square metre g/L of substratum 4) 2SO 45.0, KH 2PO 41.0, MgSO 47H 2O 1.2, MnSO 4H 2O 1.2, FeSO 47H 2O 1.5, CaCO 360; Nature pH, wherein (NH 4) 2SO 4Separately sterilization.Seed culture medium adopts 121 ℃ of sterilizations 25 minutes, inserts the wheat bran kind after the cooling, keeps 35-37 ℃ of temperature, stirring velocity 280-300r/m, and air flow 0.7vvm, tank pressure 0.01-0.02MPa cultivated 20 hours, and residual sugar 7g/L moves in the breeding jar and cultivates.
The breeding tank volume is 3m 3, dress liquid 2.4m 3Adopt the deep layer spore to produce substratum (composition is identical with seed culture medium), sterilized 20 minutes for 121 ℃, the cooling back moves into the mature seed nutrient solution, keeps 36-38 ℃ of temperature, stirring velocity 240-250r/m, air flow 0.6vvm, tank pressure 0.01-0.02MPa cultivated 18 hours, and spore output is 5.0 * 10 5Individual/milliliter, residual sugar 9g/L moves into fermentor tank.
The fermentor tank volume is 20m 3, dress liquid 16m 3, adopt the fermentation and acid substratum.Fermentation and acid substratum composition (calculating): glucose 125, (NH by every liter of contained grammes per square metre g/L of substratum 4) 2SO 45.0, KH 2PO 41.0, MgSO 47H 2O1.2, MnSO 4H 21.2, FeSO 47H 2O 1.5, CaCO 3120; Nature pH, wherein (NH 4) 2SO 4Separately sterilization.121 ℃ of sterilizations of fermentation and acid substratum 10 minutes, the cooling back moves into the seed and the sophisticated spore nutrient solution of suitable cell age, keep 35-37 ℃ of temperature, stirring velocity 140-150r/m, air flow 0.3vvm, tank pressure 0.01-0.02MPa, cultivated 120 hours, and produced sour 85.0g/L, residual sugar 5g/L, stop fermentation, carry out L MALIC ACID and extract.
Put directly vacuum filtration karusen of jar back, obtain to contain the L MALIC ACID calcium filter cake of thalline; Behind filter cake usefulness diluted acid water (no pozzuolite acid) furnishing pulpous state,, carry out acidolysis, removal of impurities and decolouring under the mild conditions of stirring velocity 90-100r/m 50 ℃ of temperature; After removing by filter gypsum tailings and thalline etc., add polyacrylamide flocculant and clarify, the consumption of flocculation agent is 1mg/L; The thick solution of L MALIC ACID carries out the essence filter and by behind A561 anion-exchange resin column and the C26 cation exchange resin column, under 70 ℃, adopts two-stage to concentrate, when being concentrated into 1300g/L, introduce in the crystallizer, under 8-10r/m stirs, be cooled to 20 ℃, add an amount of crystal seed and carry out crystallization; After the crystallization, with whizzer crystal separation is come out, mother liquor and wash water carry out concentrating once more or purifying treatment, and crystal is then packed behind inlet temperature 70-80 ℃ of following fluidized drying immediately.

Claims (18)

1, L MALIC ACID high productive mutant aspergillus (Aspergillus sp.N1-14 '), it is characterized in that this mutant strain be its sample preservation in China typical culture collection center (CCTCC), be numbered the such microorganism strains of M94013.
2, a kind of method of producing L MALIC ACID is characterized in that utilizing N1-14 ' CCTCC M94013 direct fermentation saccharine material and produces L MALIC ACID.
3, by the method for the described production L MALIC ACID of claim 2, it is characterized in that said saccharine material is glucose, amylum hydrolysate of the sugar, sucrose or starch.
4, by the method for the described production L MALIC ACID of claim 2, it is characterized in that the technological process of producing comprises:
4.1, fermentation technology process:
4.1.1,25-35 ℃ of temperature range and to add its major ingredient be glucose and KH 2PO 4The condition of product spore promotor under, make N1-14 ' CCTCC M94013 a large amount of spores that produce in the triangular flask bran mass;
4.1.2, sophisticated spore inserted the substratum that contains lime carbonate in the seeding tank carry out seed culture;
4.1.3, sophisticated seed moved into the substratum that contains lime carbonate in the breeding jar carry out the production of deep-layer liquid spore;
4.1.4, the seed of suitable cell age and sophisticated spore are moved into the substratum that contains lime carbonate in the fermentor tank, saccharine material is carried out the karusen that direct fermentation obtains containing L MALIC ACID calcium;
4.2, L MALIC ACID extraction process process:
4.2.1, directly the vacuum filtration karusen is received to such an extent that contain the L MALIC ACID calcium of thalline;
4.2.2, contain the L MALIC ACID calcium of thalline with no pozzuolite acid acidolysis, and acid hydrolysis solution is carried out removal of impurities and decolouring:
4.2.3, above-mentioned acid hydrolysis solution vacuum filtration is removed thalline and calcium sulfate, obtain the thick solution of L MALIC ACID;
4.2.4, in the thick solution of L MALIC ACID, add the smart filter in flocculation agent clarification back, smart filtrate is purified by anion-cation exchange resin;
4.2.5, the L MALIC ACID solution after will purifying concentrates, crystallization, at last with centrifugal receive wet crystal carry out drying.
5, press the method for the described production L MALIC ACID of claim 4, after the process that it is characterized in that step 4.1.1 comprises potato dextrose agar (PDA) the test tube slant inoculation of preparation newly, putting 28-32 ℃ cultivated 5-7 days, after treating the spore maturation, add sterilized water, the spore suspension that makes is inserted in the triangular flask bran mass, and being added with its major ingredient in the substratum is glucose and KH 2PO 4Product spore promotor, put 28-32 ℃ and cultivated 5-7 days down.
6, by the method for claim 4 or 5 described production L MALIC ACIDs, it is characterized in that said product spore promotor composition ratio is: glucose: KH 2PO 4=1: 0.005-0.05 (weight part).
7,, it is characterized in that the consumption of product spore promotor among the step 4.1.1 is (1～10) * 10 of bran mass by the method for the described production L MALIC ACID of claim 6 -2
8,, it is characterized in that the consumption of product spore promotor among the step 4.1.1 is (3～6) * 10 of bran mass by the method for the described production L MALIC ACID of claim 6 -2
9,, it is characterized in that the said composition (calculating by every liter of contained grammes per square metre g/L of substratum) that contains the substratum of lime carbonate is among step 4.1.2 and the 4.1.3: reducing sugar 30-70, (NH by the method for the described production L MALIC ACID of claim 4 4) 2SO 41.0-5.0, KH 2PO 40.1-1.0, MgSO 47H 2O 0.2-1.2, MnSO 4H 2O 0.2-1.2, FeSO 47H 2O 0.3-1.5, CaCO 320-60.
10,, it is characterized in that the said composition (calculating by every liter of contained grammes per square metre g/L of substratum) that contains the substratum of lime carbonate is among the step 4.1.4: reducing sugar 115-135, (NH by the method for the described production L MALIC ACID of claim 4 4) 2SO 41.0-5.0, KH 2PO 40.1-1.0, MgSO 47H 2O 0.2-1.2, MnSO 4H 2O 0.2-1.2, FeSO 47H 2O 0.3-1.5, CaCO 370-120.
11, press the method for the described production L MALIC ACID of claim 4, it is characterized in that among the step 4.1.2 that seed culture is is 30-37 ℃ in temperature, stirring velocity is 200-300r/m, and air flow is 0.4-0.7vvm, and tank pressure is to cultivate 20-30 hour under the condition of 0.01-0.1MPa.
12, press the method for the described production L MALIC ACID of claim 4, it is characterized in that the production of step 4.1.3 mid-deep strata liquid spore is is 33-38 ℃ in temperature, stirring velocity is 150-250r/m, and air flow is 0.3-0.6vvm, and tank pressure is to cultivate 18-23 hour under the condition of 0.01-0.1MPa.
13, press the method for the described production L MALIC ACID of claim 4, it is characterized in that among the step 4.1.4 that being is 32-37 ℃ in temperature, stirring velocity is 50-150r/m, and air flow is 0.05-0.3vvm, and tank pressure is under the condition of 0.01-0.1MPa saccharine material to be fermented.
14,, it is characterized in that in the step 2.2 it being to add (20-60) * 10 by the method for the described production L MALIC ACID of claim 4 -2No pozzuolite acid, and be 30-50 ℃ in temperature, stirring velocity is the L MALIC ACID calcium that acidolysis contains thalline under the condition of 50-100r/m.
15, by the method for the described production L MALIC ACID of claim 4, it is characterized in that employed flocculation agent is mainly polyacrylamide among the step 4.2.4, its consumption is 1-30mg/L.
16, by the method for the described production L MALIC ACID of claim 14, the consumption that it is characterized in that polyacrylamide is 5～10mg/L.
17, by the method for the described production L MALIC ACID of claim 4, it is characterized in that employed anionite-exchange resin is F-1 citric acid anionite-exchange resin or A561 anionite-exchange resin among the step 4.2.4.
18, by the method for the described production L MALIC ACID of claim 4, it is characterized in that employed Zeo-karb is 732 Zeo-karbs or C26 Zeo-karb among the step 4.2.4.
CN94105633A 1994-05-20 1994-05-20 High-yield saltant aspergillus N1-14' for L-malic acid and method for producing L-malic acid Expired - Fee Related CN1059700C (en)

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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
CN103642853B (en) * 2013-12-02 2015-11-04 山东阜丰发酵有限公司 A kind of L MALIC ACID novel technology for extracting
CN103789359B (en) * 2014-01-24 2016-03-16 合肥工业大学 A kind of method utilizing the fermentation of Testa Tritici hydrolysis sugar to produce oxysuccinic acid
CN107083407B (en) * 2017-06-02 2020-02-14 山东阜丰发酵有限公司 Preparation, separation, impurity removal and purification method of L-malic acid
CN110272339A (en) * 2019-07-02 2019-09-24 安徽丰原发酵技术工程研究有限公司 A kind of method of separation and Extraction high-purity malic acid
CN111909025B (en) * 2019-07-02 2022-07-15 安徽丰原发酵技术工程研究有限公司 Method for producing calcium hydrogen malate and application of method in production of malic acid

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EP0120213A2 (en) * 1983-03-25 1984-10-03 Hüls Aktiengesellschaft Process for the biotechnological production of L-malic acid

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Publication number Priority date Publication date Assignee Title
EP0120213A2 (en) * 1983-03-25 1984-10-03 Hüls Aktiengesellschaft Process for the biotechnological production of L-malic acid

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