CN105861574A - Method for producing L-malic acid through aspergillus oryzae fermentation - Google Patents
Method for producing L-malic acid through aspergillus oryzae fermentation Download PDFInfo
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- 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
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Abstract
The invention discloses a method for producing L-malic acid through aspergillus oryzae fermentation, belonging to the field of fermentation technology. The method comprises the following steps: with aspergillus oryzae as a production strain, inoculating a fermentation medium with 20% of liquid volume with 10% of spore suspension; adding 60-80g/L CaCO3; and performing ventilated culture for 120h at 30-34 DEG C. In the invention, a sugar-containing raw material is fermented by aspergillus oryzae to produce malic acid, the maximum acid yield can reach 103.2g/L, the raw materials are cheap, and the cost is low; moreover, by adopting single-bacterium fermentation, the required medium composition is simple, the fermentation conditions are simple and easy to control, and the problem of toxin generation caused by mixing aspergillus flavus for fermentation is avoided; and the technology for producing L-malic acid by fermenting the sugar-containing raw material, provided by the invention, has an important economic value.
Description
Technical field
The present invention relates to a kind of method utilizing aspergillus oryzae fermentation to produce L MALIC ACID, belong to fermentation technical field.
Background technology
Malic acid (Malic acid) has another name called dyhydrobutanedioic acid, is a kind of four important carbon platform chemicals, by USDOE
It is classified as one of 12 kinds of potential compounds.It is the important intermediate of TC organism circulation, has the tart flavour of special happiness,
Easily be absorbed by the body utilization, and therefore acid condiment and functional food as excellent performance are widely used in food service industry.With
Time there is antioxidation, the effect such as oil-control is become sour, be used for cosmetics, medicine, chemical industry.
At present, the production method of malic acid mainly has: biological catalysis, two-step fermentation and one-step fermentation.Both at home and abroad with life
Thing catalysis method is main, and wherein Japan is the main manufacturing country of L MALIC ACID and exported country.(1) biological catalysis is with fumaric acid as the end
Thing is converted into malic acid through fumarase.In its cost of material height, finished product, heteroacid is high and biological pollution is bigger；(2) two steps
Fermentation method refers to through two kinds of microorganism fermenting and producing malic acids in two stages.Its fermentation period length, by-product cost many, isolated and purified
Height and condition of culture are complicated, are currently under laboratory research level；(3) one-step fermentation refers to obtain through a kind of fermentable
Obtain 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 relevant research report is still
Few.Composting fermentation is owing to relating to two kinds of microorganisms, and condition of culture requires relatively stricter, and fermentation period is longer, and acid production rate is relatively
Low, by-product is more.One-step fermentation fado employing Aspergillus flavus is as producing bacterial strain, but its Aspergillus flavus is easily generated Huang Qu when fermentation
Mould toxin so that it is fermentation technology difficulty, it is difficult to for industrialized production.
Summary of the invention
The invention provides a kind of method fermented and produce L MALIC ACID.
Described method, is with aspergillus oryzae for producing bacterial strain, is in the fermentation medium of liquid amount 20%, accesses the spore of 10%
Suspension, adds 60～80g/L CaCO3, 30～34 DEG C of aerlbic culture 120h；Described fermentation medium, in terms of g/L, contains: 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 described spore suspension miospore is 106Individual/mL.
In one embodiment of the invention, fermentation temperature is 34 DEG C.
In one embodiment of the invention, described CaCO3Concentration is 80g/L.
In one embodiment of the invention, described aspergillus oryzae, buy in CICC (Chinese industrial Microbiological Culture Collection management
Center), numbered: CICC2005.
In one embodiment of the invention, the preparation process of described spore suspension is: aspergillus oryzae strain slant strains accessed
Seed culture medium, 30 DEG C, 200rpm, cultivates 14h.
In one embodiment of the invention, described seed culture medium contains in terms of g/L: 30 glucoses, 3 tryptones,
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 is natural.
The present invention uses aspergillus oryzae sugar fermentation raw material to produce malic acid, and its acid yield reaches as high as 103.2g/L, cost of material
Cheap, low cost；Further, the present invention is to use single bacterium fermentation, and the medium component of needs is simple, and fermentation condition is simple and easy to control
System, it is thus also avoided that with Aspergillus flavus mixed fermentation can toxigenic problem, the present invention offer with saccharine material fermenting and producing L-Herba Marsileae Quadrifoliae
The technique of fruit acid has important economic worth.
Accompanying drawing explanation
Fig. 1 different temperatures produces the impact of L MALIC ACID yield to aspergillus oryzae
Detailed description of the invention
Residual glucose method: fermented sample 8000r/min is centrifuged 10min, takes supernatant dilution certain multiple, draws 1mL and diluted
After sample in 20mL color comparison tube, add 1.5mLDNS reagent, accurate heating in water bath 5min, fixed after being quickly cooled to room temperature
Hold to 10mL, measure light absorption value record at wavelength 540nm.
L MALIC ACID assay method: use HPLC method to measure.Flowing phase: 0.1mol/L KH2PO4, pH2.8；Flow velocity:
0.6mL/min；Column temperature: 25 DEG C；Detector: UV-detector；Wavelength: 215nm；Pillar: C18.Add 1 times of volume
2mol/LHCL by the dissolution of calcium carbonate in fermentation liquid completely after, 8000r/min is centrifuged 10min, draws supernatant dilution certain
Multiple is crossed film process liquid phase and is measured.
Aspergillus oryzae used by following example, buys in CICC, numbered: CICC2005.
Embodiment 1 different carbon source produces the impact of L MALIC ACID yield to aspergillus oryzae
The culture medium used, in terms of g/L: add 120 glucoses, starch, maltose, fructose and lactose respectively and do 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 natural.Temperature 30 DEG C, under 200r/min rotating speed, send out
Ferment 120h.Result is as shown in table 1.From table 1, in addition to lactose, all kinds of saccharides can act as preferable carbon source, wherein with
Glucose is as carbon source, and it is the highest that it produces acid amount.
Table 1 different carbon source produces the impact of L MALIC ACID to aspergillus oryzae
| 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 |
Embodiment 2 different nitrogen sources produces the impact of L MALIC ACID yield to aspergillus oryzae
Use culture medium, in terms of g/L: add respectively 3 tryptones, soy peptone, carbamide, ammonium sulfate, Semen Maydis pulp and
Soybean cake powder is as nitrogen source, 120 glucoses, 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 natural.In temperature
30 DEG C, under 200r/min rotating speed, ferment 120h.Result is as shown in table 2.As shown in Table 2, tryptone and Semen sojae atricolor are used
Peptone effect is preferable, wherein best using tryptone as nitrogen source effect.
Table 2 different nitrogen sources produces the impact of L MALIC ACID to aspergillus oryzae
| Nitrogen source category | 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 |
| Carbamide | 3 | 18.1 | 38.4 |
| Ammonium sulfate | 3 | 15.4 | 48.2 |
| Semen Maydis pulp | 3 | 10.2 | 43.3 |
| Soybean cake powder | 3 | 15.6 | 38.4 |
Embodiment 3 different temperatures produces the impact of L MALIC ACID yield to aspergillus oryzae
The culture medium used, in terms of g/L: 120 glucoses, 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 is natural.Design temperature is 28 DEG C, 30 DEG C, 32 DEG C, 34 DEG C, 36 DEG C, 38 DEG C, 40 DEG C respectively.At 200r/min rotating speed
Under, ferment 120h.Shown in result Fig. 1.As shown in Figure 1, temperature before 34 DEG C the yield of L MALIC ACID along with temperature
Raising and increase, temperature begins to decline when 36 DEG C, therefore choosing optimum fermentation temp is 34 DEG C.
The different calcium carbonate addition of embodiment 4 produces the impact of L MALIC ACID yield to aspergillus oryzae
The culture medium used, in terms of g/L: 120 glucoses, 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 are natural.
Wherein, CaCO3Interpolation concentration be respectively 60g/L, 70g/L, 80g/L, 90g/L, 100g/L.Fermentation temperature 34 DEG C,
Under 200r/min rotating speed, ferment 120h.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 94.69g/L.Therefore selecting calcium carbonate to add concentration is 80g/L.
Table 3 different nitrogen sources produces the impact of L MALIC ACID to aspergillus oryzae
| 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 |
The different Fe of embodiment 52+Concentration produces the impact of L MALIC ACID yield to aspergillus oryzae
The culture medium used, in terms of g/L: 120 glucoses, 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 natural.Wherein, FeSO4·7H2O
Interpolation concentration be respectively 0.005g/L, 0.05g/L, 0.5g/L.Fermentation temperature 34 DEG C, under 200r/min rotating speed, fermentation
120h.The results are shown in Table shown in 4.As shown in Table 4, FeSO is worked as4·7H2When O concentration is 0.05g/L, the yield of L MALIC ACID
It is up to 103.2g/L.
The different Fe of table 42+Concentration produces the impact of L MALIC ACID to aspergillus oryzae
| 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 is open the most as above with preferred embodiment, but it is not limited to the present invention, any person skilled in the art,
Without departing from the spirit and scope of the present invention, all can do various changes and modification, therefore protection scope of the present invention should be with
What claims were defined is as the criterion.
Claims (7)
1. the method producing L MALIC ACID of fermenting, it is characterised in that with aspergillus oryzae for producing bacterial strain, be to liquid amount 20%
Fermentation medium in, access 10% spore suspension, add 60～80g/L CaCO3, 30～34 DEG C of aerlbic culture 120h；Institute
State fermentation medium in terms of g/L, contain: 120 glucoses, 3 tryptones, 0.15KH2PO4、0.15K2HPO4、0.10
MgSO4·7H2O、0.10CaCl2·H2O, 0.05NaCl, 0.01 citric acid, 0.05FeSO4·7H2O, pH are natural.
Method the most according to claim 1, it is characterised in that the quantity of described spore suspension miospore is 106Individual/mL.
Method the most according to claim 1, it is characterised in that fermentation temperature is 34 DEG C.
Method the most according to claim 1, it is characterised in that CaCO3Concentration is 80g/L.
Method the most according to claim 1, it is characterised in that described aspergillus oryzae, buys in CICC, numbered:
CICC2005。
Method the most according to claim 1, it is characterised in that the preparation process of described spore suspension is: by aspergillus oryzae
Strain accesses seed culture medium from inclined-plane, 30 DEG C, 200rpm, cultivates 14h.
Method the most according to claim 6, it is characterised in that described seed culture medium contains in terms of g/L: 30 glucoses,
3 tryptones, 0.56KH2PO4、0.56K2HPO4、0.925NaH2PO4·H2O、0.82Na2HPO4、0.075
MgSO4·7H2O、0.075CaCl2·H2O、0.05NaCl、0.05FeSO4·7H2O, 0.01 citric acid, pH is natural.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434369A (en) * | 2016-09-23 | 2017-02-22 | 江南大学 | Aspergillus oryzae capable of producing L-malic acid and application of Aspergillus oryzae |
| CN111876446A (en) * | 2020-08-11 | 2020-11-03 | 新拓洋生物工程有限公司 | Process for preparing L-malic acid by Aspergillus oryzae fermentation |
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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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- 2016-04-25 CN CN201610262363.1A patent/CN105861574B/en active Active
Patent Citations (3)
| 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 |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434369A (en) * | 2016-09-23 | 2017-02-22 | 江南大学 | Aspergillus oryzae capable of producing L-malic acid and application of Aspergillus oryzae |
| CN106434369B (en) * | 2016-09-23 | 2019-07-02 | 江南大学 | One plant of aspergillus oryzae for producing L MALIC ACID and its application |
| CN111876446A (en) * | 2020-08-11 | 2020-11-03 | 新拓洋生物工程有限公司 | Process for preparing L-malic acid by Aspergillus oryzae fermentation |
| 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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