RU1822411C - Process for preparing ethyl fatty acid esters - Google Patents

Abstract

Field of application: biotechnology, biochemical synthesis of organic substances, in particular esters of fatty acids and monohydric and polyhydric alcohols. SUMMARY OF THE INVENTION: the use of the Methylococcus capsulatus strain VSB-87 as a raw material for the synthesis of intracellular fatty acids without prior isolation from biomass, as well as through the use of an intracellular enzyme of the same strain without prior isolation from biomass and immobilization on an inert carrier. The method is carried out as follows. The strain is cultured in a liquid nutrient medium, the resulting biomass is separated by centrifugation, ethanol is applied in a ratio of 5: 1-5: 10 and incubated for 20-25 days at room temperature on a shaker. The resulting mixture was subjected to chloroform treatment to recover lipids. Fatty acid ethyl esters were isolated by column adsorption chromatography. next to

Description

The invention relates to the field of biochemical synthesis of organic substances, in particular esters of fatty acids and monohydric and polyhydric alcohols.

A known method for the preparation of fatty acid esters comprising the esterification of C7-C fatty acids to alcohols of the Cs composition upon heating in the presence of a catalyst. As a catalyst, polysulfophenyl ketone on aluminosilicate is used. The process is carried out at a temperature of 170-220 ° C.

The process is complex because it uses an expensive, difficult to produce catalyst. The process takes place at a sufficiently high temperature at which oxidation of fatty acids, especially polyunsaturated ones, is possible.

The closest in technical essence and the achieved result to the claimed invention is a method for producing esters, which consists in carrying out an esterification reaction with the participation of 100 parts of an aliphatic monohydric alcohol.

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25-bCO parts of an aliphatic saturated or unsaturated carboxylic acid with the number of carbon atoms from 3 to 22 and 00-100000 units (based on 1 part of carboxylic acid) of an immobilized lipase derived from a microorganism. The reaction is carried out at a temperature of 20-80 ° C (preferably at 30-60 ° C) with stirring. The resulting ester was separated from the reaction mixture.

The process requires the use of lipase immobilized on an inert carrier and the presence of only fatty acids and alcohol in the reaction mixture, which greatly complicates the stage of preparation of the synthesis.

The aim of the invention is to simplify the process for the preparation of fatty acid ethyl esters C () - C) B in saturated, mono- and di-unsaturated iodine by the action of microbial lipase.

The goal is achieved in that in the method for producing ethyl esters of fatty acids, including esterification of fatty acids with ethanol using a microbial lipase as a catalyst, the difference is that the biomass obtained by culturing the Methylococcus capsulatus BCB- strain is used as a source of fatty acids S, on which ethanol was applied in a ratio of 5: 1 - 5:10 after separation of the culture fluid, the resulting mixture was incubated for 20-25 days and treated with organic solvents to obtain no concentrate of ethyl esters of chiric acids.

The use of biomass obtained by cultivation of the H. capsulatus strain VSB-87 is due to the high content of normal structure fatty acids (20%) with the number of carbon atoms or 13 to 18, saturated, mono- and di-unsaturated. The predominant are palmitic and palmitoleic acids, the contents of which reach 9%, respectively. Oleic acid is also present in appreciable amounts (5.5).

The strain obtained at VNIISsynthesis protein was isolated from wastewater from an oil well in the LzSSR and selected by growing in a long continuous process with gradual

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lowering the pH of the medium and increasing temperature. Morphological, cultural and physiological characteristics, as well as technological characteristics are described in the passport of the strain.

The strain is acidotherm tolerant and is currently used for the industrial production of feed protein from methane. This makes it possible to organize cheap industrial production of nonionic surfactants (ethyl esters of fatty acids), since the raw materials for their synthesis can be obtained from the biomass of the strain without reducing its value as a protein feed product.

The application of ethanol to biomass in the indicated proportions is due to the need to achieve the maximum rate of synthesis of fatty acid ethyl esters. When the ratio of biomass and ethanol is less than 5: 1, the amount of synthesized ethyl esters of fatty acids does not exceed 35%. With an increase in the ratio of biomass and ethanol over 5:10, the yield of fatty acid ethyl esters remains the same (52%

Exposure of the resulting mixture for 20-25 days is due to the complete formation of fatty acid ethyl esters. With an exposure of less than 20 days, the amount of synthesized ethyl esters of fatty acids does not exceed 35-0%. The maximum amount of esters is formed on day 22-25. With further exposure, an increase in the amount of esters practically does not occur.

The treatment of the mixture after aging is carried out with organic solvents in order to obtain a concentrate of ethyl esters of fatty acids.

These properties are not only new, but also provide a positive effect, which consists in simplifying the preparation and the process of obtaining ethyl esters of fatty acids.

The method is carried out as follows. The M.capsulatus strain VSB-b was cultured in a liquid nutrient medium, the resulting biomass was separated by centrifugation, ethanol was applied in a ratio of 5: 1 5:10 and incubated for 20-25 days at room temperature on a shaker. The process of synthesis of esters and its end control

by thin layer chromatography on Silufol plates in a solvent system of hexane: diesel ethyl acetate (80: 20: 1) by the number of fractions of free peaceful acids and ethyl esters of fatty acids. The resulting mixture was subjected to treatment with chloroform (3 times twice the amount to be extracted) in order to extract lipilrv. Fatty acid ethyl esters were isolated by column adsorption chromatography.

Example 1. Biomass was obtained by deep cultivation of the bacterial strain M.capsulatus VSB-b in a continuous mode in a Fermenter with a volume of tO l (working volume of 20 l) on a nutrient medium of the following composition, g / l: 80 $ Н3Р04 - 0.05 ml, MgS04 -7N20-0.02; KC1 0.025; trace elements (mg / l): ZnS04-7P20 - 0.03, MnS04 4Н20 - 1.9, CuS04-5Н20 - 1.0, HjBOj- 1.25, FeS04 7H20 -2.1, Na ,, Mo04 2HZ0-; 0.045, CoS04-7H20 - 0.048, NiS04x 7IgO - 0.1, A12 (S04) 3-4H20 - 0.2, CrCl2-2H20 - 0.085.

The consumption of natural gas per 1 liter of medium is 15 l / h, air - 5 l / h, the cultivation temperature is 2 ° C, the pH of the medium is 5.6, (U 0.25 h 1.

The lipid content is 15% of the dry biomass.

The resulting biomass is separated by centrifugation (10 thousand rpm, 15-20 minutes) and in an amount of 50 g is placed in a conical flask with a 200 ml thin section. The biomass moisture content is 75%. 10 g of ethanol is introduced into the flask, closed with a stopper and put on the shaker (100 cycles / min, amplitude - yes - 5). The flask was kept under the indicated conditions at room temperature for 20 days.

Fatty acid ethyl esters are extracted with chloroform (3 times 100 ml) and are isolated by column adsorption chromatography.

The amount of synthesized ethyl Bbix fatty acid esters was 35%.

Example 2. Bacterial biomass was obtained analogously to example 1.

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The amount of synthesized ethyl esters of fatty acids was 52e / - ,.

Example 3. Bacterial biomass was prepared analogously to Example 1. 20 g of ethanol, which is 5: 2, was introduced into a flask with 50 g of biomass and kept for 22 days. The isolation of the synthesized esters was carried out analogously to Example 1.

The amount of synthesized ethyl. fatty acid esters amounted to 50%.

Example k. Bacterial biomass was obtained analogously to Example 1. 5 g of ethanol, which was 5: 0.5, was added to a flask with 50 g of biomass and kept for 15 days. The isolation of the synthesized esters was carried out analogously to Example 1.

The amount of synthesized fatty acid ethyl eOirosis was 30%.

Example 5. Bacterial biomass was prepared analogously to Example 1. 200 g of ethanol was added to a flask with 50 g of biomass, which was 5:20, and kept for 30 days. The synthesis of esters was carried out analogously to example I.

The amount of synthesized ethyl esters of fatty acids was 52%.

Using the proposed method in comparison with the prototype will simplify the production of ethyl ester fatty acids due to the use of raw materials for synthesis

40 intracellular fatty acids of the strain M.capsulatus VSB-87 without prior isolation from biomass, as well as through the use of an intracellular enzyme of the same strain

45 without prior isolation from biomass and immobilization on an inert carrier.

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Claims (1)

The claims A method for the production of fatty acid ethyl esters, including the esterification of fatty acids with ethanol using micron as a catalyst In a flask with 50 g of biomass, 100 g of its ropy lipase, distinguishing ethanol, which is 5:10, was introduced and kept for 25 days. The isolation of the synthesized esters was carried out analogously to Example 1. so that, in order to simplify the process, biomass obtained by cultivation is used as a source of fatty acids and lipase so that, in order to simplify the process, biomass obtained by cultivation is used as a source of fatty acids and lipase 71822 4118 strain Methylococcus capsulatus, the mixture is incubated for 20-25 days VKPM-17 3, to which, after separation and isolation of the desired product of the extra-culture liquid, it was applied by ethanolation with organic solvents in the ratio 5: 1 - 5:10 obtained by me.