DD209187A1 - PROCESS FOR OBTAINING ERGOSTEROL AND UBICHINONES - Google Patents

Abstract

The invention relates to a process for the recovery of ergosterol and ubiquinones from lipid HC extracts with HC contents> 10%, which are obtained in the microbial production of protein based on HC. According to the invention, ergosterol and ubiquinone are adsorptively enriched prior to their isolation using silica gel from the lipid HC extract or the acetone-soluble fraction of the lipid HC extract and simultaneously separated from the HC. The enrichment is carried out at a ratio of 1 part by weight of silica gel to 2 to 4 parts by weight of lipid HC extract or acetone-soluble fraction of a hydrocarbon or hydrocarbon mixture, preferably the Siedelage 50 to 160 degrees C.

Description

• Title ·. '

Process for the production of ergosterol and "übichinonen Anwendungsgebiet der Erfindung-

The invention relates to a process for the recovery of ergosterol and ubiquinones from Ldpid hydrocarbon extracts, which are obtained in the microbial production of protein based on hydrocarbons (HC). She is in the IPK C 0? C to classify »

Characteristic of the known technical solutions

Lipid K ¥ extracts are obtained in the microbial production of protein by extraction of the biomass with low-boiling solvents or Solventsgemischt. They are characterized by a content of 10 to 80 $ phosphatides, 10 to 80 $ fatty acids, which are mainly present in bound form, as well as other biogenic ingredients - sterols, fat-soluble coenzymes and vitamins - which are not exploited in the microbially exploited proportion Ergosterol and XJbicfainone are of particular interest for the biogenic ingredients »Ergosterol can be used as a starting material for the production of vitamin D or steroid hor- ones, and ubiquinones have become particularly important as coranartherapeutics.

Methods for obtaining ergosterol and / or Übichinonen from lipid K ¥ extracts are known. These methods are all associated with metabolic processes. In DD-PS 115 899 is a method for the simultaneous recovery of ergosterol

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and ubiquinone-45 from lipid extracts of microbial origin have been reported. After the alkaline-ethanolic saponification in the presence of a reducing agent, the unsaponifiable constituents are isolated from the lipid extract and separated therefrom by cooling with ergosterol and by chromatography on Al-O, - ubiquinone-45. The method mentioned has considerable disadvantages if the content of E in the lipid extract ^ 10 $> and at the same time the EW may be of very different K ¥ types, eg. The saponification, extraction of the soap solution, and the isolation of the ergosterol and ubiquinone from the gastric juice, require much more chemicals, labor, and energy than usual for the Isolation of these biologically active compounds from lipid fractions is necessary. In addition, in KW shares ^ - $ Q% ± m lipid extract and ergosterol concentrations j £ 0.5 %, the recovery of ergosterol only with insufficient yields or not at all possible »in DD-PS 151 007 is therefore proposed for recovery Ergosterol from the XAiverselfbaren make an adsorptive enrichment using silica gel. The lipid fraction obtained after the desorption of the silica gel contains, in addition to the ergosterol and other Ldpiden still another main component KF, dominate the aromatics «: Due to the similar chemical structures, these aromatics are adsorbed with the ergosterol preferably on silica gel (aromatic content of the KW-iTraktion "* 63% _f aromatics content of the petroleum distillate * s 30 %) , so that a high effort is required for the subsequent purification of the srgosterol.Especially disadvantageous is a KW content ^ 10 % in the lipid fraction for the isolation of übichinons A separation of the Übichinons from the KW can only be carried out by adsorption chromatography · The required amounts of adsorbent material and solvents and the time required for the elution are extremely high,

In DD-PS 154 448 a method is described in which a part of the K ¥ is removed by distillation. After a 2-stage film distillation a acetonlöslicben fraction containing 63% KW, while a bottom product is obtained which still A * 30% K ¥ having tmd in the further workup is also still an essential greater expenditure required in comparison to recovery from a K ¥ -free lipid extract.

Object of the invention

The aim of the invention is to develop methods in which the simple and cost-effective recovery of ergosterol and ubiquinones from lipid K ¥ -E: extracts with KST proportions> -10 $. allows.

Presentation of the essence of the invention

It is an object of the present invention to enrich ergosterol and TJbiquinones from lipid K ¥ -E: s: s: s under use-of differences in adhesion to adsorbents and at the same time to separate them from EW before isolating the biologically active compounds. According to the invention the object is achieved in that ergosterol and TTbichinone are enriched by adsorption chromatography on silica gel from the lipid oil extract or the acetone-soluble fraction of the lipid KF tract and largely separated from the oils.

More specifically, the procedure is as follows: The lipid extract or the acetone-soluble fraction of the lipid K ¥ -E, xtraktes extract be ± 3 times the volume of a K ¥ or a K ¥ mixture, preferably the Siedelage 50 bis I60 ⁰ C dissolved and treated with silica gel. The ratio of silica gel to lipid SOLTA extract is 1 wt. - Tail to 2 to 4 Get # .- parts.

3.0 After decanting, the silica gel nociaia is washed as a hit with the gleiphan solvent. After the solvent has been distilled off, the combined fractions have a composition which has changed only insignificantly compared to the starting material. Dia desorption of the compound adsorbed on the silica gel

fertilize with a polar solvent, for example, a mixture of hexane / methanol (60: ko) or Hesan / Etaanol / ¥ asser (60: 20: 20) .The residue contains ergosterol and ubiquinones in at least 5-fold concentration in comparison to The recovery of the ergosterol -and the Ubiohinone can be carried out from the K-free fraction in a known manner by crystallization or Adsorptionsschronxatographie., The silica gel can be used again after the regeneration.

The inventive method has a number of advantages. Ergosterol and ubiquinone are separated from the 3DF, which are a major component of Lipid K ¥ i extracts, and largely from the other lipophilic components

and enriched to at least the 5-course. As a result, the cost of chemicals, solvents, energy and working time for the isolation and purification of the ergosterol and the übichinone is significantly reduced. In addition, it is possible ergosterol also from such lipid K ¥ extracts

to have ergosterol content <0.5 i> with a high K ¥ share;

The after the adsorptive removal of the E _r gosterols and übichinone remaining lipid KF-Estrakt or ^1, the acetone-soluble fraction no material conversion process is subjected, and comprises essentially the same components as before separation of the biologically active compounds, and may in a known ¥ else, z «B. used in separating oils or flotation auxiliaries«

The process according to the invention is explained by the following examples:

A lipid-hydrocarbon extract was recovered from a hydrocarbon-utilizing strain of the yeast LodderoEryces elongispo · rus. The yeast was cultured in the presence of ⁰ to 380 C boiling in the range of ZHQ, serving as a carbon source Erdöldestillat3 in continuous fermentation.

The recovery of the lipid hydrocarbon extract was obtained by extraction of the Hefetrockenproduktes with a petrol / ethanol / water mixture at 4o ⁰ C. Hach distillative removal of the solvent, a lipid extract Eohlenwasserstoff the following composition was obtained:

Hydrocarbons and the like 35% by weight unsaponifiable ingredients

Phosphatides 29

free fatty acids and glycerides 34

Übichinon-45 0.24 wt .- ^

Ergosterol 0.8% by weight 1)

Non-lipids 1.0% by weight

1) ^

^/ Ergosterol ester ^ 10 %

example 1

30 g of extract are mixed with 100 ml of hexane and shaken for 15 minutes after addition of 15 g Siücagel (particle size 32 to 100 yu). It is decanted and still 3 ^ aI rit 50 ml of hexane in each case 5 minutes shaken. The hexane fractions are combined and concentrated.

The hexane fraction is similar in composition to the "starting product." For desorption, the silica gel is first shaken for 10 minutes and then for 5 minutes each with 100 ml of hexane / ethanol / water (60: 20: 20) The fractions are combined, the aqueous phase After separation, the organic phase is concentrated on a rotary evaporator over sodium sulfate. Yield: 3.2 g The enriched fraction has the following composition:

Ergosterol 4.91% by weight

Ubiquinone 1.25 wt .- ^

Hydrocarbons 2.4% by weight

Phosphatides 2.20% by weight

other lipids> 85, O (glycerides, fatty acids)

After saponification of the enriched fraction, the ergosterol is recovered from the unsaponifiable by crystallization and the ubiquinone is isolated by column chromatography on silica gel.

Yield! Ergosterol 117 Jag ubichinon 3k rag

Example 2

The silica gel obtained in Example 1 after hexane treatment is mixed twice with 100 ml of hexane / methanol (60: 40) and shaken twice to desorb all substances. The solvent is removed on a rotary evaporator removed yield: 4.0 g The enriched fraction is composed as follows:

Ergosterol: 4.4% by weight

Ubiquinone: 1j ^ wt .- ^

Hydrocarbons: 3.5 wt .- $

Phosphatides: 5 »3 wt .- $

other lipids: } & 5 % by weight (glycerides, fatty acids)

From the enriched fraction, the ergosterol is recovered directly by crystallization. The ubiquinone is isolated after saponification and column chromatography of the unsaponifiable, yield: ergosterol 115 mg ubiquinone 48 mg

Example 3

Lipid-Sohlen-Hydstoff-Extract "is stirred vigorously with twice the amount of acetone for 5 minutes, after which two phases are quenched, the upper phase is decanted off and, after removal of the solvent, an acetone-soluble fraction of the following composition is obtained:

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Hydrocarbon θ ti. a. unsaponifiable 52 ingredients

Phosphatides 4.0

free fatty acids and glycerides 42% by weight

Ergosterol 1.23% by weight -%

Ubiquinone 0.29 wt. - <$

The acetone-soluble fraction is treated further as described in Example 1 for extract. Yield: 3> 15 s

The resulting enriched fraction has the following composition:

Ergosterol 6.22% by weight

Ubiquinone 1.29 wt. '- £

Hydrocarbons. 3, ^ 0 wt .- ^

Phosphatides 2 _S wt .- ^ 5O

 "Later lipids" 85 ^

From the enriched fraction, XJbiquinone and ergosterol are isolated as described in Example 1. Yield: Ergosterol 147 rag of over 40 mg of bisichinone

The silica gel is used again after heeration.

Claims (4)

-a- invention claim A method for obtaining ergosterol and ubiquinones from lipid Klf extracts obtained by extraction of microbial K ¥ -based biomass, characterized in that from the lipid HC extract or the acetone-soluble fraction of the lipid -K ¥ extract ergosterol and ubiquinone adsorptively enriched using silica gel and simultaneously separated from the carbon, hydrogen » 2. The method according to item * 1, characterized in that the enrichment of ergosterol and ubiquinones at a ratio of 1 wt, silica gel -sTeil weight to 2 to k, £ parts by lipid extract or if acetone-soluble fraction is * 3 «Method according Pkt» 1 and 2, characterized in that silica gel of grain size 30 to 500 p, preferably 30 to 100 u, is used. 4. The method according to pkt. 1 to 3 », characterized in that the adsorption on silica gel from a KW or a K ¥ - mixture preferably the Siedelage 50 to 16O C takes place *