CA1209073A - Process for the biotechnical production of l-malic acid - Google Patents
Process for the biotechnical production of l-malic acidInfo
- Publication number
- CA1209073A CA1209073A CA000450366A CA450366A CA1209073A CA 1209073 A CA1209073 A CA 1209073A CA 000450366 A CA000450366 A CA 000450366A CA 450366 A CA450366 A CA 450366A CA 1209073 A CA1209073 A CA 1209073A
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- Canada
- Prior art keywords
- acid
- solution
- fumaric acid
- nutrient
- fumaric
- Prior art date
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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
ABSTRACT OF THE DISCLOSURE
L-Malic acid can be produced, in a concentration of more than 100 g per liter and in a high yield by microbial fermentation by means of freely mobile fungi, from neutralized fumaric acid (more than 110 g per liter) in a preferably nutrient-free solution. Pure L-malic acid in a quality suitable for food and pharmaceuticals can be obtained therefrom in an economical way with great productivity. Fermentation in nutrient-containing fumaric acid solution can be terminated after at most three days; fermentation in nutrient-free fumaric acid solution can be terminated as early as after one day.
L-Malic acid can be produced, in a concentration of more than 100 g per liter and in a high yield by microbial fermentation by means of freely mobile fungi, from neutralized fumaric acid (more than 110 g per liter) in a preferably nutrient-free solution. Pure L-malic acid in a quality suitable for food and pharmaceuticals can be obtained therefrom in an economical way with great productivity. Fermentation in nutrient-containing fumaric acid solution can be terminated after at most three days; fermentation in nutrient-free fumaric acid solution can be terminated as early as after one day.
Description
The invention relates to a process for ~-he production of pure L-isomer of malic acid from neutralized fumaric acid. The process involves microbial fermentation by means of freely mobile microorganisms in an aqueous phase. The concentration of L-malic acid at the instant of harvesting is about 100-170 g per liter o:E culture solution.
L-Malic acid is utilized in the foodstuff industry and in the pharmaceutical industry as a buffer material, a complexing agent, an acidulant, and a moisturizer. L-Malic acid, as a natural substance, is more suitable for these kinds of applications than the chemically accessible DL-isomer mixture of malic acid. The latter mixture is producible, for example, by addition of water to maleic anhydride. The D-isomer of malic acid does not occur in nature; for this reason, use of chemically synthesized DL-malic acid in the grocery and pharmaceutical areas is not entirely innocuous.
The present invention seeks to improve the economy of biotechnical manufacture of L-malic acid in pure form.
Several methods may be used for the biotechnical production of L-malic acid. Some of these processes operate with special bacteria converting fumaric acid into L-malic acid by the addition of water. United States Patent 3,922,195 proposes to immobilize the bacterial cells. According to DAS
L-Malic acid is utilized in the foodstuff industry and in the pharmaceutical industry as a buffer material, a complexing agent, an acidulant, and a moisturizer. L-Malic acid, as a natural substance, is more suitable for these kinds of applications than the chemically accessible DL-isomer mixture of malic acid. The latter mixture is producible, for example, by addition of water to maleic anhydride. The D-isomer of malic acid does not occur in nature; for this reason, use of chemically synthesized DL-malic acid in the grocery and pharmaceutical areas is not entirely innocuous.
The present invention seeks to improve the economy of biotechnical manufacture of L-malic acid in pure form.
Several methods may be used for the biotechnical production of L-malic acid. Some of these processes operate with special bacteria converting fumaric acid into L-malic acid by the addition of water. United States Patent 3,922,195 proposes to immobilize the bacterial cells. According to DAS
2,363,285, the process is carried out with free bacterial cells or with the enzyme fumarase, isolated from bacterial cells.
Furthermore, the conversion of glucose to L-malic acid by association of a fungus with a bacterium has been described [J. Ferment Technol. 54 (4) :
197-204 (1976)]. In this method, the fungus makes fumaric acid from glucose, and this fumaric acid is then converted into L-malic acid by the bacterium.
The enzyme fumarase, effecting conversion of fumaric acid to L-malic
Furthermore, the conversion of glucose to L-malic acid by association of a fungus with a bacterium has been described [J. Ferment Technol. 54 (4) :
197-204 (1976)]. In this method, the fungus makes fumaric acid from glucose, and this fumaric acid is then converted into L-malic acid by the bacterium.
The enzyme fumarase, effecting conversion of fumaric acid to L-malic
3~
9~7~
acid, can be isolated in special processes from biological material, such as the cell mass of bacteria or fungi, and then can be used, in free or immobilized form, for obtaining L-malic acid from fumaric acid ~DAS 2,415,310; Czechoslo-vakian Patent 171,990).
In several processes, a large amount of a calcium compound is added to the fermentation batch whereby the resultant L-malic acid is precipitated as early during fermentation as the calcium malate (DOS 1,417,033).
Processes for the biotechnical manufacture of L-malic acid with the aid of fungi are likewise known. In these processes, the L-malic acid is essentially obtained by biochemical degradation and/or rearrangement of the carbon source fed to the respective fungus (for example molasses, sugar, ethanol, acetic acid) [J. Ferment Technol. 55 (2) : 196-l99 (1977)]. These processes do not start directly with fumaric acid.
In the process described in United States Patent 3,063,910, the fermentation batch can contain, in addition to 10-15% of a sugar, 1-10~ of an organic acid such as, for example, pyruvic or fumaric acid. However, in this fungal method, the added organic acid does not serve as a substrate for the synthesis of L-malic acid but, rather, serves as a reaction accelerator for the biochemical degradation or rearrangement of the offered sugar into L-malic acid.
Furthermore, fermentative conversion of n-parafin into L-malic acid is possible by the association of two types of yeasts, wherein one type of yeast converts paraffin into fumaric acid and the second type of yeast converts the resultant fumaric acid into L-malic acid LAgr. Biol. Chem. 34 :
1833-1838 (1970)].
The known methods for the biotechnical manufacture of L-malic acid have, inter alia, the peculiarities set out below:
(a) When using bacteria, biotechnical processes are more difficult to handle than when using fungi. For example, separation problems can arise due to the small size of the bacteria. Furthermore, bacteria tend to form by-products, including toxic by-products. A particularly disturbing factor against use of bacteria produced L-malic acid in the pharmaceuticals area is LPS toxin (lipopolysaccharide toxin)which is also formed by bacteria. This toxin has a pyrogenic effect and can be separated from L-malic acid only through the use of expensive processes such as ultrafiltration. In the bacterial method des-cribed by United States Patent 3,922,195, succinic acid by-product occurs in small amounts. This acid can be separated from malic acid only with difficul-ties.
(b) When using the enzyme fumarase, the primary fumarase source is the microbial cell mass. Before production of L-malic acid can be started, the microbial cell mass must be grown and worked up into fumarase,with consi-derable expense.
(c) Immobilization of living cells, or of the enzyme fumarase isolated from cells, is an additional, very expensive process step.
(d) When fermenting in the presence of a large concentration of CA
ions, the thus-formed L-malic acid is precipitated as calcium malate as early as during fermentation; this leads to problems during agitation and aeration of the fermentation batch.
(e) The attainable concentration of L-malic acid, is generally, compara-tively low and lies markedly below 100 g per liter of culture solution.
The reactions described in United States Patent 3,922,195 (immobilized hacteria) and in DOS 1,417,033 (high calcium concentration) give results that are exceptions to this generality.
~Z~73 ~ f) Fermentation takes longer than three days. This low produc-tivity requires the use of large capacity biotechnical reactors. Large reactor size greatly increases their cost, especially since these reactors are of an expensive construction to allow the conduct of a contamination-free process.
The invention provides a process for production of L-malic acid which is free of D-malic acid which process comprises admixing neutralized fumaric acid, in an aqueous phase having an initial concentration of fumaric acid of 11 to 15% by weight, with a freely mobile fungus to ferment the fumaric acid to L-malic acid. Preferably, the fermentation is continued until L-malic acid concentration reaches a value in the range 100 to 170 g/liter.
In the above process~ it is preferred that the aqueous solution of the neutralized fumaric acid additionally contains nutrients required for fungal growth, the solution is innoculated with 1-20% of fungal inoculum, the inoculated solution i5 fermented at 20-50 C for 1-3 days, and L-malic acid is separated from the culture solution and cell mass.
It is further preferred that the above process comprises producing an aqueous solution of the neutralized fumaric acid without nutrients for the fungus, adding to the nutrient-free fumaric acid solution fungal cell mass resulting from incubation in fumaric-acid-free or fumaric-acid-containing nutrient solution, or from a fermentation in nutrient-containing fumaric acid solution, wherein the volume of the nutrient-free fumaric acid solution amounts to one to ten times the liquid volume of the preceding incubation in fumaric-acid-free or fumaric-acid-containing nutrient solution or of the ~ _ ~2~
preceding fermentation in nutrient-containing fumaric acid solution when substantially the entire cell mass of the preceding incubation or fermenta-tion is being utilized, fermenting for 12-24 hours at 25-60C, and separating the cell mass and L-malic acid from the culture solution.
The fumaric acid can be fermented to L-malic acid in a nutrient-containing or nutrient-free solution. It may be expedient to ferment the fumaric acid only in a nutrient-free solution and to cultivate the cell mass required for this purpose in a nutrient solution that is free of fumaric acid or that contains fumaric acid. The nutrient solution suitable for this purpose can contain substantially less than 11% fumaric acid, since in this case the fungus is merely to be adapted to fumaric acid.
Although the term "fermentation" in technical literature has a relatively large spectrum of meanings, the expression "fermentation" in the context of this invention is understood to mean the biochemical conversion of fumaric acid into L-malic acid under the action of fumarase. The word "growing" is understood to mean theproliferation of the biomass from the inoculum with consumption of nutrients. In nutrient-containing fumaric acid solution, both processes (growing and fermentation) proceed side-by-side or in succession.
For fermentation in nutrient-containing fumaric acid, the nutrients and active agents necessary for fungal growth are added to the solution with neutralized fumaric acid. Aftsr sterilization, this solution is inoculated with 1-20% inoculum of a fungus, and fermented for a period of one to three days at 20-50C. Subsequently, the culture solution is separated into cell mass and culture filtrate, and L-malic acid is isolated from the culture filtrate according to conventional methods.
The separated cell mass can be utilized for several, e.g.
approximately for three additional, fermentations in respectively freshly prepared nutrient-free fumaric acid solutions. In this connection, the volume of the fumaric acid solution can amount to one to ten times the liquid volume of the preceding fermentation in nutrient-containing fumaric acid solution, or of the preceding incubation in fumaric-acid-free or fumaric-acid-containing nutrient solution, if the entire cell mass resulting from the nutrient-containing solution is employed. In a nutrient-free fumaric-acid solution, fermenta~ion is performed for 12-24 hours at 25-60 C.
The aerokic, fermentative conversion, in a nutrient-containing solution, of fumaric acid into L-malic acid takes place under aeration in an organic nutrient salt solution of a simple composition, containing, as the carbon source for the energy and fuel metabolism of the fungus, cornmeal, a sugar, or some other carbon compound (e.g. ethanol) and, as the substrate for L-malic acid production, 11-15% hy weight of fumaric acid in the form of a salt.
e fungus utilized is, preferably, a fungus which, though capable of converting fwnaric acid into L-malic acid, uses neither fumaric acid nor L-malic acid to an appreciable extent for its energy and fuel metabolism.
Among this group are fungi stemming preferably from the general Aspergillus, Penicillium, Paecilomyces, Trichosporon, Taphrina, Helminthosporium, and Pythium. Preferably, Aspergillus wentii is used. However, the process of this invention is not restricted to fungi from these genera.
Due to the large productivity, fermentation in nutrient-containing fumaric acid solution can be terminated after three days at the latest.
Fermentation in nutrient-free fumaric acid solution can be ended even as early as after 12-24 hours. ln any event, concentration of L-malic acid in the suspension is, at the time of harvesting, preferably more than 100 g per liter.
The L-malic acid is produced extensively, or exclusively, in nutrient-free fumaric acid solution. Fungal cell mass from a preceding fermentation or growing procedure is, for this purpose, separated from the preceding culture filtra~e, transferred into a solution containing merely fumaric acid (11-15% by weight) in the form of a salt, and again incubated under slight stirring and aeration. After 12-24 hours, this fermentation yields L-malic acid in a concentration of more than 100 g per litre. Using the fungal cell mass resulting from a fermentation or growing step in a nutrient-containing solution, it is possible to conduct several, e.g. about up to three, fermen-tations in a nutrient-free fumaric acid solution in succession; in this connection, the liquid volume of each single one of these fermentations amounts to one to ten times, preferably one to three times, the volume of the preceding fermentation in nutrient-containing fumaric acid solution (or of the preceding growth in fumaric-acid-free or fumaric-acid-containing nutrient solution).
The biotechnical reactors for fermentation in nutrient-free fumaric acid solution are of a substantially simpler structure than conventional biotechnical reactors. They do not need, for example, any efficient agitation and aeration system to obtain a large oxygen transfer rate since the fungus no longer grows therein. There is no necessity for a sterili~ing apparatus, either, since these fermentations, with 12-24 hours' duration, are comparatively brief, thereby preventing any appTeciable development of infection-causing microorganisms within this short time span. The latter kinds of microorganisms also face unfavorable growth conditions due to the lack of a readily metabolizable carbon compound and due to the large osmotic pressure of the solution.
The nutrient-free solution contains, as the single ingredient, fumaric acid in the form of a salt and, if desired, activators for the fer-mentative conversion of fumaric acid into L-malic acid. In this solution, the fwlgal metabolism is reduced to conversion of fumaric acid into L-malic acid, which latter acid is therefore obtained without the components usually contained in a culture solution, for example, nutrients added for growth and organic compounds formed as by-products of the growth processes.
Fumaric acid is a mass-produced article manufactured on a large industrial scale with food quality, and can be directly utilized for the pro-cess of this invention.
The ~ollowing procedure is observed in the fermentation in nutrient-containing fumaric acid solution.
Fumaric acid is provided in a concentration of 11-15%, preferably 12-13%, in water and neutralized with an alkaline solution, preferably sodium hydroxide solution. The solution is combined with the inorganic nutrients necessary for fungal growth, for example, per li~er of solution, with 1 ml concentrated phosphoric acid ~H3PO4) 1.4 ml concentrated ammonium hydroxide solution ~N~l~OH) 0.9 g magnesium sulfate (MgSO4 7 H20) 30 g ferric chloride (FeC13 6 H20) 2 g potassium chloride (KCl) The addition of KCl is required if at least part of the fumaric acid has not been neutralized with potassium hydroxide. For batched with fungi needing specific active agents for growth (for example vitamins), the solution is furthermore mixed with these active agents.
The nutrient-containing solution is adjusted to pll 3-9, preferably 6-8, and ~hen sterilized, preferably thermally. The carbon compounds required for fungal growth are sterilized either together with the inorganic nutrients or,for example, in order to avoid caramelization phenomena in sugars, separately therefrom. Suitable carbon compounds for fungal growth are corn meal, sucrose, glucose, glycerol, ethanol, and other carbon-containing com-pounds.
After sterilization, the fermentor is inoculated with an inoculum of a fungus suitable for converting fumaric acid into L-malic acid in an amount of 1-20%, preferably in a quantity of 3-10%, of the fermentation batch, and operated under controlled conditions regarding temperature, aeration, and pH
value for 1-3 days. During this period, the fungus introduced by inoculation initially builds up its cell mass while consuming the added carbon compound.
This cell mass then converts the largest portion, by far, of the introduced fumaric acid into L-malic acid so that after three days at the latest,an L-malic acid concentration is obtained of more than 100 g per liter of culture solution. The fermentation temperature is 20-50C, preferably 25-35C. This temperature, after fungal growth is extensively completed (for example after 1.5 days of fermentation), can be raised to 25-60C, preferably 30-45C.
From this temperature increase, after termination of fungal growth, results an increase in the conversion rate of fumaric acid into L-malic acid, for, surprisingly, the optimum temperature for converting fumaric acid into L-malic acid is above the temperature optimal for fungal growth.
The pH during fermentation is maintained between 3 and 9, preferably between 6 and 8, by feeding the appropriate neutralizing medium. Agitator speed and aeration are to be adjusted to provide adequate oxygen supply for the fungus.
After fermentation is completed, the culture solution is separated by filtration or some other liquid/solid separating operation into culture filtrate and fungal cell mass. The filtrate is available for isolation of L-malic acid contained therein according to conventional processes, for example, by precipitation as Ca malate. The fungal cell mass can be utilized for sub-sequent fermentation in nutrient-free fumaric acid solution.
The following procedure is observed in fermentation in nutrient-free fumaric acid solution.
The cell mass resulting from a preceding fermentation in nutrient-free or nutrient-containing fumaric acid solution, or from a grJwing step in fumaric-acid-free or fumaric-acid-containing nutrient solution, is transferred into a solution containing merely 11-15% (preferably 12-13%) of fumaric acid in the form of a salt; the suspension is incubated in a biotechnical reactor under slight agitation and aeration for 12-24 hours. The L-malic acid concen-tration then is, just as at the end of fermentation in nutrient-containing fumaric acid solution, more than 100 g liter of suspension. The temperature during fermentation in nutrient-free fumaric acid solution is 25-60C, pre-ferably 30-45C.
Fermentation in nu~rient-free fumaric acid solution can be conducted in a biotechnical reactor of a very simple construction. This reactor requires no devices for obtaining a high oxygen transfer rate and for sterilization. An easily cleaned, thermostat-equipped vessel with devices for agitation and aeration is adequate. Sligh* agitation is necessary to prevent sedimentation of the fungal cell mass; slight aeration is required in order to supply the -` 10 -. .
~2~17~
cells with the amount of oxygen needed for sustained breathing.
The simple construction type of biotechnical reactor for fermentation in nutrient-free fumaric acid solution has a favorable effect from the viewpoint of economy, above all in the case where L-malic acid is produced predominantly or exclusively by fermentation in nutrient-free fumaric acid solution. The liquid volume can be, in this connection, one to ten times, preferably two to five times, the volume used in fermentation in a nutrient-containing fumaric acid solution or during growth in a fumaric-acid-free or fumaric-acid-containing nutrient solution. This permits a ratio of 1 : 1 to 1 : 10 with respect to the volumes of the two types of biotechnical reactors.
~sing a cell mass resulting from fermentation in nutrient-containing fumaric acid solution or from a growth step in fumaric-acid free or fumaric-acid-containing nutrient solution, it is possible to perform several, approximately up to three (preferably two) fermentations in nutrient-free fumaric acid solu-tion in succession. If the fermentation in nutrient-free fumaric acid solution is repeated several times, it is recommended, in order to inactivate any con-taminating microorganisms that may occur in such a case, to incubate the fungal cell mass before each reuse for 10-60 minutes in a solution having a low pH
value, for example, a pH of 2-3, possibly with the addition of a bactericidal agent. Such processes for combating contaminating microorganisms have been used in other biotechnical procedures (for example in the production of baker's yeast).
The invention has the following advantages:
(a) Freely mobile fungi are utilized, rather than bacteria (in free or immobilized form), and rather than an enzyme which must first be isolated from a cell mass.
(b) The fermentation batch contains calcium merely as a trace element ~e.g. from drinking water).
(c) Fermentation time is short and productivity is high.
~ d) Fumaric acid is converted predominantly or exclusively in a nutrient-free solution under optimum fermentation conditions; the optimum growth conditions of the fungus need not be cansidered herein.
~ e) The separated funga~ cell mass can be repeaLedly utilized.
~ f) During fermentation in nutrient-free fumaric acid solution, there can he no appreciahle development of contaminating microorganisms.
~ g) L-Malic acid is obtained, after fermentation in ~nutrient-con-taining or nutrient-free) fumaric acid solution, in a concentration of 100-170 g per liter. The yield is more than 0.9 g of L-malic acid per gram of fumaric acid, i.e. more than 77% of theroetical yield.
~h) L-Malic acid is obtained in a solut~on very suitable for further processing into a quality suitable for foodstuffs and pharmaceuticals, for example, regarding the absence cf LpS toxîns and succinic acid.
(i) There is no production of D-malic acid.
(j) The process is highly economical since primarily biotechnical reactors of simple construction are utilized.
The invention will now be explained with reference to the following example~,but without being limited thereto.
Fermentation in nutrient-containing fumaric acid solution proceeds as follows, for example:
Example 1 One liter of water and 125 g of fumaric acid are combined, and the acid is neutralized with a mixture of NaOH and KOII (9 parts of NaOH and 1 part of KOH). The nutrients set forth below are added:
}9~73 g sucrose 1 ml concentrated phosphoric acid (H3P04) 1.4 ml concentrated ammonium hydroxide solution (NH40H) 0.9 g magnesium sulfate (~IgS04 7 H20) 3a mg ferric chloride (FeC13 6 II20) A fermentor having an operating volume of 8 1 is charged with this nutrient-containing fumaric acid solution, heat-sterilized~ and inoculated with Q.25 1 of inoculum of the fungus Aspergillus wentii.
The fermentation conditions are:
aeration 0.5 1 of air per liter of reactor volume and minute (0.5 vvm) agitator speed: 500 rpm ~paddle stirrer in combination with a guide tube) temperature: 30 C
pH value: 7.0 (kept constant by feeding NH40H
solution with control via pH
electrode) fermentation period: 72 hours At the end of the fermentation time, 132 g of L-malic acid has been produced per liter of culture solution.
Example 2 Fermentation according to Example 1 is repeated, increasing the amount of inoculum to 0.8 1. After 48 hours of fermentation, 118 g of L-malic acid has been formed per liter of culture solution.
Example 3 -Fermentation according to Example 1 is repeated~ using 0.8 1 of inoculum of the fungus Helminthosporium sativum. After 72 hours of fermenta-tion, 108 g of L-malic acid has been produced per liter of culture solution.
Example 4 Fermentation according to Example 1 is repeated with 0.8 1 of inoculum of the fungus Penicillium nalgiovensis. After 72 hours of fermenta-tion period, the yield is 105 g of L-malic acid per liter of culture solution.
Example 5 Fermentation according to Example 1 is repeated with a nutrient solution containing, in place of sucrose, 10 g of cornmeal per liter of solution. After 72 hours of fermentation, 123 g of l-malic acid has been produced per-liter of culture solution.
Example 6 Fermentation according to Example 1 is re~eated after increasing the amount of sucrose to 30 g per liter. After 72 hours of fermentation time, the yield is 128 g of L-malic acid per liter of culture solution.
In order to isolate L-malic acid formed during fermentation, the fungal mycelium is separated from the culture filtrate by filtration, and the culture filtrate is combined with the stoichiometric amount of hydrochloric acid ~'oased on fumaric acid used). The small quantity of unreacted fumaric acid is precipitated from the hydrochloric solution and is removed by filtra-tion. The filtrate is combined, after heating to about 60C, with the stoich-iometric amount of calcium hydroxide (based on the thus-formed L-malic acid) and agitated for 60 minutes at about 90 C. Subsequently the mixture is filtered in the hot state, and the calcium malate is dried under gentle condi-tions. In this way, 1,237 g of calcium malate is obtained from 8 1 of culture filtrate.
- 1~1 -. .
0~
Fermentation in nutrient-free fumaric acid solution proceeds as follows, for example:
Example 7 The fungal mycelium obtained by filtration of the 8 liters of culture filtrate of Example 6 is suspended in 16 l of a 12.5% strength fumaric acid solution (neutralized with NaOH and KOH in a ratio of 9 : l), and incu-bated at 35C in a slightly stirred and aerated container. Aftar a fermenta-tion of 20 hours, lll g of L-malic acid has been formed per liter of culture solution.
Example S
Fermentation according to Example 7 is repeated, adding 1 g of a surfactant, for example an emulsifier, and 1 g of ammonium sulfate (NH4)2S04 per liter of solution. After a fermentation period of 16 hours, 115 g of L-malic acid has been formed per liter of culture solution.
Example 9 -Fermentation according to Example 7 is repeated with 16 l of a 14.0% strength fumaric acid solution (neutralized with NaOH and KOH -- ratio 9 : 1). After 20 hours of fermenting, 142 g of L-malic acid has been produced per liter of culture solution.
Example l_ The fungal mycelium separated from the fermentation of Example 7 is suspended in 16 1 of a 12.5% strength fumaric acid solution (neutralized with NaOH and KOH in a ratio of 9 : 1). After 20 hours of fermentation, 113 g of L-malic acid has been formed per liter of culture solution.
Example 11 The fungal mycelium separated from the fermentation of Example 10 is utilized for another fermentation analogously to Example 10. After a fermentation period of 24 hours, 108 g of L-malic acid has been produced per liter of culture solution.
In all examples, the culture solution is worked up analogously to Example 6.
9~7~
acid, can be isolated in special processes from biological material, such as the cell mass of bacteria or fungi, and then can be used, in free or immobilized form, for obtaining L-malic acid from fumaric acid ~DAS 2,415,310; Czechoslo-vakian Patent 171,990).
In several processes, a large amount of a calcium compound is added to the fermentation batch whereby the resultant L-malic acid is precipitated as early during fermentation as the calcium malate (DOS 1,417,033).
Processes for the biotechnical manufacture of L-malic acid with the aid of fungi are likewise known. In these processes, the L-malic acid is essentially obtained by biochemical degradation and/or rearrangement of the carbon source fed to the respective fungus (for example molasses, sugar, ethanol, acetic acid) [J. Ferment Technol. 55 (2) : 196-l99 (1977)]. These processes do not start directly with fumaric acid.
In the process described in United States Patent 3,063,910, the fermentation batch can contain, in addition to 10-15% of a sugar, 1-10~ of an organic acid such as, for example, pyruvic or fumaric acid. However, in this fungal method, the added organic acid does not serve as a substrate for the synthesis of L-malic acid but, rather, serves as a reaction accelerator for the biochemical degradation or rearrangement of the offered sugar into L-malic acid.
Furthermore, fermentative conversion of n-parafin into L-malic acid is possible by the association of two types of yeasts, wherein one type of yeast converts paraffin into fumaric acid and the second type of yeast converts the resultant fumaric acid into L-malic acid LAgr. Biol. Chem. 34 :
1833-1838 (1970)].
The known methods for the biotechnical manufacture of L-malic acid have, inter alia, the peculiarities set out below:
(a) When using bacteria, biotechnical processes are more difficult to handle than when using fungi. For example, separation problems can arise due to the small size of the bacteria. Furthermore, bacteria tend to form by-products, including toxic by-products. A particularly disturbing factor against use of bacteria produced L-malic acid in the pharmaceuticals area is LPS toxin (lipopolysaccharide toxin)which is also formed by bacteria. This toxin has a pyrogenic effect and can be separated from L-malic acid only through the use of expensive processes such as ultrafiltration. In the bacterial method des-cribed by United States Patent 3,922,195, succinic acid by-product occurs in small amounts. This acid can be separated from malic acid only with difficul-ties.
(b) When using the enzyme fumarase, the primary fumarase source is the microbial cell mass. Before production of L-malic acid can be started, the microbial cell mass must be grown and worked up into fumarase,with consi-derable expense.
(c) Immobilization of living cells, or of the enzyme fumarase isolated from cells, is an additional, very expensive process step.
(d) When fermenting in the presence of a large concentration of CA
ions, the thus-formed L-malic acid is precipitated as calcium malate as early as during fermentation; this leads to problems during agitation and aeration of the fermentation batch.
(e) The attainable concentration of L-malic acid, is generally, compara-tively low and lies markedly below 100 g per liter of culture solution.
The reactions described in United States Patent 3,922,195 (immobilized hacteria) and in DOS 1,417,033 (high calcium concentration) give results that are exceptions to this generality.
~Z~73 ~ f) Fermentation takes longer than three days. This low produc-tivity requires the use of large capacity biotechnical reactors. Large reactor size greatly increases their cost, especially since these reactors are of an expensive construction to allow the conduct of a contamination-free process.
The invention provides a process for production of L-malic acid which is free of D-malic acid which process comprises admixing neutralized fumaric acid, in an aqueous phase having an initial concentration of fumaric acid of 11 to 15% by weight, with a freely mobile fungus to ferment the fumaric acid to L-malic acid. Preferably, the fermentation is continued until L-malic acid concentration reaches a value in the range 100 to 170 g/liter.
In the above process~ it is preferred that the aqueous solution of the neutralized fumaric acid additionally contains nutrients required for fungal growth, the solution is innoculated with 1-20% of fungal inoculum, the inoculated solution i5 fermented at 20-50 C for 1-3 days, and L-malic acid is separated from the culture solution and cell mass.
It is further preferred that the above process comprises producing an aqueous solution of the neutralized fumaric acid without nutrients for the fungus, adding to the nutrient-free fumaric acid solution fungal cell mass resulting from incubation in fumaric-acid-free or fumaric-acid-containing nutrient solution, or from a fermentation in nutrient-containing fumaric acid solution, wherein the volume of the nutrient-free fumaric acid solution amounts to one to ten times the liquid volume of the preceding incubation in fumaric-acid-free or fumaric-acid-containing nutrient solution or of the ~ _ ~2~
preceding fermentation in nutrient-containing fumaric acid solution when substantially the entire cell mass of the preceding incubation or fermenta-tion is being utilized, fermenting for 12-24 hours at 25-60C, and separating the cell mass and L-malic acid from the culture solution.
The fumaric acid can be fermented to L-malic acid in a nutrient-containing or nutrient-free solution. It may be expedient to ferment the fumaric acid only in a nutrient-free solution and to cultivate the cell mass required for this purpose in a nutrient solution that is free of fumaric acid or that contains fumaric acid. The nutrient solution suitable for this purpose can contain substantially less than 11% fumaric acid, since in this case the fungus is merely to be adapted to fumaric acid.
Although the term "fermentation" in technical literature has a relatively large spectrum of meanings, the expression "fermentation" in the context of this invention is understood to mean the biochemical conversion of fumaric acid into L-malic acid under the action of fumarase. The word "growing" is understood to mean theproliferation of the biomass from the inoculum with consumption of nutrients. In nutrient-containing fumaric acid solution, both processes (growing and fermentation) proceed side-by-side or in succession.
For fermentation in nutrient-containing fumaric acid, the nutrients and active agents necessary for fungal growth are added to the solution with neutralized fumaric acid. Aftsr sterilization, this solution is inoculated with 1-20% inoculum of a fungus, and fermented for a period of one to three days at 20-50C. Subsequently, the culture solution is separated into cell mass and culture filtrate, and L-malic acid is isolated from the culture filtrate according to conventional methods.
The separated cell mass can be utilized for several, e.g.
approximately for three additional, fermentations in respectively freshly prepared nutrient-free fumaric acid solutions. In this connection, the volume of the fumaric acid solution can amount to one to ten times the liquid volume of the preceding fermentation in nutrient-containing fumaric acid solution, or of the preceding incubation in fumaric-acid-free or fumaric-acid-containing nutrient solution, if the entire cell mass resulting from the nutrient-containing solution is employed. In a nutrient-free fumaric-acid solution, fermenta~ion is performed for 12-24 hours at 25-60 C.
The aerokic, fermentative conversion, in a nutrient-containing solution, of fumaric acid into L-malic acid takes place under aeration in an organic nutrient salt solution of a simple composition, containing, as the carbon source for the energy and fuel metabolism of the fungus, cornmeal, a sugar, or some other carbon compound (e.g. ethanol) and, as the substrate for L-malic acid production, 11-15% hy weight of fumaric acid in the form of a salt.
e fungus utilized is, preferably, a fungus which, though capable of converting fwnaric acid into L-malic acid, uses neither fumaric acid nor L-malic acid to an appreciable extent for its energy and fuel metabolism.
Among this group are fungi stemming preferably from the general Aspergillus, Penicillium, Paecilomyces, Trichosporon, Taphrina, Helminthosporium, and Pythium. Preferably, Aspergillus wentii is used. However, the process of this invention is not restricted to fungi from these genera.
Due to the large productivity, fermentation in nutrient-containing fumaric acid solution can be terminated after three days at the latest.
Fermentation in nutrient-free fumaric acid solution can be ended even as early as after 12-24 hours. ln any event, concentration of L-malic acid in the suspension is, at the time of harvesting, preferably more than 100 g per liter.
The L-malic acid is produced extensively, or exclusively, in nutrient-free fumaric acid solution. Fungal cell mass from a preceding fermentation or growing procedure is, for this purpose, separated from the preceding culture filtra~e, transferred into a solution containing merely fumaric acid (11-15% by weight) in the form of a salt, and again incubated under slight stirring and aeration. After 12-24 hours, this fermentation yields L-malic acid in a concentration of more than 100 g per litre. Using the fungal cell mass resulting from a fermentation or growing step in a nutrient-containing solution, it is possible to conduct several, e.g. about up to three, fermen-tations in a nutrient-free fumaric acid solution in succession; in this connection, the liquid volume of each single one of these fermentations amounts to one to ten times, preferably one to three times, the volume of the preceding fermentation in nutrient-containing fumaric acid solution (or of the preceding growth in fumaric-acid-free or fumaric-acid-containing nutrient solution).
The biotechnical reactors for fermentation in nutrient-free fumaric acid solution are of a substantially simpler structure than conventional biotechnical reactors. They do not need, for example, any efficient agitation and aeration system to obtain a large oxygen transfer rate since the fungus no longer grows therein. There is no necessity for a sterili~ing apparatus, either, since these fermentations, with 12-24 hours' duration, are comparatively brief, thereby preventing any appTeciable development of infection-causing microorganisms within this short time span. The latter kinds of microorganisms also face unfavorable growth conditions due to the lack of a readily metabolizable carbon compound and due to the large osmotic pressure of the solution.
The nutrient-free solution contains, as the single ingredient, fumaric acid in the form of a salt and, if desired, activators for the fer-mentative conversion of fumaric acid into L-malic acid. In this solution, the fwlgal metabolism is reduced to conversion of fumaric acid into L-malic acid, which latter acid is therefore obtained without the components usually contained in a culture solution, for example, nutrients added for growth and organic compounds formed as by-products of the growth processes.
Fumaric acid is a mass-produced article manufactured on a large industrial scale with food quality, and can be directly utilized for the pro-cess of this invention.
The ~ollowing procedure is observed in the fermentation in nutrient-containing fumaric acid solution.
Fumaric acid is provided in a concentration of 11-15%, preferably 12-13%, in water and neutralized with an alkaline solution, preferably sodium hydroxide solution. The solution is combined with the inorganic nutrients necessary for fungal growth, for example, per li~er of solution, with 1 ml concentrated phosphoric acid ~H3PO4) 1.4 ml concentrated ammonium hydroxide solution ~N~l~OH) 0.9 g magnesium sulfate (MgSO4 7 H20) 30 g ferric chloride (FeC13 6 H20) 2 g potassium chloride (KCl) The addition of KCl is required if at least part of the fumaric acid has not been neutralized with potassium hydroxide. For batched with fungi needing specific active agents for growth (for example vitamins), the solution is furthermore mixed with these active agents.
The nutrient-containing solution is adjusted to pll 3-9, preferably 6-8, and ~hen sterilized, preferably thermally. The carbon compounds required for fungal growth are sterilized either together with the inorganic nutrients or,for example, in order to avoid caramelization phenomena in sugars, separately therefrom. Suitable carbon compounds for fungal growth are corn meal, sucrose, glucose, glycerol, ethanol, and other carbon-containing com-pounds.
After sterilization, the fermentor is inoculated with an inoculum of a fungus suitable for converting fumaric acid into L-malic acid in an amount of 1-20%, preferably in a quantity of 3-10%, of the fermentation batch, and operated under controlled conditions regarding temperature, aeration, and pH
value for 1-3 days. During this period, the fungus introduced by inoculation initially builds up its cell mass while consuming the added carbon compound.
This cell mass then converts the largest portion, by far, of the introduced fumaric acid into L-malic acid so that after three days at the latest,an L-malic acid concentration is obtained of more than 100 g per liter of culture solution. The fermentation temperature is 20-50C, preferably 25-35C. This temperature, after fungal growth is extensively completed (for example after 1.5 days of fermentation), can be raised to 25-60C, preferably 30-45C.
From this temperature increase, after termination of fungal growth, results an increase in the conversion rate of fumaric acid into L-malic acid, for, surprisingly, the optimum temperature for converting fumaric acid into L-malic acid is above the temperature optimal for fungal growth.
The pH during fermentation is maintained between 3 and 9, preferably between 6 and 8, by feeding the appropriate neutralizing medium. Agitator speed and aeration are to be adjusted to provide adequate oxygen supply for the fungus.
After fermentation is completed, the culture solution is separated by filtration or some other liquid/solid separating operation into culture filtrate and fungal cell mass. The filtrate is available for isolation of L-malic acid contained therein according to conventional processes, for example, by precipitation as Ca malate. The fungal cell mass can be utilized for sub-sequent fermentation in nutrient-free fumaric acid solution.
The following procedure is observed in fermentation in nutrient-free fumaric acid solution.
The cell mass resulting from a preceding fermentation in nutrient-free or nutrient-containing fumaric acid solution, or from a grJwing step in fumaric-acid-free or fumaric-acid-containing nutrient solution, is transferred into a solution containing merely 11-15% (preferably 12-13%) of fumaric acid in the form of a salt; the suspension is incubated in a biotechnical reactor under slight agitation and aeration for 12-24 hours. The L-malic acid concen-tration then is, just as at the end of fermentation in nutrient-containing fumaric acid solution, more than 100 g liter of suspension. The temperature during fermentation in nutrient-free fumaric acid solution is 25-60C, pre-ferably 30-45C.
Fermentation in nu~rient-free fumaric acid solution can be conducted in a biotechnical reactor of a very simple construction. This reactor requires no devices for obtaining a high oxygen transfer rate and for sterilization. An easily cleaned, thermostat-equipped vessel with devices for agitation and aeration is adequate. Sligh* agitation is necessary to prevent sedimentation of the fungal cell mass; slight aeration is required in order to supply the -` 10 -. .
~2~17~
cells with the amount of oxygen needed for sustained breathing.
The simple construction type of biotechnical reactor for fermentation in nutrient-free fumaric acid solution has a favorable effect from the viewpoint of economy, above all in the case where L-malic acid is produced predominantly or exclusively by fermentation in nutrient-free fumaric acid solution. The liquid volume can be, in this connection, one to ten times, preferably two to five times, the volume used in fermentation in a nutrient-containing fumaric acid solution or during growth in a fumaric-acid-free or fumaric-acid-containing nutrient solution. This permits a ratio of 1 : 1 to 1 : 10 with respect to the volumes of the two types of biotechnical reactors.
~sing a cell mass resulting from fermentation in nutrient-containing fumaric acid solution or from a growth step in fumaric-acid free or fumaric-acid-containing nutrient solution, it is possible to perform several, approximately up to three (preferably two) fermentations in nutrient-free fumaric acid solu-tion in succession. If the fermentation in nutrient-free fumaric acid solution is repeated several times, it is recommended, in order to inactivate any con-taminating microorganisms that may occur in such a case, to incubate the fungal cell mass before each reuse for 10-60 minutes in a solution having a low pH
value, for example, a pH of 2-3, possibly with the addition of a bactericidal agent. Such processes for combating contaminating microorganisms have been used in other biotechnical procedures (for example in the production of baker's yeast).
The invention has the following advantages:
(a) Freely mobile fungi are utilized, rather than bacteria (in free or immobilized form), and rather than an enzyme which must first be isolated from a cell mass.
(b) The fermentation batch contains calcium merely as a trace element ~e.g. from drinking water).
(c) Fermentation time is short and productivity is high.
~ d) Fumaric acid is converted predominantly or exclusively in a nutrient-free solution under optimum fermentation conditions; the optimum growth conditions of the fungus need not be cansidered herein.
~ e) The separated funga~ cell mass can be repeaLedly utilized.
~ f) During fermentation in nutrient-free fumaric acid solution, there can he no appreciahle development of contaminating microorganisms.
~ g) L-Malic acid is obtained, after fermentation in ~nutrient-con-taining or nutrient-free) fumaric acid solution, in a concentration of 100-170 g per liter. The yield is more than 0.9 g of L-malic acid per gram of fumaric acid, i.e. more than 77% of theroetical yield.
~h) L-Malic acid is obtained in a solut~on very suitable for further processing into a quality suitable for foodstuffs and pharmaceuticals, for example, regarding the absence cf LpS toxîns and succinic acid.
(i) There is no production of D-malic acid.
(j) The process is highly economical since primarily biotechnical reactors of simple construction are utilized.
The invention will now be explained with reference to the following example~,but without being limited thereto.
Fermentation in nutrient-containing fumaric acid solution proceeds as follows, for example:
Example 1 One liter of water and 125 g of fumaric acid are combined, and the acid is neutralized with a mixture of NaOH and KOII (9 parts of NaOH and 1 part of KOH). The nutrients set forth below are added:
}9~73 g sucrose 1 ml concentrated phosphoric acid (H3P04) 1.4 ml concentrated ammonium hydroxide solution (NH40H) 0.9 g magnesium sulfate (~IgS04 7 H20) 3a mg ferric chloride (FeC13 6 II20) A fermentor having an operating volume of 8 1 is charged with this nutrient-containing fumaric acid solution, heat-sterilized~ and inoculated with Q.25 1 of inoculum of the fungus Aspergillus wentii.
The fermentation conditions are:
aeration 0.5 1 of air per liter of reactor volume and minute (0.5 vvm) agitator speed: 500 rpm ~paddle stirrer in combination with a guide tube) temperature: 30 C
pH value: 7.0 (kept constant by feeding NH40H
solution with control via pH
electrode) fermentation period: 72 hours At the end of the fermentation time, 132 g of L-malic acid has been produced per liter of culture solution.
Example 2 Fermentation according to Example 1 is repeated, increasing the amount of inoculum to 0.8 1. After 48 hours of fermentation, 118 g of L-malic acid has been formed per liter of culture solution.
Example 3 -Fermentation according to Example 1 is repeated~ using 0.8 1 of inoculum of the fungus Helminthosporium sativum. After 72 hours of fermenta-tion, 108 g of L-malic acid has been produced per liter of culture solution.
Example 4 Fermentation according to Example 1 is repeated with 0.8 1 of inoculum of the fungus Penicillium nalgiovensis. After 72 hours of fermenta-tion period, the yield is 105 g of L-malic acid per liter of culture solution.
Example 5 Fermentation according to Example 1 is repeated with a nutrient solution containing, in place of sucrose, 10 g of cornmeal per liter of solution. After 72 hours of fermentation, 123 g of l-malic acid has been produced per-liter of culture solution.
Example 6 Fermentation according to Example 1 is re~eated after increasing the amount of sucrose to 30 g per liter. After 72 hours of fermentation time, the yield is 128 g of L-malic acid per liter of culture solution.
In order to isolate L-malic acid formed during fermentation, the fungal mycelium is separated from the culture filtrate by filtration, and the culture filtrate is combined with the stoichiometric amount of hydrochloric acid ~'oased on fumaric acid used). The small quantity of unreacted fumaric acid is precipitated from the hydrochloric solution and is removed by filtra-tion. The filtrate is combined, after heating to about 60C, with the stoich-iometric amount of calcium hydroxide (based on the thus-formed L-malic acid) and agitated for 60 minutes at about 90 C. Subsequently the mixture is filtered in the hot state, and the calcium malate is dried under gentle condi-tions. In this way, 1,237 g of calcium malate is obtained from 8 1 of culture filtrate.
- 1~1 -. .
0~
Fermentation in nutrient-free fumaric acid solution proceeds as follows, for example:
Example 7 The fungal mycelium obtained by filtration of the 8 liters of culture filtrate of Example 6 is suspended in 16 l of a 12.5% strength fumaric acid solution (neutralized with NaOH and KOH in a ratio of 9 : l), and incu-bated at 35C in a slightly stirred and aerated container. Aftar a fermenta-tion of 20 hours, lll g of L-malic acid has been formed per liter of culture solution.
Example S
Fermentation according to Example 7 is repeated, adding 1 g of a surfactant, for example an emulsifier, and 1 g of ammonium sulfate (NH4)2S04 per liter of solution. After a fermentation period of 16 hours, 115 g of L-malic acid has been formed per liter of culture solution.
Example 9 -Fermentation according to Example 7 is repeated with 16 l of a 14.0% strength fumaric acid solution (neutralized with NaOH and KOH -- ratio 9 : 1). After 20 hours of fermenting, 142 g of L-malic acid has been produced per liter of culture solution.
Example l_ The fungal mycelium separated from the fermentation of Example 7 is suspended in 16 1 of a 12.5% strength fumaric acid solution (neutralized with NaOH and KOH in a ratio of 9 : 1). After 20 hours of fermentation, 113 g of L-malic acid has been formed per liter of culture solution.
Example 11 The fungal mycelium separated from the fermentation of Example 10 is utilized for another fermentation analogously to Example 10. After a fermentation period of 24 hours, 108 g of L-malic acid has been produced per liter of culture solution.
In all examples, the culture solution is worked up analogously to Example 6.
Claims (9)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for production of L-malic acid which is free of D-malic acid which process comprises admixing neutralized fumaric acid, in an aqueous phase having an initial concentration of fumaric acid of 11 to 15% by weight, with a freely mobile fungus to ferment the fumaric acid to L-malic acid.
2. The process defined in claim 1 wherein the fermentation is contin-ued until L-malic acid concentration reaches a value in the range 100 to 170 g/liter.
3. The process as defined in claim 1, wherein the aqueous solution of the neutralized fumaric acid additionally contains nutrients required for fungal growth, the solution is inoculated with 1-20% of fungal inoculum, the inoculated solution is fermented at 20-50° C for 1-3 days, and L-malic acid is separated from the culture solution and cell mass.
4. The process as defined in claim 1, which comprises producing an aqueous solution of the neutralized fumaric acid without nutrients for the fungus, adding to the nutrient-free fumaric acid solution fungal cell mass resulting from incubation in fumaric-acid-free or fumaric-acid-containing nutrient solution, or from a fermentation in nutrient-containing fumaric acid solution, wherein the volume of the nutrient-free fumaric acid solution amounts to one to ten times the liuqid volume of the preceding incubation in fumaric-acid-free or fumaric-acid-containing nutrient solution or of the preceding fermentation in nutrient-containing fumaric acid solution when substantially the entire cell mass of the preceding incubation or fermentation is being utilized, fermenting for 12-24 hours at 25-60° C, and separating the cell mass and L-malic acid from the culture solution.
5. The process as defined in claim 1,2 or 3 wherein the separated cell mass is used again in one or more further fumaric acid solutions.
6. The process as defined in claim 4 wherein the separated cell mass is used again in one or more further nutrient-free, fumaric acid solutions.
7. The process as defined in claim 1, 3 or 4 wherein the fungus is selected from one of the genera A-pergillus, Penicillium, Paecilomyces, Trichlosporon, Taphrina, Helminthosporium, Pythium.
8. The process as defined in claim 1, 3 or 4, wherein the fungus is Aspergillus wentii.
9. The process as defined in claim 1, 3 or 4 wherein the fumaric acid is neutralized by sodium hydroxide.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3310849.8 | 1983-03-25 | ||
| DE19833310849 DE3310849A1 (en) | 1983-03-25 | 1983-03-25 | METHOD FOR THE BIOTECHNOLOGICAL PRODUCTION OF L-APEPLIC ACID |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1209073A true CA1209073A (en) | 1986-08-05 |
Family
ID=6194600
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000450366A Expired CA1209073A (en) | 1983-03-25 | 1984-03-23 | Process for the biotechnical production of l-malic acid |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0120213B1 (en) |
| JP (1) | JPS59187789A (en) |
| AT (1) | ATE25266T1 (en) |
| CA (1) | CA1209073A (en) |
| DE (2) | DE3310849A1 (en) |
| DK (1) | DK78184A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| JP4860180B2 (en) * | 2005-05-17 | 2012-01-25 | Ｎｅｃカシオモバイルコミュニケーションズ株式会社 | Timer device and program |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1417033A1 (en) * | 1959-12-16 | 1969-03-13 | Kyowa Hakko Kogyo Kk | Process for the production of L-malic acid |
| US3063910A (en) * | 1960-02-03 | 1962-11-13 | Kyowa Hakko Kogyo Kk | Method of producing l-malic acid by fermentation |
| IT981777B (en) * | 1973-03-30 | 1974-10-10 | Snam Progetti | PROCEDURE FOR THE ENZYMATIC PRODUCTION OF MALIC ACID |
| US3922195A (en) * | 1974-10-17 | 1975-11-25 | Tanabe Seiyaku Co | Process for preparing L-Malic acid |
| JPS5290691A (en) * | 1976-01-27 | 1977-07-30 | Shiyouichi Takao | Production of llmalic acid by fermention |
-
1983
- 1983-03-25 DE DE19833310849 patent/DE3310849A1/en not_active Withdrawn
-
1984
- 1984-01-21 EP EP84100768A patent/EP0120213B1/en not_active Expired
- 1984-01-21 AT AT84100768T patent/ATE25266T1/en not_active IP Right Cessation
- 1984-01-21 DE DE8484100768T patent/DE3462271D1/en not_active Expired
- 1984-02-20 DK DK78184A patent/DK78184A/en not_active Application Discontinuation
- 1984-03-22 JP JP59053665A patent/JPS59187789A/en active Pending
- 1984-03-23 CA CA000450366A patent/CA1209073A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| EP0120213A2 (en) | 1984-10-03 |
| JPS59187789A (en) | 1984-10-24 |
| EP0120213B1 (en) | 1987-01-28 |
| DK78184D0 (en) | 1984-02-20 |
| DE3462271D1 (en) | 1987-03-05 |
| DE3310849A1 (en) | 1984-09-27 |
| ATE25266T1 (en) | 1987-02-15 |
| EP0120213A3 (en) | 1985-10-02 |
| DK78184A (en) | 1984-09-26 |
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