DD151007A3 - PROCESS FOR OBTAINING PHOSPHATIDES, FATTY ACIDS AND ERGOSTERIN - Google Patents

Abstract

Process for obtaining phosphatides, fatty acids and ergosterol from lipid HC fractions of microbial origin. The aim of the invention was a process which allows the simultaneous recovery of major and minor components from lipid HC fractions obtained by extraction with non-polar or combinations of polar and non-polar solvents from feed-based yeasts produced on a hydrocarbon basis. The object of the invention was to prepare lipid HC extracts, which have HC contents> 50% and solubilities of ergosterol in the unsaponifiable fraction> 5 mg / g, in stages by means of material conversion processes. A method has been developed which provides for the separation of lipid HC fractions by means of material conversion processes, in particular taking advantage of the different solubilities in organic solvents or solvent mixtures, the different vapor pressures and the different adhesive strength to adsorbents, and the recovery of all main and secondary components allowed. All products of the workup - phosphatides, fatty acids, glycerine water, HC and ergosterol - are used for economic purposes. The invention can be used in the processing of lipid HC extracts, which are obtained in the microbial fermentation of KW.

Description

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title

Process for obtaining phosphatides, fatty acids and ergosterol

Field of application of the invention

The invention relates to a process for obtaining phosphatides, fatty acids and ergosterol from lipid-hydrocarbon extracts which are obtained in the microbial production of hydrocarbons based on hydrocarbons.

Characteristic of the known technical solutions

Lipid HC extracts are obtained in the microbial production of protein by extraction of the biomass with low-boiling solvents or solvent mixtures. »They contain 10 to 80 % of phosphatides, 10 to 80 % of fatty acids, which are predominantly in bound form. as well as other biogenic ingredients sterols, fat-soluble coenzymes and vitamins - characterized in the laikrobiell not recycled portion of the KW fraction are solved. Consequently, the lipid HC extracts consist of a mixture of lipids and possibly very

different hydrocarbon types, eg »B. the boiling point 240 to 380 ⁰ C, The hydrocarbons may be present in significant kengen (> 50 %).

For lipid extracts of microbial origin, which contain no substrate-specific hydrocarbons, the simultaneous extraction of several lipophilic components from the

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Lipid extract known. Thus, for example, DE-PS 916847 describes a process which provides for the recovery of fatty acids, ergosterol and cerebrin. On lipid HC extracts these known solutions are not transferable due to the high HC content.

Therefore, methods have been developed which provide for the recovery of individual lipophilic components - fatty acids, ergosterol, ubiquinone - from the lipid Kl extracts. In most of the known methods, either only the main components-preferably fatty acids-or the biologically active substances-ergosterol and ubiquinone are isolated. In DE-OS 2756977 wii * ^d, for example, a method is described which provides for the isolation of the fatty acids. In this case, to reduce the phosphorus content and to release the fatty acids of the lipid HC extract at a temperature> 200 ⁰ C and pressures of *** 20 atm. subjected to a limited hydrolysis, which also leads to the chemical destruction of the light, air, alkali and temperature-sensitive biologically active substances. On the other hand, there are described in SU-PS 568676 and in DD-PS 115899 methods exclusively for obtaining the biplogically active substances ergosterin bzv /. Ergosterol and ubiquinone-45 - are aligned. The alkaline saponification of the lipid HC extract leads to complete chemical hydrolysis of the phosphatides, so that their extraction is no longer possible. In DD-PS 110037 a method for the production of ergosterol is described, which allows in addition to the recovery of ergosterol the isolation of phosphatides and fatty acids. However, at low ergosterol concentrations, the process is limited to work-up of lipid HC extracts obtained by extraction of feed biomasses with non-polar solvents. If combinations of polar and non-polar solvents are used for the extraction, the range of lipophilic components present in the lipid HC fractions and their cleavage products is due to destruction

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the lipid-protein complexes or cleavage of covalent bonds by polar solvents or hydrolysis, oxidation and polymerization in the Miszellaaufkonzentrierung in the presence of reactive solvents such as alcohols, Lasser, much more diverse.

This leads to the fact that the amount of extract and the solubility of ergosterol in the unsaponifiable fraction greatly increased * Thus, for lipid HC extracts (gasoline extraction), a solubility of ergosterol in the unsaponifiable fraction of 0.5 mg / g, for lipid KW- Extracts (gasoline / ethanol / water extraction) of 5 mg / g determined at 0 C · / At low ergosterol concentrations «0.5 %) therefore described in DD-PS 110037 crystallization of ergosterol only with insufficient yields or gaiinicht more is possible"

Another disadvantage of the described in DD-PS 110037 Verfahrers is that - due to the high proportion of HC, especially in the acetone-soluble phase - the consumption of energy and excipients in saponification, extraction and crystallization is extremely high ·

Aim of the invention;

The aim of the invention is to develop a process which comprises the simultaneous recovery of major and minor components from lipid HC fractions obtained by extraction of microbially generated biomass with KW. nonpolar or combinations of polar and non-polar solvents have been obtained and hydrocarbons y 50 % and solubilities of ergosterol in the unsaponifiable portion> 5 mg / g may have, with minimal excipients and energy consumption allows and the waste product works.

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Presentation; the essence of the invention

The invention is based on the object, lipid HC extracts, the hydrocarbon fractions> 50 % and solubilities of ergosterol in the unsaponifiable fraction> 5 mg / g may have to gradually work up using their physicochemical and chemical properties «

Features of the invention

According to the invention, the object is achieved by separating the lipid HC extract with acetone into an acetone-insoluble, the phosphatides-containing, and an acetone-soluble, free fatty acids, Pette and the biologically active compounds-containing fraction. The acetone-soluble fraction is subjected to distillative concentration of an alcoholic or aqueous saponification. From the saponification mixture, the ergosterol is separated directly by crystallization or the saponification mixture is separated into soap solution and unsaponifiable fraction, worked up the soap solution to fatty acids and possibly glycerol and from the unsaponifiable fraction, the ergosterol directly or after distillative removal of a part dei * hydrocarbons or Adsorptive enrichment by crystallization at -10 ⁰ C to +20 ⁰ C, preferably at -5 ⁰ C to +5 ⁰ C isolated.

In detail, the procedure is as follows:

The phosphatides are prepared in a known manner by acetone

v '

treatment of the lipid HC extract, which may contain up to 30% by weight of solvent, separated and refined according to the requirements of the purity of the phosphatides by further acetone treatment and, if necessary, stabilized by dissolution in mineral oil. The acetone-soluble phase is heated after distillative removal of the solvent to 160 to 200 ⁰ C at ^ 2 Torr to separate a portion of the hydrocarbons.

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As a result, an enrichment of the sterols in the sump while reducing the amount to be enforced in the saponification acetone-soluble phase to 1/3 to 1/4 is achieved. The bottom product is saponified with alcoholic or aqueous KOH and the ergosterol from the saponification mixture by cooling to -10 ⁰ C to + 20 ⁰ C, preferably -5 to +5 C, separated, washed the resulting amorphous crystals with aqueous alcohol and the residue purified from acetone or alcohol in the stated temperature range by crystallization. "The soap solution is worked up after addition of mineral acid to fatty acids, hydrocarbons and possibly glycerol. The saponification mixture can also be separated into an unsaponifiable fraction and soap solution by extraction with organic solvents or solvent mixtures, preferably gasoline, gasoline / alcohol and gasoline / acetone mixtures. From the soap solution, the fatty acids are deposited after acid addition, which can be purified by distillation in a known manner. The aqueous, glycerol-containing residue obtained in the work-up of the soap solution can be recycled as a carbon source in the fermentation or worked up in a known manner to glycerol

From the unsaponifiable fraction is Ergosterinsehalten> 0.5 % recovery of ergosterol by crystallization at -10 to +20 ⁰ C, preferably +5 ⁰ C, possible. The HC fraction (distillate or unsaponifiable fraction after solvent removal) can be used advantageously as heating oil.

It is also possible to saponify the acetone-soluble, solvent-free phase directly, to separate off the unsaponifiable fraction by extraction and to remove a portion of the hydrocarbons by distillation at 16O to 200 ⁰ G and ^ 2 Torr prior to Ergosterinkristallisation or separate the ergosterol by adsorption on silica gel ,

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The essential advantage of the method according to the invention is that all components of the lipid HC extract can be recycled and no waste products are produced. In addition, it is also possible to work up such lipid HC extracts which, at high HC contents, ( Λ 50 %) have a low ergosterol concentration ( K 0.5%) and a solubility of ergosterol in the unsaponifiable fraction of ^ 5 mg / g unsaponifiable fraction. A particular advantage of the method is further seen in the fact that at each stage an intermediate stop is possible. In addition, the quantities of acetone-soluble phase to be passed through, the extraction agent, etc. are substantially reduced by the distillative enrichment before the saponification stage.

The fractions and pure substances that can be extracted from the lipid HC extracts are all raw materials and intermediates desperately needed in the economy ·

The process according to the invention is illustrated by the following examples.

Example 1 :

A lipid HC extract was obtained from a hydrocarbon-utilizing strain of the yeast Candida guilliermondii which resulted from a fermentation in which the yeast was cultured in the presence of a carbon dioxide-boiling petroleum distillate boiling in the range of 240 to 380 ^° C. The recovery of the lipid HC extract was carried out after extraction of the yeast dry product with aitff gasoline / ethanol / water mixture at 40 ⁰ C and distillative removal of the main amount of the extractant. The lipid HC extract is characterized as follows:

extracting agent 15 5 weight .56 Hydrocarbons and the like unsaponifiable shares 30 5 weight 35 phosphatides 40 weight -56 free fatty acids and glycerides 7 weight

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Ergosterol 0.6% by weight

Non-lipids and the like 6.4% by weight

100 g of lipid HC extract are mixed intensively with 200 g of acetone at room temperature for 10 min. The upper phase containing the acetone-soluble components is decanted after a settling phase, the acetone is removed by distillation and the residue is used as an acetone-soluble component (ALK) for saponification. The lower, acetone-insoluble phase after removal of the solvent represents the crude phosphatide. Yield: 55 g (phosphatide content 70 %) For stabilization, it is added after taking up in petrol in mineral oil (1: 1 parts by weight) and then the gasoline removed in vacuo.

Pure phosphatides (HC content <1.5 %) are obtained by injecting the crude phosphatides taken up in petrol (1: 1 parts by weight) into 3 to 5 times the amount of acetone. Yield: ca. 40 g (phosphatide content 95 %) The ALK obtained by acetone precipitation is characterized as follows:

Saponification 58 mg KOH / g ALK

Neutralization number 22 mg KOH / g ALK

Ergosterol 0.75% by weight

Hydrocarbons 65% by weight

Phosphatides 5.5% by weight

It was distilled before the saponification to remove a portion of the interfering hydrocarbons in vacuo at 160 ⁰ C and 0.5 to 1 Torr

 Yield: 22 g of distillation residue The distillation residue is characterized as follows:

Saponification number 95 mg KOH / g ALK

Neutralization number 22 mg KOH / g ALK

Ergosterol 1.3% by weight

Hydrocarbons 35% by weight

Phosphatides and split 10% by weight of products.

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22 g of distillation residue are mixed with 30 g of ethanol. KOH (10 g KOH in 100 ml of solution) saponified for 30 min with intensive stirring at reflux, then 20 g V / asser added and the saponification mixture cooled to 5 ⁰ C, the precipitate separated, with 30 percent strength. Alcohol washed and the residue dissolved in 10 to 15 ml of acetone at ca, 50 ⁰ C, the insoluble residue separated by filtration and the ergosterol from the acetone solution at 5 ⁰ C like,

Ergosterol yield: 150 to 170 mg The fatty acids are removed from the soap solution after addition of 25 per cent. Sulfuric acid (or hydrochloric acid) separated in a separating funnel and purified by distillation, yield of crude fatty acids: 15 g (fatty acid content ca, 65 %) Hydrocarbon yield: (distillate ALK and fatty acid)

27 g

The aqueous glycerol-containing residue is recycled to the fermentation after distillative removal of the solvent as a carbon source.

Example 2 :

The starting material was the same saponification mixture as in Example 1. Instead of direct crystallization, the saponification mixture is extracted 3 to 4 times with 30 g of gasoline each. The gasoline extracts are combined, washed twice with 20 g of water and concentrated under reduced pressure (10 to 15 torr, 50 to 60 ⁰ C) to about 35 ml. The ergosterol is separated from the gasoline / K 2 / solution at 5 C and recrystallized from about 10 to 15 ml of acetone,

Ergosterol yield: 200 to 220 mg The workup of the soap solution is carried out according to Example 1,

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Example 3 :

The starting material was the same ALK as in Example 1. The ALK is directly with 6 g of a 50 percent. aqueous KOH for 20 min at vigorous stirring to 125 ⁰ C bath temperature saponified · The unsaponifiable fraction is, after addition of 50 g of acetone / water (1: 1) by 3 to 4 repeated extraction (4 each with 30 g of a petrol / acetone mixture: 1 part by volume) separated. The solvent is removed in vacuo and the unsaponifiable portion is then distilled under vacuum at 0.7 to 1.5 Torr and 170 C. From him after addition of gasoline and cooling to 5 ⁰ G, the ergosterol is separated. Ergosterol yield: 200 to 220 mg The further work-up is carried out according to Example 1.

Example 4 :

The starting material used was the unsaponifiable action prepared according to Example 1, 2 and 4. After addition of 2 to 2.5 parts by weight of gasoline and 1/2 part by weight of silica gel, the mixture is extracted intensively (shake flask), the solution is decanted and the extraction is repeated 3 times with 1 part by weight of gasoline Sterols containing practice is desorbed by a hexane / ethanol / water mixture (60: 20: 20) from the silica gel, the organic phase separated, dried and distilled. The ergosterol is then separated by crystallization

 Ergosterol yield: about 200 to 220 mg

Claims (14)

21 72 6 9 - "ο- Claim * f 1. A process for obtaining phosphatides, fatty acids, and ergosterol from lipid hydrocarbon extracts obtained by extraction of microbial to hydrocarbon 5. biomass obtained with nonpolar or combinations of polar and nonpolar solvents and separated with acetone into an acetone-insoluble, the phosphatides-containing and an acetone-soluble, free fatty acids, fats and biologically active compounds containing fraction, characterized in that the acetone-soluble fraction directly or after distillative concentration of an alkaline saponification, the saponification mixture is separated into extract soap and unsaponifiable fraction, the soap solution to fatty acids and aqueous glycerol worked up, the ergosterol directly or after adsorptive or distillative enrichment by crystallization from the hydrocarbons or separates the ergosterol by crystallization separated from the saponification mixture. 2. The method according to item 1, characterized in that the acetone-soluble, solvent-free phase at a pressure of = 5 Torr, preferably = * 2 Torr, and bottom temperatures between 140 and 250 ⁰ C, preferably between 16O and 200 C, concentrated. ϊ. 2 ί 72-69 -H- 3, Process according to item 1 to 2, characterized in that the saponification with 1.1 to 1.5 parts of ethanolic lye, preferably 1.2 parts of alkali, and at a temperature of 80 to 90 ⁰ C 'within 20 to 60 min, preferably 30 min, takes place, .4 · Process according to item 1 to 3, characterized in that the saponification with 1.1 to 1.5 parts of aqueous alkali, preferably 1.2 parts of alkali, and at a temperature of 120 to 125 C within 10 to 60 min , preferably 20 min, takes place. 5 · The method of item 1 to 4, characterized in that the ergosterol, immediately separated from the hydrolysis mixture by crystallisation at -10 to +20 ⁰ C, preferably -5 to +5 C and purified by crystallization from acetone or alcohol · 6 * Process according to point 1 to 5 »characterized in that the fatty acids are separated off by decantation from the soap solution at a pH of 2 to Process according to items 1 to 6, characterized in that the aqueous glycerol-containing phase is recycled to the process of fermentation, 8, process according to item 1 to 7, characterized in that the unsaponifiable fraction at a temperature in the range of 140 to 250 ⁰ C, preferably 16O to 200 ⁰ C, and a pressure of = 5 Torr, preferably = 2 Torr, distilled · 9, method according to item 1 to 8, characterized in that an enrichment of ergosterol on silica gel at a ratio of 1 part of silica gel to 2 parts unsaponifiable share · 217269 10th method according to item 1 to 9, characterized in that the crystallization of ergosterol at -10 to +20 ⁰ C, preferably -5 to + 5 ° C, takes place · 11 .. Process according to item 1 to 10, characterized in that the hydrocarbon fraction is used as heating oil · Process according to items 1 to 11, characterized in that the lipid HC extract is obtained by extraction with non-polar or combinations of non-polar and polar solvents in the microbial fermentation of hydrocarbons. 13 «method according to item 12, characterized in that the microorganism is a yeast grown on hydrocarbons. 14, method according to item 1 to 13, characterized in that a lipid HC extract is used which contains 0 to 30 % _t, preferably 15% »solvent.