HU207276B - Process for separating oil from fishes - Google Patents

Abstract

Fish oils contg. omega-3-fatty acids are extracted in step(s) by an extractant mixt. of methanol and 1,2-dichloroethane. (Dwg.0/0)

Description

The present invention relates to a process for the extraction of oils containing omega-3 fatty acids by extraction from marine or freshwater fish.

Numerous literature data refer to the so-called. two unsaturated fatty acids of the omega-3 group, all-cis -5,8,11,14,17, ecosapentenoic acid [32839-30-8] (hereinafter EPA) and all-cis-4,7,10,13,16, 19, the beneficial pharmacological effects of docosahexaenoic acid [32829-18-2] (hereinafter DHA). Among these, the anti-inflammatory [J. A. Salmon, T. Terano, Proc. Nutr. 44, 385 (1985)], the prevention of hypotension and hypertension (Y, Kasuya et al., 1986, J. PharmacoDynamics 9, 239) and the protection against thrombosis and infarction [K. A. Jorgensen et al., Acta Med. Scand. 219, 473 (1986)], the most significant. (The numbers in square brackets are the Chemical Abstracts Identification Numbers.) More recently, the assumption has been made that ecosapentenoic acid can be used in the prevention of AIDS, among other essential fatty acids [Μ. Begin, U.N. Das, Med. Hypotheses 20, 1 (1986)]. The effects on inflammation and vascular disease are likely to be marked reductions in serum triglyceride and cholesterol levels, overexpression of leukotriene and thromboxane metabolites acting as inhibitors of thrombosis and consequent changes in monocyte and neutrophil leukocyte inhibitory effect. The mechanism of action is also likely to be influenced by the beneficial effects of the two omega-3 fatty acids on erythrocyte membrane composition and increased erythrocyte deformability.

It is a well-known fact that seafood oils contain significant amounts of EPA and DHA. Such oils can be obtained from fish muscle and internal organs (eg liver). Fish species differ in oil yield. The proportion of these two omega-3 unsaturated fatty acids in the composition is usually also a function of the latitude of the habitat. North Sea habitats are particularly favorable in this respect. Mackerel and salmon are the two "fatty" fish with the highest oil yields, and in particular the content of polyunsaturated fatty acids.

Freshwater fish have long been overshadowed by EPA and DHA sources because no freshwater fish species could be found that could be economically harvested. However, an article published in 1978 (Chenged I et al., Aquacultura Hungarica (Szarvas), Vol. I, 35-43.) Describes the fact that the native species of bush fish, also introduced in Hungary, stand out from the high EPA and DHA.

Methods to isolate total lipid from fish are very diverse. The appropriate procedure is developed taking into account the type of fish, the source of the fish, the organs of the fish (eg liver, muscle, mesenteric fat, etc.) and the lipid components (eg phospholipids, triglycerides, special fatty acids, etc.). the purpose of the process (e.g., small experiments or a simple and inexpensive operational procedure).

Lipids are found in fish in two ways. On the one hand, there are so-called. fat deposits, which, independently of other tissues, form deposits as adipose tissue, e.g. under the skin or on the intestines and connective tissue (intestinal fat or mesenteric fat) that support the intestines, etc. The function of these adipose tissues is twofold. They store some of the excess energy in the body, which can be mobilized when needed (the specific energy content of fats is the highest among the nutrients), and partly it fulfills other physiological functions, such as: thermal insulation or mechanical protective function. These lipids are mainly neutral fats, mostly triglycerides.

Another class of lipids is the so-called lipid. functional lipids that are present in virtually every tissue and are involved in important life functions. Blood, nerve tissue, cell membranes, etc. are all parts of the body where lipids play a significant role. Usually for other materials, e.g. they are bound to proteins in the body (lipoproteins), or are encapsulated in other cell types as components of fat cells. In other (non-fatty) tissues, lipids are generally found in lower amounts than other substances (eg, protein, carbohydrate) but can be extracted (extracted) by special methods. To do this, the tissue structure must be revealed and the lipids dissolved in suitable solvents or solvent mixtures.

Of the fish tissues, the muscle tissue is characterized by its high lipid content compared to the muscle tissue of other animals, so our method focuses on extracting lipid from the muscle.

The known processes can also be partially grouped in such a way that they are more suitable for the production of depot fats or tissue fats.

Deposition fat is relatively easy to process because it consists of homogenization and dissolution, yielding almost 100%, since the fat tissue is virtually pure fat. Neutral fats are highly apolar compounds, so apolar solvents can be used alone to extract or dissolve them.

In fish, depot fats contain lower amounts of polyunsaturated acids when viewed in the proportion of total fatty acids than functional fats, so that products derived from depot fats usually need to be enriched separately with costly processes for the portion containing the more valuable components. In addition, the presence of depot fat in the intestines and mesenteric connective tissue may be dependent on the movement of the fish, the season, etc. It is usually present in three-month old fish, but their use is impractical for fish farming, since three-year old fish are mostly mother fish.

Intestinal fat is the subject of British Patent No. 2023634, which extracts glyceride oils with a mixture of aliphatic alicyclic hydrocarbons having a boiling range of 60 to 101 ° C. Contains extractant

EN 207 276 Β glaze also contains 1% v / v formic acid, mainly for antimicrobial protection of the product. After extraction at high temperature, the extra juice is cooled and the separated oil is separated.

Hydrocarbons, in particular hexane, are also used in Japanese Patent Application No. 85170700, wherein the whole fish (e.g. Eel) is ground, mixed with potato starch, then extracted with hexane, and the extract is washed with sodium sulfate and evaporated.

Extraction of functional fats found in tissues requires other types of solvents. It is advantageous to use mixtures of non-polar and polar solvents, partly because the tissues need to be chemically uncovered and the lipids often need to be liberated from the chemically bound state and partly because the tissue lipids contain not only apolar but also polar lipids. are rich and aim to extract all useful lipids (including free fatty acids) with good yields.

Lipid extraction procedures from various tissues are summarized in Methods in Enzymology. Manual III. 299, Vol. 299, by C. Entenman (Acad Press, New York, 1957. Ed. S. P. Colowick, N. 0 Chaplain). Describes procedures for whole blood, serum or plasma, erythrocytes, brain, skin, muscle, arteries, whole body, bone, lymph. It describes the use of a wide variety of solvents and solvent mixtures that can be used in various variants under laboratory conditions to achieve good lipid yields. Generally, prolonged treatments with reflux are carried out at a higher temperature, which is not advantageous for chemically sensitive polyunsaturated fatty acids. The solvents used are alcohol ether, followed by ether or chloroform, petroleum ether, chloroform-methanol (1: 1).

It should be noted that there are several suitable methods for muscle tissue mentioned in the monograph, such as extraction with an alcohol-ether mixture followed by chloroform or an alcohol-ether mixture followed by ether or a mixture of methanol-chloroform on a Soxhlet apparatus. An important factor is the exploration of muscle tissue prior to extraction, that is, the physical structure of the tissue being broken down so that it is readily accessible to solvents.

The use of the chloroform-methanol (2: 1) mixture describes the composition on brain tissue. This was first described in 1951 by Folch et al. (J. Bioi. Chem. 191, 883, 1951), and later became widespread as the most preferred method.

Limitations and various modifications to the use of the chloroform-methanol (2: 1) mixture are discussed in N.S. Radin (Methods in Enzymology XIV, p. 245, ed. J. M. Lowenstein, Acad. Press, New York, 1969). It is also described as a commonly used method in Lipid Analysis. Handbook, 2nd Edition, (ed. W. W. Christie, Pergamon Press, Oxford, 1982, Chapter 2, p. 19).

From the bush fish starting methods, in the previously cited article written by István Csengeri et al. (Aquacultura Hungarica, Vol. I, 35-43), extraction with chloroform-methanol is also carried out from the liver and muscle tissue of the bush species. The procedure is for the preparation of samples for analytical testing and yield data are not provided.

Another extraction process based on bush fish is known in the art. Hungarian Patent Application Publication No. T / 46211 discloses bush fish grease. It uses petroleum ether, also a hydrocarbon fraction, as a solvent. It produces an oil product which has long been known for its enrichment by urea (urea) complex purification. It also mentions the solvent mixture of chloroform-methanol (2: 1) as a possible extraction agent. In order to provide comparative data, the procedure described in the application also involved extraction from bush muscle starting at 3% by weight of lipid.

The disadvantage of the exclusive use of petroleum ether and other non-polar solvents is that they are less suitable for liberating and dismantling the fat content of the tissues from other tissue components and poorly soluble in the more polar lipids. In fish, for example. precious fatty acids are also found in their free state, and they are not well soluble in petroleum ether only if methyl esters, apolar compounds, are prepared from them. Therefore, the use of polar and apolar solvent mixtures is much more preferred.

A further disadvantage of these processes is that they use highly volatile and explosive solvents, which require hazardous or very costly preventive safety measures to be applied in an industrial process.

Chloroform and diethyl ether commonly used for extraction purposes are dangerous sleeping pills and narcotics, and are highly volatile. In addition, chloroform can produce highly toxic phosgene in the presence of light in moist air. In addition, chloroform is a non-combustible material and is therefore environmentally disadvantageous. To neutralize it by thermal decomposition is very expensive.

It is an object of the present invention to provide an extraction process for recovering omega-3 unsaturated fatty acids having valuable healing properties from bush fish using a sufficient amount of fish tissue. Our other goal was to make our new process easily industrially feasible and as environmentally friendly as possible.

The present invention therefore relates to a process for the extraction of oil containing omega-3 fatty acids from marine or freshwater fish by performing a four-step extraction process comprising a 35: 2 by volume mixture of methanol and 1,2-dichloroethane in the first extraction step. in a second extraction step with methanol: 1,2-dichloroethane (2: 3-5 by volume), in a third extraction step with methanol and

Extraction is carried out with a mixture of 1,2-dichloroethane (3-5: 2 by volume) and in the fourth extraction step a mixture of methanol and 1,2-dichloroethane (1: 11.5 by volume).

Our process uses 1,2-dichloroethane as the apolar component and methanol as the polar component in the solvent mixture used for extraction. In this way, ki3

Harmful effects of the use of chloroform have been eliminated. Dichloroethane is a readily available, widely used solvent, much cheaper, and more advantageous than chloroform. Methanol is also a cheaper and more readily available solvent such as methanol. ethanol.

Since the physical properties of the solvent mixture during the extraction process are decisive, it was not anticipated by those skilled in the art that the above mixture could be used in the process of the invention.

The use of dichloroethane-nrethanol solvent mixture has not yet been described in the literature. The two solvents are not highly explosive, they are combustible, so the environmental damage can be reduced.

The lipid content can also be extracted in very good yield from muscle tissue, especially when multiple extraction steps are used and different ratios of methanol to 1,2-dichloroethane are used.

The previously known industrial process of using bush, which has formed a substantial proportion of the meat of the fish, has not been considered as a starting material. In contrast, the oil content of the oil obtained by our process is high enough to be marketed directly in encapsulated form or otherwise, no further enrichment is required. The EPA and DHA content is 8-18% depending on the starting material. This value is 3 to 6 times the corresponding value of the bush muscle tissue extraction procedure described in the literature.

It is noted, however, that many methods are known for further enriching the valuable components in the oil obtained by our process.

In a most preferred embodiment of the process according to the invention, fish meal containing the bulk of muscle tissue, prepared after cooking or raw after washing, which is free of useless parts (head, tail, bones, etc.), is subjected to a four-step extraction. and 1,2-dichloroethane in a 3-5: 2, preferably 2: 1 by volume ratio, in a second extraction step a mixture of methanol and 1,2-dichloroethane in a ratio of 2: 3-5, preferably 1: 2 by volume; in a step of methanol: 1,2-dichloroethane (3-5: 2), preferably 2: 1 (v / v); in the fourth extraction step (essentially purification), methanol: 1,2-dichloroethane (1: 1-1.5), preferably 1: 1 A mixture of 1.25 by volume is used as an extraction agent. In order to avoid decomposition of the valuable components, it is advisable to carry out the extraction at a temperature of 0 to 20 ° C and of the evaporation at a temperature of 25 to 45 ° C, preferably 30 ° C, under vacuum. A further advantage is that the solvent mixture obtained during the evaporation after the first, second and third extraction steps can be reused for extraction after dehydration, which has a very significant positive economic effect.

In the first three extraction steps, the extraction mixture also contains a small amount of antioxidant, preferably at a concentration of 10-500 mg / l of 2,6-di-tert-butyl-p-cresol.

The amounts of EPA and DHA, respectively, represent the proportion of total fatty acids in the oil extracted from the bush muscle.

According to our studies, based on fatty acid assays, W. W. Lipid Analysis, Pergamon Press, Oxford, 1982 (2nd ed.), P. 65.] The most common fatty acids that occur in addition to polyunsaturated fatty acids are as follows.

Significant amounts (10-30%);

16: Hexadecanoic acid [57-10-3] (palmitic acid)

16: 1 (n9) 7-Hexadecenoic acid [25 447-95-4] (7-dehydro-palmitic acid)

18: 1 (n9 Cis-9-octadecenoic acid [112-80-1] (oleic acid)

In medium amounts (3-10%):

18: 0 Octadecanoic acid [57-11-4-] (stearic acid)

18: 2 (n6) Cis, cis-9,12-octadecadienoic acid [6033-1] (linoleic acid)

18: 3 (n3) Cis, Cis-9,12,15-Octadecatrienoic acid [463-40-1] linolenic acid

20: 4 (n6) Cis, cis, cis-5-8, 11,14-ejcatetrenic acid [506-32-1] (arachidonic acid)

In small quantities (%):

16: 2 (n6) 7,10-Hexadecadienoic acid [25,377-52-0]

20: 3 (n3) 11,14,17-Eccathioic acid [27070-56-0]

22: 4 (n6) 7,10,13,16-Docetracetic acid [209125-0]

22: 5 (n3) 7,10,13,16,19-Docosapentenoic acid [2234-74-4]

The therapeutic use of the oil obtained according to the invention in its original form is advantageous, e.g. capsules for oral administration. The lipase enzymes present in the intestinal tract break down the glycerides, and their components are used to re-synthesize useful and specific lipids for the body. It is therefore not necessary to separate EPA and DHA from other fatty acids.

These are generally trivalent glycerol esters which are mixed triglycerides. Thus, the same molecule may contain one, two, or three polyunsaturated fatty acids in various forms, among other fatty acids.

Thus, the oil obtained during the extraction can be used directly. Contains no harmful fatty acids. As described in the literature, there are indications that 20: 1 (n9) and 22: 1 (n9) mono-fatty acids (11-ecosenoic acid [2462-94-4] and cis-3-docosenoic acid [112-867 ], also known as erucasic acid). Cis-13-docosenoic acid and its trans variant, trans-13-docosenoic acid [50633-2], cause reversible lipidosis in the myocardial membranes of experimental animals. [Astorg, P. O., Ann. Nutr. Metab. 25 (4), 201, 1981; Chien, K.R. et al. al., Am. J. Pathol. 112 (1), 68, 1983]. Although, as described above, only high doses were used, and even for a short period of time, adverse effects were found, but it was considered necessary to investigate the presence of these fatty acids in the extracted oils. According to the literature, Christie, W. W., Lipid Analysis, Pergamon Press, Oxford, 1982 (2nd ed.), P. 65.] retention time ratios for the two fatty acids in question are as follows:

Retention time ratios

Concerned. basis: palmitic stearic oleic 20: 1 (n9) 3.59 2.08 1.75 22: 1 (n9) 6.19 3.59 3.02

HU 207 276 B

Fat analysis of the oils described in the Examples has shown that they do not contain the aforementioned mono-fatty acids.

The process of the present invention will be described in more detail below.

Example 1

Procedure starting with white bush harvested in July (average live weight: 1.2 kg)

Place about 5-6 kg of partially cleaned fish in boiling water and boil for 10 minutes, then remove the fish and meat debris. We squeeze the water in a cloth or filter paper. Weigh 2 kg of cooked fish meat on the scales for further processing.

45 L of methyl alcohol (MeOH) and 35 L of distilled 1,2-dichloroethane (DCE) are prepared at 4 ° C containing 50 mg of 2,6-di-tertiary-butyl-p-cresol (trade name) : butylhydroxytoluene (BHT).

The 2 kg fish meat prepared as above is homogenized in 5 L of MeOH. (After homogenization, the meat gives the appearance of a fluffy precipitate without lumps.) The resulting mixture is poured into a mixture of 11 L of MeOH and 8 L of DCE. The material was allowed to stand cold for 30 minutes after homogenization, ca. Stirring again every 5 minutes.

After 30 minutes, the material was vacuum filtered through paper (a mirror solution was obtained) and stored refrigerated. (First extraction step.)

A 1: 2 mixture of MeOH and DCE was added to the precipitate under vigorous stirring in a total volume of 241 volumes. In the following, the procedure of the first extraction step is followed. (Second extraction step.)

The third extraction step was carried out as described for the second extraction step, but using MeOH: DCE 2: 1 as the extraction agent in a total volume of 241 volumes.

The combined filtrates were mixed and concentrated on a 10 L Rotavapor using an external water bath at 40 ° C. The residue, which is partly oily to protein-like, slightly sticky gray, is mixed and evaporated to dryness three times with 200 ml of anhydrous ethanol.

An off-white oil is obtained which mixes with the precipitate. For this precipitation 2.8 1 kb. 15 ° C 1: 1.25 MeOH-DCE was added. (Fourth extraction step.) The yellowish oily phase dissolves and most of the precipitate forms a suspension. The mixture is cca. After 1 hour at 4 ° C, it is vacuum filtered through a double filter paper. The filtrate was made up to 3.2 L with 1: 1.25 MeOHDCE while washing the dishes. The mixture is cooled to 13-15 ° C, if necessary, and then combined with 640 ml of 0.1 M KCl in 13-15 ° C in a shaking funnel. The contents of the shaking funnel were shaken vigorously and then allowed to stand at room temperature. After reaching a sharp phase boundary (about 10 minutes), the lower phase (about 1800 ml) was separated and evaporated in a Rotavapor at 30 ° C, solvent-free. Dehydration is facilitated by adding twice 100 ml of anhydrous ethanol as above. Solvent-free is assured by measuring weight constant.

152 g of an oil are obtained, the EPA + DHA content of which is determined by gas-liquid chromatography. The chromatogram obtained is illustrated in Figure 1. The assay was started from the methylated derivative of the final product of Example 1. The measurement was performed under the following conditions:

Charge: Gas Chrom Q 10% Ethylene Succinate Ethyl Silicone (EGSS-X)

Carrier gas N ₂ (flow rate: 50 ml / min).

The column is approx. 2.1 m, internal diameter 2 mm, column temperature 178 ° C, injector temperature 240 ° C, detector (flame ionization) temperature 250 ° C.

Program 80 min isotherm; integration start: 2.7 minutes.

Solvent: light petroleum.

Eicosapentenoic acid: retention time 23.76 min, 6.3% based on total fatty acid.

Docosahexaenoic acid: retention time 49.37 min., Relative to total fatty acids: 4.85%.

Retention ratios relative to palmitic acid (16: 0): eicosapentenoic acid 7.95 to docosahexaenoic acid 16.51

That is, the oil obtained has an EPA + DHA content of 11.2%.

The active ingredient content of the oils obtained in the following examples was determined according to the method described above.

Example 2

October whip harvest (average live weight: 1.0 kg)

In each case, the procedure of Example 1 was followed. 250 g of an oil are obtained which contains EPA + DHA

8.3%.

Example 3

October spotted bush (average live weight: 0.8 kg).

In each case, the procedure of Example 1 was followed. 156 g of an oil are obtained which contains EPA + DHA

15.9%.

Example 4

This is a white fish bus harvested in November (average live weight: 1.4 kg).

In each case, the procedure of Example 1 was followed. 178 g of an oil are obtained which contains EPA + DHA

9.6%.

Example 5

Spotted bush harvested in November (average live weight: 1.8 kg)

In each case, the procedure of Example 1 was followed. 210 g of an oil are obtained which contains EPA + DHA

11.8%.

HU 207 276 Β

Example 6

Procedure starting with white bush harvested in November (average live weight: 1.4 kg)

We start with raw fish meat, which is obtained after the fish skin has been removed by removing bones and bones. The muscle is minced in a meat grinder and then proceeded as described in Example 1 below.

140 g of an oil are obtained with an EPA + DHA content of 11.8%.

Example 7

Spotted bush harvested in November (average live weight: 1.8 kg)

In each case, the procedure of Example 6 was followed.

168 g of an oil are obtained with an EPA + DHA content of 15

11.4%.

Example 8

Starting from a speckled bush harvested in December (average live weight: 2.1 kg) 20

In each case, the procedure of Example 6 was followed.

117 g of an oil are obtained with an EPA + DHA content of 15.0%.

Example 9 25

Procedure for marine mackerel originating in Germany (average live weight 0.36 kg).

The procedure of Example 6 was followed except that Example 1 was performed with 100 g of minced muscle, reducing the volume of solvent used for extraction and other operations in proportion to the weight of the muscle.

9.88 g of an oil are obtained having an EPA + DHA content of 28.5%.

Example 10

Procedure for sea hake originating in German imports (average live weight: 0.85 kg).

In each case, the procedure of Example 9 was followed.

2.32 g of an oil were obtained with an EPA + DHA content of 40 41.25%.

Example 11

Process based on domestic freshwater carp (average live weight 1.25 kg). 45

All of the procedures of Example 9 were followed. 11.45 g of an oil were obtained with an EPA + DHA content of 2.99%.

Example 12

Process based on domestic freshwater bream (average live weight 0.18 kg).

In each case, the procedure of Example 9 was followed. 2.8 g of an oil was obtained which had an EPA + DHA content

26.26%.

Example 13

Import from sea herring (average live weight 0,15 kg).

In each case, the procedure of Example 9 was followed. 7.6 g of an oil are obtained with 26.69% EPA + DHA.

Example 14

A procedure based on domestic freshwater amur (average live weight: 1.47 kg).

In each case, the procedure of Example 9 was followed. 7.14 g of an oil was obtained which had an EPA + DHA content

3.08%.

Claims (3)

PATENT CLAIMS A process for the extraction of oil containing omega-3 fatty acids from marine or freshwater fish, characterized in that the extraction is carried out in four steps, in which the first extraction step comprises a 3-5: 2 by volume mixture of methanol and 1,2-dichloroethane. in a second extraction step with methanol: 1,2-dichloroethane (2: 3-5 by volume), in a third extraction step with methanol and Extraction is carried out with a mixture of 1,2-dichloroethane (3-5: 2 by volume) and the fourth extraction step is carried out with a mixture of methanol and 1,2-dichloroethane (1: 1-1.5 by volume). 2. The method of claim 1, wherein the marine fish is mackerel, hake or herring, and the freshwater fish is carp, bream, amur or bush. 3. A process according to any one of claims 1 to 3, characterized in that the extractant also contains an antioxidant, preferably 2,6-di-tert-butyl-p-cresol.