PROCEDURE FOR EXTRACTION OF LIPIDS FROM BIOLOGICAL MATERIAL The present invention is concerned with a method as stated in the ingress of claim 1, for extracting lipids from a biological material. In short, the method is concerned with extraction of lipids by means of an organic solvent, in the presence of or after a pretreatment of the raw material with an acid in order to effect a partial dissolution of the structure of the material before extraction. Thereby, and by the use of known organic solvents (such as ethanol), which without pretreatment of the raw material gives low recovery of lipids, a signifikant increase in the yield of lipids, such as phospholipids, is achieved.

By use of a raw material, for instance a marine raw material, for the production of inter alia biochemicals, such as proteins, peptides, nucleic acids (DNA), fats (including phospholipids) and extractives, a common feature is that the substance (s) of interest is (are) present in small amounts. The applied methods for the production of these substances do not always consider the possibility of production of additional components from the same raw material. The result is inter alia that the cost for the raw materials per kg produced goods will be higher than would be the case if the cost for the raw material could be divided between several products from the same raw material.

Another common feature for the production of single components from a biological material is the necessity of removing fats fromo the preparations to be produced. In addition, it might be desirable to utilize the fats separately.

Thus, there is a need to develop gentle methods for the removal of lipids (including phospholipids) from biological materials, making possible a subsequent preparation of

other products (including biochemicals) from the same raw material.

In a marine raw material, the fats are particularly valuable due to the advantageous composition of the polyunsaturated fatty acids. These are localized both in the triglycerides and for instance in the phospholipids.

While the triglycerides can be extracted relatively easily from biological materials, phospholipids are much more difficult to extract. This is due to the fact that the phospholipids are the main components in all biological membranes and that they when"liberated"form vesicles which are easily suspended in the aqueuos phase and not necessarily dissolved in the triglycerides.

For the preparation of triglycerides there are several methods in use, for instance heating with a resulting coagulation of the raw material with subsequent centrifugation or pressing (for instance common fishmeal and preparation of oil from herrings), use of acid and subsequent heating and separation of the fats, or use of enzymes in order to hydrolyze the raw material under acidic or neutral conditions, with a subsequent separation of the lipids from the aqueuos solution. For separation of phospholipids from a biological raw material, it is, however, necessary to use organic solvents in order to obtain the highest possible yield, or optionally to use supercritical extraction with carbon dioxide. The latter method is, however too expensive in relation to the other methods.

In addition to the fact that the above methods can extract phospholipids only in a limited degree, said methods will to a varying degree alter the other components which might be of interest. It might not be desirable to coagulate the proteins, further, an enzymatic hydrolysis will lead to the formation of short-chained peptides and free amino acids, which will restrict the use of the protein containing

material present in the starting material. Said methods are thus of limited interest in respect to the aim of removing the fats in an effective and gentle manner.

When extracting lipids from biological raw material by means of organic solvents, hexan is for instance used (extraction of soya), and for example a mixture of chloroform and methanol is used for extraction from aqueuos biological raw material. Common for said extraction agents are that they are a health hazard. This is particularly true for chloroform. Alcohols, such as ethanol, can also be used as an extraction medium, however the yield is reduced when the extraction is performed from an aqueuos biological material due to the limited solubility of the neutral lipids in an aqueuos ethanol solution.

It is known that by the use of acid, a partial hydrolysis of biological raw material can be effected. The degree of hydrolysis will of course vary with the choice of acid, the amount of acid, temperature and time of exposure. We have investigated the possibility of extracting lipids, including phospholipids with solvents by treating the raw material with acid, hereby obtaining a higher yield than when using solvents more acceptable for use of lipids in areas such as nutrition, health, and pharmacology.

Experiments have shown that treatment with acid gives a partial change in the biological structure, and by extraction we obtained a substantial increase in the yield compared to when not treating with acid. With use of e. g. ethanol, the total lipid yield surprisingly increased with a factor in the range 2.0-2.8, whereas the phospholipids increased with a factor in the range 2.7-3.4 and more, which is an increase in lipid yield in the range 57-80% and 70-88% for total lipids and phospholipids, respectively, compared to the control without acid treatment.

The process is thus characterized in what is specified in the characterizing clause of claim 1. Further features are apparent from claims 2-4.

EXAMPLES The following experiments demonstrate the effect of the acidic treatment.

Example 1, pH 2.0 To one kg of rough-grinded cod roe was added 50% HC1 until pH 2 was reached. Then 1 1 of ethanol was added, followed by homogenisation until partial dissolution of the roe structure was achieved (5 min). The mixture was left for a few minutes, then decantered and filtered. The filter mass was washed twice with 1 1 of 96% ethanol. The filtrates were combined, evaporated to dryness and analysed for the total lipid and for phospholipids by precipitation with aceton according to standard methods. The results are shown in Table 1.

Table 1 Extraction with ethanol after treatment with acid, pH 2.0 With HC1, Chloroform + pH 2 methanol Total lipid, % 10.0 g 20.0 g 35.0 g Phospholipids, 4.4 g 12.0g 17.0 g % Example 2, pH 4 To rough grinded cod roe (100 g) was added 50% acetic acid until pH 4 was reached. The mixture was homogenized until a partial dissolution of the roe structure (5 min). Then 100 ml of ethanol (96%) was added, followed by 5 min of stirring. The mixture was left for a few minutes, then

decantered and filtered. The filter mass was washed twice with 100 ml of 96% ethanol. The filtrates were combined, evaporated to dryness and analysed for total lipids and for phospholipids by precipitation with acetone according to standard methods. The results are shown in Table 2.

Table 2 Extraction of roe with ethanol after treatment with acid, pH4. 0 without 1 With acetic Chloroform + Lipid, acetic acid I acid (pH 4) methanol Total lipids, % 1.0 g 2.8 g 3.5 g Phospholipids, % 0.44 g 1.5 g 1.7 g