CN105861574B - A method of it is fermented using aspergillus oryzae and produces L MALIC ACID - Google Patents
A method of it is fermented using aspergillus oryzae and produces L MALIC ACID Download PDFInfo
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- CN105861574B CN105861574B CN201610262363.1A CN201610262363A CN105861574B CN 105861574 B CN105861574 B CN 105861574B CN 201610262363 A CN201610262363 A CN 201610262363A CN 105861574 B CN105861574 B CN 105861574B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
Abstract
The invention discloses a kind of methods for producing L MALIC ACID using aspergillus oryzae fermentation, belong to fermentation technical field.The present invention is production bacterial strain with aspergillus oryzae, into the fermentation medium of liquid amount 20%, accesses 10% spore suspension, 60~80g/LCaCO is added3, 30~34 DEG C of aerlbic culture 120h.The present invention produces malic acid using aspergillus oryzae fermentation saccharine material, and acid yield reaches as high as 103.2g/L, and low in raw material price is at low cost；And, the present invention is fermented using single bacterium, and the medium component needed is simple, and fermentation condition is simple and easy to control, also avoid with aspergillus flavus mixed fermentation can toxigenic problem, the technique provided by the invention with saccharine material fermenting and producing L MALIC ACID have important economic value.
Description
Technical field
The present invention relates to a kind of methods for producing L MALIC ACID using aspergillus oryzae fermentation, belong to fermentation technical field.
Background technique
Malic acid (Malic acid) also known as dyhydrobutanedioic acid are a kind of four important carbon platform chemicals, by the U.S.
Ministry of Energy is classified as one of 12 kinds of potential compounds.It is the important intermediate of TC biology body circulation, is had special pleasant
Tart flavour, be easily absorbed by the body utilization, therefore is widely used in food as the acid condiment and functional food haveing excellent performance
Industry.Have the effects that anti-oxidant, inhibition is spoiled by rancid oil or fat simultaneously, is used for cosmetics, medicine, chemical industry.
Currently, the production method of malic acid mainly has: biological catalysis, two-step fermentation and one-step fermentation.Both at home and abroad
Based on biological catalysis, wherein Japan is the main producing country of L MALIC ACID and exported country.(1) biological catalysis is with fumaric acid
Malic acid is converted into through fumarase for substrate.Heteroacid is high in its cost of material height, finished product and biological pollution is larger；(2)
Two-step fermentation refers to through two kinds of microorganisms fermenting and producing malic acid in two stages.Its fermentation period is long, by-product is more, isolates and purifies
At high cost and condition of culture is complicated, is currently under laboratory research level；(3) one-step fermentation refers to sends out through a kind of microorganism
Ferment obtains malic acid.Its fermentation condition is simple, and production cost is low.
In saccharine material Fermentation, one-step fermentation and composting fermentation have greater advance, but related research
It reports still few.Composting fermentation is due to being related to two kinds of microorganisms, and condition of culture requirement is stringenter, and fermentation period is longer, produces
Sour rate is lower, and by-product is more.One-step fermentation mostly uses aspergillus flavus as production bacterial strain, but its aspergillus flavus easily produces in fermentation
Raw aflatoxin keeps its zymotechnique difficult, it is difficult to be used for industrialized production.
Summary of the invention
The present invention provides a kind of methods that fermentation produces L MALIC ACID.
The method is with aspergillus oryzae for production bacterial strain, is to access 10% into the fermentation medium of liquid amount 20%
60~80g/L CaCO is added in spore suspension3, 30~34 DEG C of aerlbic culture 120h；The fermentation medium is contained in terms of g/L:
120 glucose, 3 tryptones, 0.15KH2PO4、0.15K2HPO4、0.10MgSO4·7H2O、0.10CaCl2·H2O、
0.05NaCl, 0.01 citric acid, 0.05FeSO4·7H2O, pH are natural.
In one embodiment of the invention, the quantity of the spore suspension miospore is 106A/mL.
In one embodiment of the invention, fermentation temperature is 34 DEG C.
In one embodiment of the invention, the CaCO3Concentration is 80g/L.
In one embodiment of the invention, the aspergillus oryzae is bought in CICC (Chinese industrial Microbiological Culture Collection
Administrative center), number are as follows: CICC2005.
In one embodiment of the invention, the preparation process of the spore suspension are as follows: by aspergillus oryzae strain inclined-plane bacterium
Kind access seed culture medium, cultivates 14h by 30 DEG C, 200rpm.
In one embodiment of the invention, the seed culture medium is contained in terms of g/L: 30 glucose, 3 tryptoses
Peptone, 0.56 KH2PO4、0.56 K2HPO4、0.925 NaH2PO4·H2O、0.82 Na2HPO4、0.075 MgSO4·7H2O、
0.075 CaCl2·H2O、0.05 NaCl、0.05 FeSO4·7H2O, 0.01 citric acid, pH are natural.
The present invention produces malic acid using aspergillus oryzae fermentation saccharine material, and acid yield reaches as high as 103.2g/L, former
Expect it is cheap, it is at low cost；Also, the present invention is fermented using single bacterium, and the medium component needed is simple, and fermentation condition is simple
It is easy to control, it is thus also avoided that with aspergillus flavus mixed fermentation can toxigenic problem, it is provided by the invention with saccharine material ferment give birth to
The technique for producing L MALIC ACID has important economic value.
Detailed description of the invention
Influence of Fig. 1 different temperatures to aspergillus oryzae production L MALIC ACID yield
Specific embodiment
Residual glucose method: fermented sample 8000r/min is centrifuged 10min, and supernatant is taken to dilute certain multiple, and it is dilute to draw 1mL
In 20mL colorimetric cylinder 1.5mLDNS reagent is added, accurate heating water bath 5min is fixed after being rapidly cooled to room temperature in sample after releasing
Hold 10mL, measure light absorption value in wavelength 540nm and records.
L MALIC ACID measuring method: it is measured using HPLC method.Mobile phase: 0.1mol/L KH2PO4, pH2.8；Flow velocity:
0.6mL/min；Column temperature: 25 DEG C；Detector: UV detector；Wavelength: 215nm；Pillar: C18.The 2mol/ of 1 times of volume is added
LHCL by the dissolution of calcium carbonate in fermentation liquid it is complete after, 8000r/min be centrifuged 10min, draw supernatant dilution certain multiple cross film
Liquid phase measurement is used in processing.
Aspergillus oryzae used in following embodiment, purchase is in CICC, number are as follows: CICC2005.
Influence of 1 different carbon source of embodiment to aspergillus oryzae production L MALIC ACID yield
The culture medium of use, in terms of g/L: 120 glucose, starch, maltose, fructose and lactose are added respectively does carbon source, 3
Soy peptone, 0.15 KH2PO4、0.15 K2HPO4、0.10 MgSO4·7H2O、0.10 CaCl2·H2O、0.05 NaCl、
0.01 citric acid, 0.005 FeSO4·7H2O、60 CaCO3, pH it is natural.Under 30 DEG C of temperature, 200r/min revolving speed, fermentation
120h.The results are shown in Table 1.Seen from table 1, in addition to lactose, all kinds of carbohydrates can act as preferable carbon source, wherein with glucose
As carbon source, acid amount highest is produced.
Influence of 1 different carbon source of table to aspergillus oryzae production L MALIC ACID
| Carbon source kind | Concentration (g/L) | Residual sugar (g/L) | L MALIC ACID (g/L) |
| Glucose | 120 | 15.1 | 77.0 |
| Starch | 120 | 5.5 | 69.5 |
| Maltose | 120 | 15.6 | 55.2 |
| Fructose | 120 | 40.2 | 43.0 |
| Lactose | 120 | 55.8 | 10.8 |
Influence of 2 different nitrogen sources of embodiment to aspergillus oryzae production L MALIC ACID yield
The culture medium of use, in terms of g/L: adding 3 tryptones, soy peptone, urea, ammonium sulfate, corn pulp respectively
With soybean cake powder as nitrogen source, 120 glucose, 0.15 KH2PO4、0.15 K2HPO4、0.10 MgSO4·7H2O、0.10
CaCl2·H2O, 0.05 NaCl, 0.01 citric acid, 0.005 FeSO4·7H2O、60 CaCO3, pH it is natural.At 30 DEG C of temperature,
Under 200r/min revolving speed, ferment 120h.The results are shown in Table 2.As shown in Table 2, using tryptone and soy peptone effect
Preferably, wherein it is best using tryptone as nitrogen source effect.
Influence of 2 different nitrogen sources of table to aspergillus oryzae production L MALIC ACID
| Nitrogen source type | Concentration (g/L) | Residual sugar (g/L) | L MALIC ACID (g/L) |
| Tryptone | 3 | 13.6 | 72.7 |
| Soy peptone | 3 | 9.2 | 65.1 |
| Urea | 3 | 18.1 | 38.4 |
| Ammonium sulfate | 3 | 15.4 | 48.2 |
| Corn pulp | 3 | 10.2 | 43.3 |
| Soybean cake powder | 3 | 15.6 | 38.4 |
Influence of 3 different temperatures of embodiment to aspergillus oryzae production L MALIC ACID yield
The culture medium of use, in terms of g/L: 120 glucose, 3 tryptones, 0.15 KH2PO4、0.15 K2HPO4、0.10
MgSO4·7H2O、0.10 CaCl2·H2O, 0.05 NaCl, 0.01 citric acid, 0.005 FeSO4·7H2O、60 CaCO3、pH
It is natural.It is 28 DEG C, 30 DEG C, 32 DEG C, 34 DEG C, 36 DEG C, 38 DEG C, 40 DEG C that temperature, which is set separately,.Under 200r/min revolving speed, fermentation
120h.Shown in result figure 1.As shown in Figure 1, the yield of temperature L MALIC ACID before 34 DEG C increases as the temperature rises,
Temperature is begun to decline at 36 DEG C, therefore choosing optimum fermentation temp is 34 DEG C.
Influence of the different calcium carbonate additive amounts of embodiment 4 to aspergillus oryzae production L MALIC ACID yield
The culture medium of use, in terms of g/L: 120 glucose, 3 tryptones, 0.15 KH2PO4、0.15 K2HPO4、0.10
MgSO4·7H2O、0.10 CaCl2·H2O, 0.05 NaCl, 0.01 citric acid, 0.005 FeSO4·7H2O, pH is natural.Wherein,
CaCO3Addition concentration be respectively 60g/L, 70g/L, 80g/L, 90g/L, 100g/L.At 34 DEG C of fermentation temperature, 200r/min turns
Under speed, ferment 120h.It the results are shown in Table shown in 3.As shown in Table 3, when calcium carbonate concentration is 80g/L, the yield of L MALIC ACID reaches
To 94.69g/L.Therefore selecting calcium carbonate addition concentration is 80g/L.
Influence of 3 different nitrogen sources of table to aspergillus oryzae production L MALIC ACID
| CaCO3Concentration (g/L) | Residual sugar (g/L) | L MALIC ACID (g/L) |
| 60 | 10.8 | 77.0 |
| 70 | 12.9 | 88.0 |
| 80 | 10.3 | 94.6 |
| 90 | 12.3 | 88.7 |
| 100 | 16.5 | 76.6 |
5 difference Fe of embodiment2+Influence of the concentration to aspergillus oryzae production L MALIC ACID yield
The culture medium of use, in terms of g/L: 120 glucose, 3 tryptones, 0.15 KH2PO4、0.15 K2HPO4、0.10
MgSO4·7H2O、0.10 CaCl2·H2O, 0.05 NaCl, 0.01 citric acid, 80 CaCO3, pH it is natural.Wherein, FeSO4·
7H2The addition concentration of O is respectively 0.005g/L, 0.05g/L, 0.5g/L.Under 34 DEG C of fermentation temperature, 200r/min revolving speed, hair
Ferment 120h.It the results are shown in Table shown in 4.As shown in Table 4, work as FeSO4·7H2When O concentration is 0.05g/L, the yield highest of L MALIC ACID
For 103.2g/L.
4 difference Fe of table2+Influence of the concentration to aspergillus oryzae production L MALIC ACID
| FeSO4·7H2O concentration (g/L) | Residual sugar (g/L) | L MALIC ACID (g/L) |
| 0.005 | 8.8 | 90.6 |
| 0.05 | 5.3 | 103.2 |
| 0.5 | 10.8 | 90.4 |
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (6)
1. a kind of method that fermentation produces L MALIC ACID, which is characterized in that be to liquid amount 20% with aspergillus oryzae to produce bacterial strain
In fermentation medium, 10% spore suspension is accessed, 60~80g/L CaCO is added3, 30~34 DEG C of aerlbic culture 120h；It is described
Fermentation medium is contained in terms of g/L: 120 glucose, 3 tryptones, 0.15 KH2PO4、0.15 K2HPO4、0.10 MgSO4·
7H2O、0.10 CaCl2·H2O, 0.05 NaCl, 0.01 citric acid, 0.05 FeSO4·7H2O, pH are natural；The aspergillus oryzae, purchase
It buys in CICC, number are as follows: CICC2005.
2. the method according to claim 1, wherein the quantity of the spore suspension miospore is 106A/mL.
3. the method according to claim 1, wherein fermentation temperature is 34 DEG C.
4. the method according to claim 1, wherein CaCO3Concentration is 80g/L.
5. the method according to claim 1, wherein the preparation process of the spore suspension are as follows: by aspergillus oryzae
Strain accesses seed culture medium from inclined-plane, and 30 DEG C, 200rpm cultivate 14h.
6. according to the method described in claim 5, it is characterized in that, the seed culture medium is contained in terms of g/L: 30 glucose, 3
Tryptone, 0.56 KH2PO4、0.56 K2HPO4、0.925 NaH2PO4·H2O、0.82 Na2HPO4、0.075 MgSO4·
7H2O、0.075 CaCl2·H2O、0.05 NaCl、0.05 FeSO4·7H2O, 0.01 citric acid, pH are natural.
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| CN111876446B (en) * | 2020-08-11 | 2023-10-13 | 新拓洋生物工程有限公司 | Process for preparing L-malic acid by fermenting aspergillus oryzae |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102597224A (en) * | 2009-09-01 | 2012-07-18 | 诺维信股份有限公司 | Methods for improving malic acid production in filamentous fungi |
| CN102618589A (en) * | 2012-04-12 | 2012-08-01 | 苏州百趣食品有限公司 | Method for producing L-malic acid by means of rhizopus oryzae one-step fermentation |
| CN103627738A (en) * | 2013-12-02 | 2014-03-12 | 山东阜丰发酵有限公司 | Direct fermentation method for producing L-malic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102597224A (en) * | 2009-09-01 | 2012-07-18 | 诺维信股份有限公司 | Methods for improving malic acid production in filamentous fungi |
| CN102618589A (en) * | 2012-04-12 | 2012-08-01 | 苏州百趣食品有限公司 | Method for producing L-malic acid by means of rhizopus oryzae one-step fermentation |
| CN103627738A (en) * | 2013-12-02 | 2014-03-12 | 山东阜丰发酵有限公司 | Direct fermentation method for producing L-malic acid |
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