HU202406B - Process for producing fat mixture or fatty acid mixture containing eicosapentaenic acid ester or occasionally eicosapentaenic acid from rain-worms of lubriciade - Google Patents

Abstract

Water-washed worms are kept between wet paper sheets at 4 to 10 DEG C for 40 to 80 hours in order to clean them internally then treated with 0.1 to 1.5 per cent by weight of enzyme calculated for the dry weight of worms, for about 1 to 4 hours. The treated mass is dried in a conventional manner then extracted with a lipophilic solvent or with a supercritical fluid. The extract is separated from the residual solid and exempted from the solvent by a conventional method. Optionally, fatty acids are released from the fat compounds obtained in a conventional manner and the mixture is enriched in eicosapentaenoic acid by any known method.

Description

FIELD OF THE INVENTION The present invention relates to a process for preparing an eicosapentaenoic fatty acid ester, and optionally an eicosapentaenoic fatty acid fatty acid mixture thereof, from the Lumbricidae family.

In recent years, the production of humus from stable manure has become a widespread process.

The process is done with earthworms. In their diet, the giltheads produce humus from the farmyard manure and at the same time reproduce themselves. The wheat stock multiplies six to ten times in one year. Thus, the processing and utilization of bulk earthworms for purposes other than the production of humus is a problem worldwide.

Japanese Patent Application Laid-Open No. 81,144,703 discloses a process for processing earthworms for fish feed. During the process, earthworms are washed, dried and put up as fish feed.

Chinese Patent Publication No. 85,109,664 discloses a method of washing cultivated earthworm, hydrolyzing it with hydrochloric acid at 110 ° C for 48 hours, and preparing amino acids from its hairs by electrolysis and hydrogen sulphide removal.

German Patent Publication No. 3 306 944 discloses a process for the aqueous extraction of earthworms with a solution containing at least 50% by weight of water in order to obtain an anticoagulant.

The wet or dried, preferably defatted, gelatine is extracted during the process with a mixture of an aqueous solvent, preferably physiological sodium chloride or other aqueous buffer solution having a pH of 5 to 10 and a polar solvent, preferably ethyl alcohol. Thus, a protein or "quasi" protein can be obtained which has fibrinolytic activity and is capable of indirectly solubilizing fibrin.

In our experiments, we have found that a significant proportion of the fat content of the Lumbricidae family, particularly Eiseniafoetida, Eisenia andrei (Eisenia foetida var Andrei), Lumbricus rubellus, Lumbricus terrestis, is good with eicosapentenoic acid ester. In particular, Eisenia foetida has a significant content of eicosapentenoic acid ester.

However, part of the eicosapentenoic acid content of the globose is present in the form of a glycerol ester wherein a hydroxyl group of the glycerol is esterified with a phosphoric acid derivative. The recovery of this is a major problem and cannot be achieved by aqueous-solvent extraction or a simple extraction method.

It is an object of the present invention to provide a process for the preparation of a good yield of an eicosapentaenoic acid ester fatty acid and, if desired, an eicosapentaenoic fatty acid rich fatty acid mixture thereof.

In our experiments, we have found that in good yields, eicosapentaenoic acid can be obtained from the Lumbricidae family by first digesting the washed, internally purified gilthead with an enzyme.

For enzymatic digestion, the protease and / or the phospholipase C enzyme is preferably used.

After several hours of enzymatic digestion, the mixture is dried if desired, e.g. lyophilization, spray drying and then extracting the resulting product with a fat solvent or carbon dioxide. The latter extraction is carried out using known supercritical techniques.

Such supercritical extraction is described, for example, in U.S. Patent No. 4,675,132.

The process of the present invention provides a fat blend having 15-22% by weight of glycerol ester of eicosapentaenoic acid (EPA).

The extraction residue obtained as a by-product of the process can be used as a protein-rich mixture after solvent removal for feeding purposes.

In the process of the invention, the digestive enzymes are used in an amount of 0.1-1.5% by weight based on the dry weight of the meal.

The role of the enzyme is to rupture the cell membrane of the earthworm and thus release the structural oil encapsulated in the cells. In addition, phospholipase-C also degrades phospholipids. Preferably the fat solvent used in the extraction is chloroform, alcohols, preferably methanol, dichloromethane, diethyl ether, cyclohexane, hexane, and the like. employed.

Preferably, the extraction is performed with liquid carbon dioxide under supercritical conditions.

The fat blend obtained during the extraction contains a C16-C18 saturated fatty acid fraction and a C18-C20 unsaturated fatty acid fraction both in the photo of the glycerol ester.

Eicosapentaenoic acid is designated as 2Ο: 5: ω3, where 20 represents the number of carbon atoms, 5 represents the number of double bonds and 3 represents the number of carbon atoms of the saturated carbon chain behind the last double bond.

The earthworm has a dry matter content of 8-9% by weight of fat and 15-22% by weight of the EPA content in the form of glycerol ester.

Preferably, the method comprises processing Eisenia foeitida.

This propagation is conveniently carried out as follows.

Litter mixed with cattle manure is placed on prisms 0.8-1.0 m high and 1.5 m wide on a concrete surface or 15 cm high and covered with foil. Wait until the temperature of the fertilizer prism has cooled to 20 ° C.

Then, one million glues flour is spread evenly into 80 g of manure. If necessary, irrigate the manure stack to maintain the required moisture. The earthworms fertilized with fertilizers process 80 lbs of fertilizer into humus within 6 months, while their numbers multiply to 6-10 million.

We separate the compost and earthworms by covering the work surface with foil. After covering, the earthworms escape from the upper compost layer to the lower compost layer, and thus a considerable amount of compost can be removed without the use of deep-water. This operation is repeated several times. When this operation can no longer be carried out, the remaining deep-water compost is separated on a drum sieve into earthworms and compost.

The acrylate thus obtained is processed in the process of the invention.

The present invention relates to a process for the preparation of an eicosapentaenoic fatty acid mixture and, optionally, an eicosapentaenoic fatty acid mixture thereof, from the Lumbricidae family, by washing, drying, mad extraction and de-solventing of the extract.

The process is characterized in that the aqueous gel-purified gelatin is held between wet paper sheets at 4-10 ° C for 40-48 hours and then internally cleaned.

-2 H

The animals are treated with 0.1 to 1.5% by weight, based on their dry weight, of enzyme, preferably protease and / or phospholipase C, for 1 to 4 hours, and the pre-treated mass is then dried, ground and then extracted with an organic fat solvent or followed by carbon dioxide supercritical extraction, the extraction residue is separated from the extract, and the extract is de-solvented in a known manner, optionally liberating fatty acids from the fat mixture and enriching the eicosapentaenoic acid content.

Post-enzymatic drying may be carried out by heat treatment at 80-100 ° C, lyophilization, spray drying, etc. Preferably, the organic fat solubilizer is a 2: 1 by weight mixture of chloroform: methanol.

The fatty acids are preferably liberated from the fat blend by alkaline decomposition.

Preferably, the EPA content of the fatty acid mixture is enriched by known urea adduct formation.

The method was described by H.P. Kaufman, "Analyze dér Fette und Ferrprodukte" (Vol. I, p. 76, Springer Verlag, 1958), but described in U.S. Patent No. 4,377,526. See also U.S. Pat.

The essence of the method is that saturated or mono-diene fatty acids form an adduct (complex) with urea, which is precipitated.

Higher unsaturated fatty acids do not form an adduct with urea.

One product of the process according to the invention is the Epaester-rich fatty acid mixture or the EPA-rich fatty acid mixture. This oil product is conveniently marketed in capsules or mixed with food.

EPA-rich, oil-containing capsules or food supplements are preferably used for therapeutic purposes to reduce cholesterol deposition, to reduce vasoconstriction, atherosclerosis, and occlusion, and to treat certain skin inflammations.

Furthermore, the extraction residue rich in the protein produced by the process of the invention, after solvent removal, is preferably used as feed, since it contains the proteins in a digested, digested state.

Solvent removal of the solid residue is conveniently accomplished by drying at 40-50 ° C.

Advantages of the process according to the invention are:

- Both products of the grain processing industry are well utilized and valuable materials.

- No waste material is produced during processing.

- The resulting fat blend has a high EPA content.

The following examples illustrate the process of the invention.

/. example

Live gillyweed (Eisenia foeitida) is separated from the compost on a drum sieve. The resulting 100 parts by weight of the earthworms are washed with water and the washed gelatine is stored in wet papers at 6 ° C for 48 hours, during which time the intestinal tract is emptied.

The resulting mass is placed in 4 parts by weight of a 0.2 M aqueous phosphate buffer solution containing 3% by weight of protease enzyme.

The phosphate buffer has a pH of 7.6 and contains a mixture of KH ₂ PO ₄ and K ₂ HPO ₄ . The enzymatic digestion was carried out at 30 ° C for 2.5 hours.

The pretreated guillamate mass is then separated from the solution and dried on 5 cm layers in trays in an oven at 95 ° C for 24 hours until constant weight is achieved. This gives 17 parts by weight of dry weight which is milled to an average particle size of 0.9 mm. To the milled mass was added 1.5 parts by weight of a 2: 1 by weight mixture of chloroform: methanol.

The extraction mixture was allowed to stand for 3 hours with stirring.

The extract was collected by filtration from the solid residue and evaporated. The solid residue was dried at 50 ° C.

The concentrated concentrate is 1.5 parts by weight, of which 20% by weight is an EPA glycerol ester and 80% is the glycerol ester of other saturated and unsaturated fatty acids.

The result of the fat analysis is as follows:

Fatty acids (in the form of glycerol esters) Weight% 12: 0-16: 0 37.2 18: 0 4.9 18: 1 12.5 18: 2 14.4 18: 3 3.4 20: 2 1.4 20: 3 0.2 20: 4 5.1 20: 5 (EPA) 20.0

The fat blend is encapsulated and used as a nutritional supplement.

The solid residue containing 67.5% by weight of degraded protein is used for feeding purposes.

Example 2

All proceed as in Example 1, but using dichloromethane as the extra fish.

The resulting de-solvented 1.4 parts by weight of the fat blend had an EPA ester content of 19.5% by weight.

The fatty acids in the fat blend were liberated by dissolving 1.4 parts by weight of petroleum ether in 14 parts by weight of potassium hydroxide in ethanol and allowing the mixture to stand at room temperature for 12 hours. Then, 18 parts by weight of water are added, the mixture is homogenized and the separated phases are separated.

The upper phase is the petroleum ether phase, which is collected as a contaminated solvent. The lower phase contains the fatty acid potassium salt. The pH of the phase is adjusted to 4.0 with HCl and the fatty acids are liberated. The fatty acids are separated from the water by extraction with petroleum ether. After evaporation of the solvent, the fatty acid phase is obtained.

The 1.0 parts by weight mixture was then dissolved in 20% methanol. To this is added 6 parts by weight of urea in the form of a 12% solution of methanol. The mixture was allowed to stand at 3 'C for 24 hours. In this case, the urea forms the adduct of the saturated fatty acids and the methyl ester of the monoene, diene fatty acids and crystallizes out of the system. The crystals were filtered off with cooling. The filtrate was diluted with water and the fatty acids were extracted with petroleum ether.

After solvent removal, a mixture of fatty acids containing 64% by weight of EPA or other omega-3 fatty acids was obtained.

HU 202406 A

Example 3

Wash as in Example 1 and purify the gelatin of Example 1 (Éisenia foetida). Subsequently, 100 g of shredded cilantro are added to a stirred vessel and 0.01 parts by weight of butyl hydroxytoluene antioxidant are added.

The mixture was adjusted to pH 8.0 with ammonium hydroxide. Then 1 part by weight of proteinase and 1 part by weight of phospholipase C (Bacillus cereus) is added. The contents of the vessel were homogenized and stirred for 4 hours at 37 ° C.

After this time, the mass is lyophilized. Extract 15 parts by weight of dry material with hexane by shaking the dry powder with 100 parts by weight of hexane, and after 3 hours, separate the extraction residue and extract.

The extraction residue is used as feed after solvent removal. The solvent was removed from the extract on a rotary evaporator.

The product thus obtained had a weight of 1.2 g, with an EPA content of 15.5% by weight based on the total weight of the fatty acids.

Example 4

The procedure of Example 1 was followed, but instead of the enzyme mixture, 2 parts by weight of phospholipase C were added to the gelatine and the digested gelatine mass was spray dried rather than lyophilized.

The fat content is then recovered by carbon dioxide supercritical extraction.

The extraction is carried out according to the method described in U.S. Patent No. 4,675,132.

The parameters of supercritical extraction are as follows:

Extraction temperature: 75 'C, pressure: 17.6 MPa. Yield: 1.2 g fat blend, 15% EPA by weight based on total fatty acid.

Example 5 (Comparative)

All procedures were carried out as in Example 1, but the purified animals were not soaked in protease solution but were subjected to direct extraction. The extraction was carried out in doubled time for 6 hours. Hereinafter, the procedure of Example 1 is followed.

Weight of concentrated fat blend: 0.7 parts by weight. 15% by weight of this is in the form of the glycerol ester of EPA.

The example shows that, if the protease treatment of the invention is omitted, the yield of EPA is significantly reduced.

Example 6 (Comparative)

Animals washed and internally cleaned according to Example 1 were freeze-dried and ground to a mean particle size of 0.9 mm. The milled gillytus is extracted with an aqueous solution of a mixture of 20% ethyl alcohol and 80% isotonic sodium chloride solution.

The extract was worked up as in Example 1. The fat blend is 0.2 parts by weight and has an EPAester content of 11% by weight.

Comparative Example shows that omitting the 5 enzymatic pretreatment and extraction with aqueous alcohol greatly reduces the amount of recoverable EPA ester.

Claims (5)

CLAIMS 10 1. Process from a mixture of groundworms of the Lumbricidae family containing eicosapentaenoic acid ester and, optionally, producing a fatty acid mixture containing eicosapentaenoic acid by washing, drying, then extracting and removing the extract solvent from the aqueous extract of glycol, characterized in that the gelatin purified by water washing is kept between 4 and 10 ° C between 40 and 40 ° C. The animals which have been cleaned internally for 48 hours and subsequently on the inside are 0,1-1,5% by weight on a dry weight basis. The enzyme is treated with 20 enzymes for 1-4 hours, then the pre-kneaded mass is dried in a known manner, then milled with a fat solvent or subjected to carbon dioxide supercritical extraction, and the extract and extraction residue are separated, The extract is deprotected in a known manner and, if desired, the fatty acids are liberated from the fat mixture in a known manner and the eicosapentaenoic acid content of the mixture is enriched in a known manner. (Priority: October 16, 1990) 30 2. Procedure for the mixture of Eicosapentaenoic Acid Ester Fats from the Lumbricidae Family and, optionally, producing a fatty acid mixture containing eicosapentaenoic acid by washing, drying, then extracting and removing the extract solvent from the aqueous extract of glycol, characterized in that the water-cleaned gelatin is held between wet paper sheets at 4-10 ° C. after which the animals, which have been so cleaned internally, have a percentage of dry matter of between 0,1 and 1,5% by weight. After treatment with 40 protease enzymes for 1-3 hours, the pre-treated mass is dried in a known manner, ground, then extracted with a fat solvent, the extract and the extraction residue are separated off, the extract is then de-solvented in a known manner, The fatty acids are then liberated from the fat blend in a known manner and the eicosapentaenoic acid content of the blend is enriched in a known manner. (Priority: 13/11/1989) The process of claim 1, wherein The enzyme used is protease and / or phospholipase C. 4. A process according to claim 1, wherein the pre-treated mass is spray dried or lyophilized. (Priority: 1990. 55 10. 16) The process of claim 2, wherein the pre-treated mass is dried by heat treatment at 80-100 ° C. (Priority: 13/11/1989)