SK44699A3 - Process for the production of l-malic acid - Google Patents

Abstract

The preparation is based on that the yeast of Dipodascus, preferably of the species oventis, tetrasperma, aggregatus, magnusii are cultivated in the aqueous solution with contents 5 to 20 wt. % of fumaric acid, with the temperature 30 - 50 C, pH value 4.5 - 8.0.

Description

Technical field

The invention relates to a process for the production of L-malic acid

BACKGROUND OF THE INVENTION

Malic acid (2-hydroxybutanedioic acid, GtHeOs, Mw-134.09) is produced by a number of microorganisms, in particular bacteria, yeasts and filamentous fungi. Malic acid is naturally found in fruits, grapes, bananas and also in other higher plants, animals, microorganisms, etc. L-malic acid has better organoleptic properties than citric acid, it is used in the food industry as an acidifier, flavoring in sweets, fruit and canned vegetables, soft drinks, condensed fruits, yoghurts, starch puddings, but also in wines. It is also of use in medicine as a preparation against hyperaemia and is one of the components for infusions (Peleg, Y. et al., Appl. Microbiol. Biotechnol. 28, 1988, pp. 69-75). In addition to food and pharmacy, it is also used in the production of detergents (belongs to good plasticizers), metal processing, as a chemical intermediate for organic syntheses (eg 24, 25-dihydroxycholecalciferol). Malic acid is present in three different modifications. D (+) and L (-) - malic acids show optical activity, the racemic (D, L) -malic mixture is optically inactive.

The chemical process for the preparation of malic acid consists in catalytic isomerization of maleic acid, respectively. its anhydride. This procedure produces a racemic mixture of D- and L-malic acid forms, being able to cleave into the two active (+) - and (-) - isomers. (Ng, T.K et al., Biotechnol. Bioeng. 17, 1986, pp.335-363). By combining the chemical and biotechnological methods, it is possible to prepare O (+) malic acid from a racemic mixture when the L (-) form of the acid is utilized from the mixture by some strains of the genera Acinetobacter, Pseudomonas, Bacillus, Corynebacterium, Serratia and the like. (Nakayama et al., Pat. Jap. 02242699, 1990).

Biotechnological procedures allow the preparation of the L-form of malic acid. Fumarate malate bioconversion is performed by microorganisms with a high fumarase enzyme content, such as bacteria from the genera Lactobacillus, Brevibacterium, Bacillus, yeasts from the genera Saccharomyces, Candida, Pichia and filamentous fungi from the genera Aspergillus, Rhizopus, Paecilomyces. These microorganisms can be used as free or immobilized cells (with the need for cell permeabilization), or they can be used for bioconversion cell-free extracts from these cells, or purified fumarase, however, which makes production more expensive. The formation of by-products, in particular succinic acid, must also be avoided by pre-incubating the cells in sodium maionate solution or by adding bile extract, which is also disadvantageous.

Conventional carbohydrate sources such as glucose, sucrose, starchy raw materials can also be used to prepare L-malic acid, since filamentous fungi are capable of forming fumaran and subsequently L-malic acid in the aerobic culture mode. This fact can also be used in mixed cultures where filamentous fungi produce fumaran and bacteria, respectively, from the saccharide substrate. yeast converts it to the malate. However, the disadvantage of using a carbohydrate substrate is the long fermentation time, aerobic culture regime, lower yields and by-product formation (Takao S. et al .: J. Ferm.Technol. 85,1976, pp.197-204).

The use of yeast in the conversion of fumarate-malate has long been the focus of attention mainly on their availability (with the possibility of using waste yeast from food production) and food acceptability.

SUMMARY OF THE INVENTION

The essence of the preparation of L-malic acid according to the invention is that yeasts of the genus Dipodascus are cultured in an aqueous solution containing 5-20% by weight of filmaric acid, at a temperature of 30-50 ° C, a pH of 4.5-8.0. As production microorganisms, e.g. Dipodascus strains, oventis, tetrasperma, aggregatus, magnusii, prepared by submerged culture on fumarate-added media. The advantage of using yeasts of the genus Dipodascus is an 8-10-fold higher level of fiunarase activity at the level of intact cells compared to other yeasts, e.g. Saccharomyces cerevisiae. This fact greatly simplifies the preparation of cells with higher fumarase enzyme activity and does not impose artificial permeabilization of the cells. Also noteworthy is that the bioconversion of fumarate to L-malate by yeasts of the genus Dipodascus is selective without the formation of succinate as a by-product.

Mixed reactors are used to prepare the cells during aeration. To inoculate the production fermenter, it is preferable to use a vegetative inoculum, which is obtained by cultivating the producer for 12 to 28 hours and dosed into the production fermenter in an amount of 5 to 25% of the total volume. It is preferred to add fumaric acid to the culture medium to induce the fumarase enzyme. Cultivation is performed for 24 to 48 hours, and after fermentation, the cells are separated and used for bioconversion alone.

For the bioconversion alone, a medium containing 5-20% (w / w) of filmaric acid or its salts, which also fall within the term "fumaric acid", is used as the substrate. The product of bioconversion is L-malic acid in the form of the corresponding salts, which also fall within the term "malic acid".

The medium is stirred gently without the need for aeration, and the temperature is preferably maintained between 30-50 ° C, preferably 37 ° C, and the pH between 4.5-8.0, preferably 7.5. The biotransformation is carried out until equilibrium of fumaran - L-malate is stabilized. E.g. for 30 hours Cultivation can convert 160g / L of fumaran to L-malic acid in a yield of 9598% of theoretical. Some variations in reaction times and yields should be considered depending on the equipment used and the culture conditions.

L-Malic acid can be prepared from fermented medium after separation of biomass, de-separation, decolourisation by crystallization, or other industrial processes (cation exchangers, reverse osmosis, electrodialysis, bipolar membranes, etc.).

DETAILED DESCRIPTION OF THE INVENTION

Example 1

120 ml of culture medium is prepared in a 500 ml culture flask (sterilized for 20 min at 121 ° C). The culture medium contains 5 g yeast extract per liter of distilled water, 2 g peptone for bacteriology, (NH4) 2HPO4 0.2 g, KC10.2 g, (NH4) 2SO4 0.5 g, MgSC> 4.7H2O 0.2 g, glucose 20 g (pH 6.0-6.5). The medium thus prepared is inoculated with a yeast culture of Dipodascus magnusii CCM 8235 and cultured for 24 hours on a rotary shaker at 28 ° C. After cultivation, the yeast biomass is separated from the medium by centrifugation (5500 rpm, 15 min), washed twice with 0.05 M phosphate buffer (pH 7.5) and centrifuged again (5500 rpm, 15 min). The biomass is suspended in 30 ml of 0.05 M phosphate buffer (pH 7.5) and used to convert IM of sodium (ammonium) fumarate to sodium (ammonium) fumarate, respectively. The bioconversion of the fumaran prepared in 0.05 M phosphate buffer (pH 7.5) is carried out in a bioreactor at 37 ° C with stirring. After 42 hours of conversion, the reaction mixture contained 111.2 g / L of L-malic acid, which represents 83.1% yield.

Example 2

The bioconversion of fumaric acid to malic acid is carried out according to Example 1, except that a 1 M ammonium fumarate solution is used for the conversion. After 42 hours of conversion, 85.5% malic acid recovery was achieved.

The bioconversion of fumaric acid to malic acid is carried out according to Example 1.

The composition of the culture medium and the conditions are analogous to Example 1 except that the strain Dipodascus ovetensis CCY 30-2-6 is used for bioconversion. After 48 hrs. yield of malic acid of 105.2 g / l is achieved, which represents 78.5% yield.

Example 4

The bioconversion of filmaric acid to malic acid is carried out according to Example 1. The composition of the culture medium and the conditions are analogous to Example 1 except that the strain Dipodascus tetrasperma CCY 52-4-1 is used for bioconversion. The fermentation time and the yields obtained are analogous to Example 2.

Example 3

Example 5

The bioconversion of filmaric acid to malic acid is carried out according to Example i. The composition of the culture medium and the conditions are analogous to Example 1 except that the strain Dipodascus aggregatus CCY 52-5-1 is used for bioconversion. After 42 hours of cultivation, the yield of malic acid was 81.5%.

Industrial usability

The invention is useful in the production of L-malic acid, or salts thereof, which are widely used in the food, pharmaceutical, chemical, and other fields.

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PATENT CLAIMS

Claims (6)

Process for the production of L-malic acid or its salts, characterized in that yeasts of the genus Dipodascus are cultivated in an aqueous solution containing 5-20% filmaric acid or its salts, at a temperature of 30-50 ° C, a pH of 4 , from 5 to 8.0. Production method according to claim 1, characterized in that a strain of Dipodascus magnusii is preferably used for production. Production method according to claim 2, characterized in that the strain Dipodascus magnusii CCM 8235 is preferably used for production. Production method according to claim 1, characterized in that a strain of Dipodascus arventis is preferably used for production. Production method according to claim 1, characterized in that a strain Dzpoďascuy tetrasperma is preferably used for production. Production method according to claim 1, characterized in that a strain of Dipodascus aggregatus is preferably used for production