FIELD OF THE INVENTION

The present invention relates to a method for production of fatty acids with more than three double bonds as defined in the preamble of claim 1 and to a method for the cultivation of Gammaridae. The present invention also relates to a fish feed comprising Gammaridae, a lipid product extracted or produced from a Gammaridae, a use of the lipid product, as well as to a feed for the production of Gammaridae and the use of the feed.

BACKGROUND TO THE INVENTION There is a large need for marine lipids comprising highly unsaturated fatty acids such as omega-3 fatty acids on the world market. Fish meals and fish oils are the traditional major sources for these lipids. They are mainly derived from wild caught marine fish such as capelin, herring, anchovy, sardine fisheries etc. A major application of marine lipids is their use as feed ingredients in fish farming. Especially marine fish have a high requirement for the essential omega-3 fatty acids for normal growth and development, which is largely covered by fish oil addition to the feed or by enrichment of prey organisms used in the cultivation. Increasing aquaculture production and thus demand for fish feed comprising marine lipids and stagnating global fisheries lead to a situation where demand is exceeding the sustainable supply of this raw material. An increasing awareness of the beneficial effects of omega-3 fatty acids in other sectors such as for human consumption, pharmaceutical compositions and for health purposes in humans and other animals than fish further increases the quantitative demand for these fatty acids.

It is therefore a great challenge to find alternative resources independently of wild catches for marine lipids comprising omega-3 fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and for omega-6 fatty acids such as arachidonic acid (ARA).

Marine lipids comprising the highly unsaturated fatty acids (HUFA) such as omega-3 fatty acids (e.g. DHA, EPA) can also be found at lower fauna levels of the marine food web such as in copepods and krill. However, these organisms are in general known to be dependent on an external supply of these essential fatty acids in their diet which often consists of other smaller marine organisms comprising these fatty acids or marine microalgae with a high level of these omega-3 fatty acids and omega-6 fatty acids. Species of Gammaridae, which are also Crustaceans present in the lower food web and are belonging to the family of amphipods, were shown to comprise omega-3 and omega-6 fatty acids. However, Gammaridae are not effectively exploitable by wild catches and only very recently efforts have been done to cultivate some marine species under controlled conditions.

The main objective of the present invention is to develop a new method for producing omega-3 fatty acids such as DHA and EPA and long-chain omega-6 fatty acids such as ARA without the need for marine lipids.

Another objective of the invention is to make use of vegetable resources as a raw material basis for the production of marine lipids.

Yet another objective of the invention is to develop a new alternative source for marine lipids such as for human consumption or for aquaculture purposes which is independent of the addition of marine lipids during the production process.

SUMMARY OF THE INVENTION According to a first aspect of the invention, it is provided a method for production of fatty acids with more than three double bonds in Gammaridae whereby said Gammaridae are fed a feed comprising saturated and/or unsaturated fatty acids which have no more than 2 double bonds and wherein a portion of said supplied fatty acids is converted to fatty acids with more than three double bonds.

According to a second aspect of the invention, it is provided a method for the cultivation of Gammaridae whereby said Gammaridae are fed a feed which comprises unsaturated fatty acids having no more than 2 double bonds and whereby the Gammaridae endogenously produce polyunsaturated fatty acids having more than three double bonds.

Preferably, said fatty acids are produced through unsaturation and/or elongation of the supplied fatty acids in the feed.

Preferably, the produced fatty acids have 20 carbon atoms or more.

It is also preferred that the feed further comprises unsaturated fatty acids with 3 double bonds.

It is further preferred that the feed does not comprise any highly unsaturated fatty acids chosen from the group of DHA, EPA and/or ARA.

Preferably, the fatty acids in the feed are derived from a non-marine lipid material, preferably derived from or provided as seeds, fruits and/or other plant material from a terrestrial plant chosen from the group of soya bean, sun flower, raps, palm, coconut, corn, peanut and/or flax, most preferable from soya.

Preferably, the used feed is free of any marine lipid material.

It is also preferred that the produced unsaturated fatty acids have 20 carbon atoms or more and wherein the produced fatty acids have 4, 5 or 6 double bonds.

Preferably, the produced fatty acids are belonging to the group of omega-3 and/or omega-6 fatty acids, preferably fatty acids chosen from DHA, EPA and ARA.

It is further preferred that the comprised polyunsaturated fatty acids in the feed are substantially only C18 omega-6 fatty acids and if present the other omega-6 and/or omega-3 fatty acids account for less than 0.5 %, preferably less than 0.05%, most preferably 0.02% or less of the total fatty acids.

In another preferred method the content of C18 omega-6 fatty acids in the feed is no more than 3% of the total fatty acids. Preferably, the said cultivated Gammaridae is the species Gammarus oceanicus which has a content of omega-3 fatty acids of at least 22 mg/g dry weight and an omega-6 content of at least 11 mg/g dry weight, preferably comprising at least 7 mg DHA/g dry weight, 10 mg EPA/ g dry weight and at least 2.5 mg ARA per g dry weight.

It is also preferred that said cultivated Gammaridae is the species Gammarus locusta, which has a content of omega-3 fatty acids of at least 12 mg/g dry weight, preferably more than 24 mg/g dry weight and an omega-6 content of at least 3 mg/g dry weight, preferably more than 6 mg/g dry weight, wherein preferably at least 2 mg /g dry weight, more preferred at least 7 mg/ g dry weight is DHA, at least 6 mg/ g dry weight, more preferred at least 11 mg/g dry weight is EPA and at least 0.5 mg per g dry weight is ARA, more preferred at least 0.7 mg/g dry weight.

Preferably, the said cultivated Gammaridae is G. locusta, which has at least 3% of the total fatty acids as DHA, preferably at least 9% DHA of the total fatty acids, at least 7 % of the total fatty acids as EPA, preferably at least 15 % EPA of the total fatty acids and/or at least 0.9% of the total fatty acids as ARA, preferably at least 1.9 % ARA of the total fatty acids.

In another preferred method Gammaridae are cultivated using a feed according to any the paragraphs following below.

It is also preferred that the produced Gammaridae have a lipid content of at least 125 mg lipid/ g dry weight, preferably of at least 135 mg lipid/g dry weight, more preferably of at least 140 mg/ dry weight.

In a third aspect of the present invention, it is provided a lipid product extracted or produced from a Gammaridae whereby said Gammaridae are fed a feed which comprises unsaturated fatty acids having no more than 2 double bonds and whereby the Gammaridae endogenously produce polyunsaturated fatty acids having more than three double bonds. In a further aspect of the invention, it is provided a lipid product which is extracted or produced from a Gammaridae wherein said Gammaridae are produced according to any of the methods described in the previous paragraphs.

Preferably, the fatty acid composition of the lipid product is corresponding to the composition of the produced Gammaridae according to any of the preceding paragraphs or to claim 42, preferably the fatty acids composition of DHA, EPA and/or ARA is according to any of the Tables 4-7.

The invention also relates to a lipid product extracted or produced from a Gammaridae wherein said Gammaridae are fed a feed according to the below following during cultivation.

In another aspect of the invention it is provided a fish feed comprising Gammaridae wherein said Gammaridae are produced according to any of the preceding paragraphs.

In yet another aspect, the invention relates to the use of a lipid product produced from Gammaridae obtainable by a method according to any of the preceding claims as an ingredient in feed for animal production, preferably aquaculture farming, most preferably for farming of salmon or marine fish.

In yet another aspect, the invention relates to the use of a lipid product produced from Gammaridae obtainable by a method according to any of preceding claims for human consumption, preferably as nutrient supplement, as a health product, as a food ingredient or for the preparation of a pharmaceutical composition.

The invention further relates to a use of a lipid product produced from Gammaridae obtainable by a method according to any of preceding paragraphs for manufacturing of a pharmaceutical composition for the treatment and/or prevention of a cardiovascular disease, cerebral infarction, hyperlipidemia, arthrosclerosis, hypertension, tumor illness, diabetes, rheumatic arthritis, multiple sclerosis, psoriasis, arthritis, asthma, and/or cancer. In another aspect, the invention relates to a feed for the production of Gammaridae wherein said feed comprises non-marine lipid material.

Preferably said lipid material comprises unsaturated fatty acids having no more than 2 double bonds and wherein said lipid material is preferably from a terrestrial plant.

It is also preferred that the lipid material further comprises unsaturated fatty acids with 3 double bonds.

It is further preferred that the feed does not comprise any highly unsaturated fatty acids chosen from the group of DHA, EPA and/or ARA or wherein the sum of omega-3 fatty acids is less than 0.1mg/g dry weight, preferably 0.05 mg/g or less.

Preferably, said lipids are comprised in or derived from seeds, fruits and/or other plant material from a terrestrial plant chosen from the group of soya bean, sun flower, raps, palm, coconut, corn, peanut and/or flax, most preferable from soya.

It is also preferred that the comprised lipids are solely derived from a non-marine lipid material, preferably from a terrestrial plant.

It is further preferred that the feed further comprises other ingredients from a plant.

Preferably, the feed is for a marine Gammaridae, preferably chosen from the species Gammarus oceanicus and Gammarus locusta.

It is also preferred that said feed further comprises other common feed ingredients such as proteins, carbohydrates, vitamins and/or minerals.

Preferably, the comprised polyunsaturated fatty acids are substantially only omega-6 fatty acids with 18 carbon atoms and if present fatty acids with 20 or more carbon atoms belonging to the group of omega-6 and/or omega-3 fatty acids are no more than 0.5 %, preferably no more than 0.05%, most preferably 0.02% or less of the total fatty acids.

It is also preferred a feed wherein less than 15 % of the total fatty acids are unsaturated fatty acids, and wherein the unsaturated fatty acids solely consist of monounsaturated fatty acids and polyunsaturated omega-6 fatty acids having 18 carbon atoms.

Preferably, the comprised polyunsaturated fatty acids account for less than 3 % of the total fatty acids.

Preferably, the sum of saturated fatty acids, monounsaturated fatty acids, C18:2n-6 and C18:3n-6 account for more than 95%, preferably for at least 99% of the total fatty acids in the comprised lipids.

It is also preferred a feed wherein the composition of the fatty acids in said lipid is substantially as shown in any of the Tables 1 , 2 or 3.

In a preferred embodiment said feed further comprises lipids or other material derived from an animal, preferably from an aquatic organism, more preferable form a marine animal chosen from fish, zooplankton or a crustacean and/or from a marine plant chosen from a macroalgae such as a sea weed species or a microalgae.

Another aspect of the present invention relates to the use of a feed according to any preceding paragraphs for the cultivation of Gammaridae, preferably for the intensive cultivation.

Yet another aspect of the invention is related to a use of a feed according to any of the preceding paragraphs for increasing the lipid content of the produced Gammaridae, preferably to at least 125 mg lipid/ g dry weight, more preferably to at least 135 mg lipid/g dry weight, most preferably to at least 140 mg/ dry weight and/or for delaying the spawning/reproduction of Gammaridae.

Yet another aspect of the invention is related to a use of a feed according to any of the preceding paragraphs for the endogenous production of omega-3 fatty acids, preferably of DHA and/or EPA and/or of the omega-6 fatty acids ARA in Gammaridae when used in their cultivation process.

Preferably, the sum of the fatty acids DHA, EPA and ARA in Gammaridae cultivated with said feed is at least 8.5 mg/g dry weight, preferable at least 19.5 mg/ g dry weight, most preferable at least 20.5 mg/ g dry weight of the Gammaridae, and/or the sum of said fatty acids in the Gammaridae is at least 12 % of the total fatty acids, preferably at least 17.5 % of the total fatty acids, most preferably at least 25.5 % of the total fatty acids.

Another aspect of the present invention is related to the use of a feed according to any of the preceding paragraphs for the production of a lipid product comprising highly unsaturated long chain omega-3 fatty acids and/or arachidonic acid by cultivating Gammaridae with said feed.

The present invention also relates to the use of a feed in a method according to any of the preceding paragraphs.

Preferred embodiments are also defined in the dependent claims.

Thus, the present invention surprisingly allows to produce omega-3 fatty acids with more than 3 double bonds such as DHA and EPA and long chain omega-6 fatty acid e.g. ARA through Gammaridae by using a terrestrial plant, fruit or vegetable seed based diet, which is free or substantially free of these highly unsaturated omega-3 fatty acids or omega-6 fatty acids with more than 18 carbon atoms. By the present invention it was also possible to show that Gammaridae can produce highly unsaturated omega-3 fatty acids and omega-6 fatty acids, when practically no polyunsaturated fatty acids were present in the diet. Gammaridae are a family of amphipods inhabiting fresh to marine waters. Many members of the family are largely benthic. Gammaridae have not been exploited for aquaculture purposes so far. The applicant was able to show that members of this family have the found unexpected and surprising abilities to synthesise highly unsaturated fatty acids. Thereby, the present invention provides a new method for the production of highly unsaturated fatty acids.

The new feed according to the present invention can thus be used for the production of Gammaridae and of a derived lipid fraction which has a high degree of the omega- 3 fatty acids DHA and EPA and omega-6 fatty acid ARA ₁ in spite of the fact that the diet used in the Gammaridae production can be substantially free of these fatty acids and furthermore even practically free any omega-6 fatty acid such as linoleic acid (18:2n-6). By using the feed according to the present invention based on terrestrial lipids such as vegetable seeds or fruits as lipid source in the diet for Gammaridae according to the present invention, a new alternative production method and source for marine omgea-3 fatty acids and omega-6 fatty acids is provided. The products of the invention represent a totally new source and method for the production of marine lipids.

The diet according to the present invention can also be used to achieve a higher lipid content in Gammaridae than in wild caught animals or animals fed a fish raw material based diet with a comparable lipid content, which is a further advantage for the production of a lipid product.

The lipids produced according to the present invention can be used in nutritional compositions, as well as in health products and in pharmaceutical compositions. Highly unsaturated fatty acids such as DHA and EPA are shown to have beneficial effects in the treatment and/or prevention of certain diseases such as cardiovascular diseases, cerebral infarction, hyperlipidemia, arthrosclerosis, hypertension, tumor illness, diabetes, rheumatic arthritis, multiple sclerosis, psoriasis, arthritis, asthma, cancer as e.g. described in US 7462643 B1 , US07595341 , EP01157692 Bl Due to their high degree of highly unsaturated fatty acids as DHA, EPA and ARA, it can be assumed that the highly unsaturated fatty acids produced according to the present invention will have similar effects when used in a pharmaceutical composition.

It will be appreciated that features of the invention described in the foregoing can be combined in any combination without departing from the scope of the invention.

DESCRIPTION OF THE INVENTION

Embodiments of the invention will now be described, by the way of examples with reference to the following diagrams, wherein

Figure 1 shows the fatty acid profiles of the diets used in the feeding experiments (AL; salmon filet, SB; soya beans, CO; coconut (data for CO from Nagaraju and Belur, 2008)) and in Gammaridae fed the respective diets. ALI(GO); Gammarus oceanicus fed salmon filet, SBI (GO); G. oceanicus fed soya beans, SB2(GL); Gammarus locusta fed soya beans and COI(GL); G. locusta fed coconut. A: Illustrates the absolute values (mg/g dry weight) and B the relative distribution of fatty acids (% of total amount). Abbreviations used in the legend: MUFA; monounsaturated fatty acids, SFA; saturated fatty acids, NFAL; lipids that are not fatty acids.

EXPERIMENTAL SECTION

Feeding experiments:

All feeding experiments were carried out using black tanks with a water volume of 20 I, a water temperature of 10 - 12 ⁰ C, a salinity of 34 ppt and a water exchange of 1-2 l/min. The light regime applied was 4 h light (1000 lux) and 20 h darkness. The densities of the Gammaridae varied between 120 and 260 individuals per I. The oxygen concentration ranged between 7.2 and 8.1 mg O ₂ 1 ^"1 . The pH was 7.2 - 7.5. The animals were harvested after they have reached the adult stage (mature) or as pre-adults (directly before maturation).

Experiment 1: Gammarus oceanicus fed a diet based on Salmon filet or soya

The effect of a soya bean based diet fed to the Gammaridae species Gammarus oceanicus was studied on the lipid and fatty acids composition of the animals. As a control a group was fed meat of Atlantic salmon.

Two different groups of the marine amphipod G. oceanicus were cultivated as described above in separate units. One group of G. oceanicus was fed a soya bean diet and the other, the control group, was fed meat from Atlantic salmon which has a high content of marine lipids, in particular omega-3 (n-3) fatty acids (Figure 1 , Table 1 and 2). The experiment started when the Gammaridae were newly hatched and ended when they reached the adult stage (46 days post hatching). Thereafter, the Gammaridae were harvested from the rearing tanks and together with the diets (soya and salmon) analyzed for total lipid and fatty acid composition. Remaining feed in the rearing tanks was removed every second or third day to avoid bacterial and algal growth on the food particles and was thereafter replaced by new food particles (size between 0.5 and 1.5 mm). The lipid content and the distribution of fatty acids in the two diets are shown in Table 1 and 2.

Table 1 : Relative composition of fatty acids in the diets (% of total fatty acids, means± standard deviation): AL= Atlantic salmon filet, SB = Soya beans.

FEED

AL SB

Mean STD Mean STD

Fatty acids (%):

C12:0 0,02 0,01 0,00 0,00

C14:0 3,29 0,01 0,07 0,01

C14:1n5 0,05 0,01 0,00 0,00

C16.O 11,44 0,03 13,73 0,35

C16:1n7 3,56 0,00 0,09 0,02

C18:0 1 ,96 0,02 4,03 0,17

C18:1n9 34,14 1 ,59 25,20 0,52

C18:1n7 5,37 0,30 0,00 0,00

C18:2n6 13,45 0,02 48,66 0,06

C18:3n6 0,04 0,02 7,50 0,23

C18:3n3 6,02 0,05 0,00 0,00

C18:4n3 0,58 0,01 0,00 0,00

C20:0 0,18 0,02 0,25 0,00

C20:1 3,84 0,10 0,09 0,03

C20:2π6 0,86 0,07 0,00 0,00

C20:3n6 0,08 0,01 0,00 0,00

C20:4n6 0,50 0,02 0,00 0,00

C20:3n3 0,46 0,00 0,00 0,00

C20:4n3 0,73 0,01 0,00 0,00

C20:5n3 3,86 0,10 0,00 0,00

C22:0 0,12 0,01 0,30 0,04

C22:1 2,21 0,17 0,06 0,08

C22:5n3 1 ,44 0,11 0,00 0,00

C24:0 0,00 0,00 0,00 0,00

C22:6n3 5,47 1 ,33 0,00 0,00

C24:1n9 0,33 0,02 0,00 0,00

Sum saturated FA 17,02 0,02 18,39 0,55

Sum monounsaturated FA 49,50 1,61 25,44 0,39

Sum polyunsaturated FA 33,48 1 ,63 56,17 0,94 sum omega 3 18,55 1 ,61 0,02 0,02

Sum omega-6 14,93 0,02 56,16 0,92

Hufa n-3 11 ,50 1 ,55 0,02 0,02 n-3/n-6 1 ,24 0,11 0,00 0,00

DHA/EPA 1 ,41 0,31 non Hufa n-3 7,05 0,06 0,00 0,00 Table 2: Content of lipids (mg/g dry weight, means ± standard deviation) and composition of fatty acids in the diets: AL= Atlantic salmon filet, SB = Soya beans.

Experiment 2: Gammarus locυsta fed a diet consisting of soya bean or coconut.

The effect of a diet based on vegetable raw material was also tested in another species belonging to the Gammaridae family being the species Gammarus locusta. In this experiment two different groups of G. locusta were fed a diet either solely consisting of soya beans or of coconut fruit (Cocos nucifera). In the coconut diet, fruits were cut in pieces having a size of about 0.5 - 1.5 mm. G. locusta were collected in the Northern Norway (area west of Tromsø).

The lipid content and fatty acid composition of the soya beans are given in Table 1 and 2 and of coconut in Table 3, respectively. The feeding experiment 2 and sampling was carried out as described for experiment 1. The experiment lasted for 44 days.

Table 3. Fatty acid composition of coconut oil (ND means non detectable)

(Data from Nagaraju and Belur, 2008)

Fatty acids % of total fatty acids

C 6:0 ND

C 8:0 5.1

C 10:0 5.0

C 12:0 49.2

C 14:0 18.0

C 16:0 10.0

C 16.1 ND

C 18:0 ND

C 18:1 10.0

C 18:2 2.4

C 18:3 ND

C 20 ND

C 22 ND

C 24 ND

Analysis Samples of the Gammaridae for lipid and fatty acid analysis were collected at the end of the feeding experiments. Samples were rinsed in fresh water to remove salt and immediately frozen and stored at -80 ⁰ C until analysis. Feed samples of salmon filet, soya beans and coconut were also analyzed.

Lipids and fatty acids were analyzed according to Rainuzzo et al. (1992) by using a modified method of Bligh and Dyer (1959) for extraction of lipids and a method of Metcalfe et al. (1966) for methylation of fatty acids. FAMEs were quantified in a gas chromatograph (Carlo Erba HRGC 5160) connected to a Shimadzu-Chromatopac C R3A integrator. The total lipid content was analyzed gravimetrically (mg/g dry weight). Results and discussion

Feeding of G. oceanicus a diet solely consisting of soya beans resulted in a final lipid content of 140.4 mg per g dry weight which was more than the double of that in animals fed a diet consisting of salmon filet (68.2 mg lipid per g dry weight; Fig. 1 A and Table 4) when fed for a comparable period of time. This was the case in spite of the fact that the soya bean diet had a lower lipid content than the salmon filet being 87.2 mg/g and 238.8 mg/g, respectively. As a reference G. oceanicus sampled from the wild have a typical lipid content of 65-82 mg/g (not shown). Thus, by feeding the new diet consisting of soya beans, it was surprisingly possible to increase the lipid content of G. oceanicus compared to what was achieved by feeding of the salmon filet, or a natural diet in the wild. The higher lipid content in Gammaridae fed the soya bean diet can be related to an observed delay in spawning and thus reproductive activity, which was probably caused by the diet. Spawning is assumed to reduce the lipid content of the animals, since the lipid reserves are potentially used in egg production.

At the end of the feeding experiment 1 G. oceanicus fed the vegetable seed diet soya beans, which was substantially free of omega-3 fatty acids, had 10.59 mg EPA, 7.25 mg DHA, and 2.97 mg ARA per g dry weight, respectively (Table 4; Figure 1A). Gammaridae fed salmon filet had 6.75 mg EPA, 4.69 mg DHA and 1.8 mg ARA per g dry weight, respectively. The content of polyunsaturated fatty acids was thus almost the double in G. oceanicus receiving the vegetable diet compared to G. oceanicus receiving the Salmon diet (33.3 mg/g versus 17.72 mg/g).

Table 4: Content of lipids and fatty acids (mg/g dry weight; means ± standard deviation) in G. oceanicus fed Atlantic salmon filet (AL) and soya beans (SB). FA Fatty acids.

Gammarus oceanicus

AL SB

Total lipid 68.20 ±0.90 140.4 ±0.17

Fatty acids

C12:0 0,03 ±0,00 0,20 ±0,01

C14:0 0,47 ±0,02 2,08 ±0,01

C14:1n5 0,01 ±0,00 0,03 ±0,04

C16:0 6,48 ±0,22 16,89 ±0,08

C16:1n7 1,77 ±0,14 6,36 ±0,18

C18:0 0,36 ±0,02 0,76 ±0,00

C18:1n9 15,66 ± 0,2t 42,97 ±0,81

C18:1n7 3,85 ±0,15 9,85 ±0,18

C18:2n6 2,43 ±0,12 7,50 ±0,06

C18:3n6 0,37 ±0,49 0,07 ±0,01

C18:3n3 0,36 ±0,51 2,71 ±0,01

C18:4n3 0,09 ±0,01 0,34 ±0,02

C20:0 0,05 ±0,00 0,08 + 0,01

C20:1n9 0,84 ±0,00 2,15 ±0,02

C20:1n7 0,17 ±0,02 0,50 ±0,00

C20:2n6 0,19 ±0,00 0,56 ±0,02

C20:3n6 0,02 ±0,00 0,04 ±0,01

C20:4n6 1,80 ±0,01 2,97 ±0,03

C20:3n3 0,08 ±0,01 0,22 ±0,06

C20:4n3 0,12 ±0,01 0,38 ±0,03

C20:5n3 6,75 ±0,07 10,59 ±0,15

C22:0 0,04 ±0,01 0,04 ±0,01

C22:1n11 0,21 ±0,02 0,71 ±0,03

C22:1n9 0,17 ±0,01 0,28 ±0,03

C22:5n3 0,30 ±0,01 0,70 ±0,05

C24:0 0,00 ±0,00 0,00 ±0,00

C22:6n3 4,69 ±0,07 7,25 ±0,21

C24:1n9 0,16 ±0,01 0,27 ±0,08

Sum saturated FA 7,43 ±0,27 20,04 ±0,06

Sum monounsaturated FA 22,84 ±0,50 63,12 ±1,15

Sum polyunsaturated FA 17,19 ±0,27 33,33 ±0,56 sum omega 3 12,39 ±0,35 22,19 ±0,47

Sum n-6 4,81 ±0,62 11,13 ±0,09

Hufa n-3 11,86 ±0,16 18,92 ±0,38 n-3/n-6 2,60 ±0,41 1,99 ±0,03

DHA/EPA 0,69 ±0,00 0,68 ±0,01 non Hufa n-3 0,53 ±0,51 3,28 ±0,09 Table 5: Relative composition of fatty acids (% of total amount, means ± standard deviation) in G. oceanicus fed Atlantic salmon filet (AL) and soya beans (SB).

Gammarus oceanicus

AL SB

Fatty acids

C12:0 0,07 ±0,00 0,17 ±0,01

C14:0 0,99 ±0,03 1,78 ±0,02

C14:1n5 0,03 ±0,00 0,02 ±0,03

C16:0 13,65 ±0,16 14,50 ±0,28

C16:1n7 3,73 ±0,22 5,46 ±0,07

C18:0 0,76 ±0,02 0,65 ±0,01

C18:1n9 32,99 ±0,28 36,89 ±0,18

C18:1n7 8,12 ±0,13 8,45 ±0,03

C18:2n6 5,12 ±0,14 6,44 ±0,04

C18:3n6 0,77 ±1,01 0,06 ±0,01

C18:3n3 0,78 ±1,10 2,33 ±0,03 c18:4π3 0,19 ±0,02 0,29 ±0,01

C20:0 0,11 ±0,00 0,07 ±0,00

C20:1n9 1,76 ±0,03 1,84 ±0,01

C20:1n7 0,35 ±0,05 0,43 ±0,01

C20:2n6 0,41 ±0,02 0,48 ±0,01 c20:3n6 0,03 ±0,00 0,04 ±0,01

C20:4n6 3,79 ±0,06 2,55 ±0,01

C20:3n3 0,16 ±0,02 0,19 ±0,05 c20:4n3 0,25 ±0,01 0,32 ±0,03

C20:5n3 14,21 ±0,16 9,09 ±0,00

C22:0 0,08 ±0,01 0,03 ±0,01 c22:1n11 0,43 ±0,03 0,61 ±0,04

C22:1n9 0,37 ±0,00 0,24 ±0,03 c22:5n3 0,64 ±0,00 0,60 ±0,04

C24:0 0,00 ±0,00 0,00 ±0,00

C22:6n3 9,88 ±0,07 6,22 ±0,09

C24:1n9 0,34 ±0,02 0,23 ±0,07

Sum saturated FA 15,66 ±0,22 17,21 ±0,30

Sum monounsaturated FA 48,12 ±0,01 54,18 ±0,22

Sum polyunsaturated FA 36,23 ±0,23 28,61 ±0,08 sum omega 3 26,11 ±1,31 19,05 ±0,13

Sum n-6 10,12 ±1,08 9,56 ±0,06

H ufa n-3 24,98 ±0,21 16,24 ±0,10 n-3/n-6 2,60 ±0,41 1,99 ±0,03

DHAVEPA 0,69 ±0,00 0,68 ±0,01 non Hufa n-3 1,13 ±1,10 2,81 ±0,03 Gammarus locusta solely fed a coconut based diet in experiment 2 had as much as 27.7 mg polyunsaturated fatty acids per g dry weight compared to about 19 mg/g dry weight in the treatment fed soya beans (Table 6, Figure 1A). The lipid content obtained was fairly similar for both treatments and in the range of what was achieved for G. oceanicus fed soya beans in experiment 1. Gammaridae belonging to the coconut treatment comprised 9.5 % of the total fatty acids as DHA, 15.2 % as EPA and 0.9 % ARA (Table 7, Figure 1B), while the diet did not include any of these fatty acids (Table 3). This corresponds to a DHA content of 7.4 mg/g and EPA of 11.7 mg/g and 0.72 mg ARA /g dry weight in the animals fed coconut fruits. Feeding of the soya bean diet resulted in 3.3 % DHA, 7.2 % EPA and 1.9 % ARA of the total fatty acids (Table 7), corresponding to 2.37 mg DHA per g dry weight, 6.04 mg EPA /g dry weight and 0.53 mg ARA /g dry weight.

Table 6: Content of lipids and fatty acids (mg/g dry weight; means ± standard deviation) in G. locusta fed soya beans (SB) and coconut (CO). FA = Fatty acids.

Gammarus locusta

CO SB

Total lipid 125.9 ± 0.48 136.4 ± 0.48

Fatty acids

C12:0 0,15 0,00 0,16 0,02

C14:0 2,78 0,01 5,33 0,48

C14:1n5 0,12 0,01 0,07 0,01

C16:0 11,37 0,07 13,24 0,57

C16:1n7 7,98 0,07 5,54 0,50

C18:0 0,64 0,02 0,56 0,00

C18:1n9 16,32 0,25 24,43 0,47

C18:1n7 3,51 0,01 2,08 0,05

C18:2n6 1 ,96 0,10 4,32 0,18

C18:3n6 0,21 0,01 0,10 0,00

C18:3n3 1 ,59 0,01 2,10 0,06

C18:4n3 3,00 0,04 1 ,42 0,03

C20:0 0,07 0,02 0,06 0,01

C20:1n9 3,63 0,03 1 ,10 0,01

C20:1n7 0,45 0,02 0,13 0,00

C20:2n6 0,18 0,01 1 ,29 0,01

C20:3n6 0,02 0,01 0,29 0,01

C20:4n6 0,72 0,02 0,53 0,01

C20:3n3 0,14 0,01 0,03 0,02

C20:4n3 0,48 0,01 0,26 0,00

C20:5n3 11 ,74 0,30 6,04 0,14

C22:0 0,04 0,00 0,00 0,00

C22:1n11 1 ,99 0,05 0,00 0,00

C22:1n9 0,33 0,04 0,00 0,00

C22:5n3 0,30 0,01 0,22 0,03

C24:0 0,00 0,00 0,00 0,00

C22:6n3 7,35 0,41 2,37 0,01

C24:1n9 0,14 0,02 0,00 0,01

Sum saturated FA 15,06 0,06 19,34 1 ,07

Sum monounsaturated FA 34,47 0,28 33,35 1 ,05

Sum polyunsaturated FA 27,69 0,84 18,97 0,26 sum omega 3 24,59 0,72 12,43 0,09

Sum n-6 3,09 0,12 6,53 0,17

H ufa n-3 19,87 0,70 8,89 0,10 n-3/n-6 7,95 0,09 1 ,90 0,04

DHA/EPA 0,63 0,02 0,39 0,01 non Hufa n-3 4,73 0,02 3,54 0,00 Table 7. Relative composition of fatty acids (% of total amount, means ± standard deviation) in G. locusta fed soya beans (SB) and coconut (CO).

Gammarus locusta

CO SB

Fatty acids

C12:0 0,20 ±0,00 0,22 ±0,02

C14:0 3,61 ±0,07 7,43 ±0,42

C14:1n5 0,16 ±0,01 0,10 ±0,02

C16:0 14,73 ±0,13 18,48 ±0,18

C16:1n7 10,34 ±0,06 7J2 ±0,44

C18:0 0,84 ±0,04 0,78 ±0,02

C18:1n9 21,14 ±0,00 34,11 ±0,48

C18:1n7 4,55 ±0,06 2,90 ±0,02

C18:2n6 2,54 ±0,09 6,03 ±0,06

C18:3n6 0,28 ±0,00 0,15 ±0,01

C18:3n3 2,06 ±0,05 2,93 ±0,18

C18:4n3 3,88 ±0,01 1,98 ±0,02

C20:0 0,08 ±0,02 0,08 ±0,01

C20:1n9 4,70 ±0,03 1,53 ±0,04

C20:1n7 0,58 ±0,02 0,18 ±0,00

C20:2n6 0,23 ±0,01 1,81 ±0,07

C20:3n6 0,03 ±0,01 0,40 ±0,03

C20:4n6 0,93 ±0,01 0,74 ±0,01

C20:3n3 0,18 ±0,01 0,04 ±0,03

C20:4n3 0,62 ±0,00 0,36 ±0,01

C20:5n3 15,21 ±0,15 8,44 ±0,09

C22:0 0,05 ±0,00 0,00 ±0,00

C22:1n11 2,58 ±0,11 0,00 ±0,00

C22:1n9 0,43 ±0,05 0,00 ±0,00

C22:5n3 0,38 ±0,02 0,31 ±0,05

C24:0 0,00 ±0,00 0,00 ±0,00

C22:6n3 9,52 ±0,38 3,30 ±0,14

C24:1n9 0,18 ±0,03 0,00 ±0,00

Sum saturated FA 19,50 ±0,22 26,99 ±0,60

Sum monounsaturated FA 44,64 ±0,32 46,54 ±0,08

Sum polyunsaturated FA 35,85 ±0,55 26,48 ±0,52 sum omega 3 31,85 ±0,45 17,35 ±0,46

Sum n-6 4,01 ±0,10 9,12 ±0,07

Hufa n-3 25,73 ±0,52 12,41 ±0,29 n-3/n-6 7,95 ±0,09 1,90 ±0,04

DHA/EPA 0,63 ±0,02 0,39 ±0,01 non Hufa n-3 6,12 ±0,07 4,94 ±0,17 The results obtained demonstrate that a pure vegetable diet (i.e. a non-marine lipid material) based on vegetable seeds of soya or on coconut was surprisingly suitable as a feed for Gammaridae. This surprising effect was demonstrated for the first time in the present invention and was exemplified with the two marine species Gammarus oceanicus and Gammarus locusta. Feeding of these diets surprisingly resulted in a fatty acid profile typical for marine species fed diets comprising lipid of marine origin with a high content of omgea-3 fatty acids with 4 or more double bonds as it is the case for the salmon filet.

Close to 90 % of the total fatty acids in coconut oil extracted from the fruit of coconut are saturated fatty acids. The amount of monounsaturated fatty acids is about 10 %, while the unsaturated fatty acids are very low with about 2.4% (C18:2). Furthermore, no polyunsaturated fatty acids with more than 18 carbon atoms are detectable in this diet. Thus, Gammaridae are surprisingly able to synthesize polyunsaturated fatty acids even though when only fed a diet which does not comprise polyunsaturated fatty acids of any significant amount. Furthermore, the results also surprisingly demonstrate that Gammaridae are able to synthesize omega-3 fatty acids when they are feed a diet which almost does not include any omega-6 fatty acids such as C18:2 n-6 as was the case for the coconut diet where the amount of C18:2 was as low as 2.4% of the total fatty acid.

By using a non-marine lipid material such as soya beans or coconut as feed it was possible to produce Gammaridae as well as a lipid fraction derived from Gammaridae comprising omega-3 fatty acids e.g. DHA, EPA as well as the omega-6 fatty acid ARA. The lipid product obtainable from Gammaridae produced according to the invention can be used as a feed ingredient for aquaculture purposes such as for salmon farming. It can also be used as a nutrient supplement, health product or pharmaceutical composition for humans or animals. Thus, the produced lipids according to the present invention can be used as a replacement for lipids from other known marine sources such as farmed or wild caught fish, krill, copepods, marine microalgae etc.

Thus, not only a new feed has been invented in the present invention but also a new method for producing a marine amphipod belonging to the family of Gammaridae comprising the omega-3 fatty acids DHA and EPA and ARA while being fed a diet which was substantially free of omega-3 fatty acids and ARA. The surprisingly achieved results according to the invention also allow a new in vivo production method for highly unsaturated omega-3 and omega-6 fatty acids such as DHA; EPA and ARA by use of species of Gammaridae fed a diet not comprising any of these fatty acids such as a vegetable or vegetable seed based diet.

The exact mechanism for the production of the omega-3 fatty acids in Gammaridae is not clear yet, although it is seems like that the animals have a surprising ability and capacity to synthesize the highly unsaturated fatty acids DHA ₁ EPA and ARA without being fed any of these fatty acids. It seems like the organisms can make use of a diet comprising only typical terrestrial fatty acids found in some vegetable seeds as in soya beans, fruits or vegetables to produce these long chain omega-3 and long chain omega-6 fatty acids. Moreover, the results obtained when using the coconut based diet show that Gammaridae are surprisingly able to synthesize marine polyunsaturated fatty acids de novo. Lipids comprised in the coconut diet do mainly consist of saturated fatty acids. Oleic acid is found as a monounsaturated fatty acid but only in an amount of 10 % of the total fatty acid (see Table 3). The only polyunsaturated fatty acid present in coconut is linoleic acid. However, the content of linoleic acid is reposted as being as low as only as 2.4 % (Table 3) of the total fatty acids. Wilson et al (2005) even reported only 1.8 % as linoleic acid in coconut oil. This amount can be considered as insignificant from a biomass balance view.

It will be appreciated that the features of the invention described in the foregoing can be modified without departing from the scope of the invention.

References:

Bligh, E. G. and Dyer, W. J. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911 - 917.

Metcalfe, L. D., Schimtz, A. A. and Pelka, J. R. 1966. Rapid preparation of fatty acids esters from lipids for gas chromatography. Anal. Chem. 38, 514 - 515.

Nagaraju, A., and Belur, L. R. 2008. Rats fed blended oils containing coconut oil with groundnut oil or olive oil showed an enhanced activity of hepatic antioxidant enzymes and a reduction in LDL oxidation. Food Chemistry 108 (2008) 950-957. Rainuzzo, J. R., Reitan, K. I. and Jørgensen, L. 1992. Comparative study on the fatty acid and lipid composition of four marine fish larvae. Comp. Biochem. Physiol. 103B, 21 - 26.

Wilson, T. A., Nicolosi, R. J., Kotyla, T., Sundram, K., and Kritchevsky, D. 2005. Different palm oil preparations reduce plasma cholesterol concentrations and aortic cholesterol accumulation compared to coconut oil in hypercholesterolemic hamsters. Journal of Nutritional Biochemistry 16 (2005) 633-640.

Definitions of terms and abbreviations:

ARA: Arachidonic acid (20:4n-6)

DHA: Docosahexaenoic acid (22:6n-3)

EPA: Eicosapentaenoic acid (20:5n-3)

FA: Fatty acids

Gammaridae: Amphipods belonging to the family of Gammaridae including species such as Gammarus oceanicus, Gammarus lawrencianus, Gammarus locusta, Gammarus lacustris, Gammarus duebeni, Gammarus obstusatus, Gammarus finnmarchicus, Gammarus stoerensis, Gammarus tigrinus, Gammarus setosus, Gammarus wilkitzkii, Gammarus sensu, or Gammarus salinus.

HUFA: Highly unsaturated fatty acids such as DHA, EPA and ARA

MUFA: Monounsaturated fatty acids

NFAL: Lipids that are not fatty acids

Polyunsaturated fatty acids: Fatty acid having more than one double bond in the molecule. Production: The term production refers to the intensive cultivation of

Gammaridae e.g. in aquaculture cultivation.

SFA: Saturated fatty acids

Marine lipid material: Lipid material comprising highly unsaturated fatty acids especially chosen from the group of DHA, EPA and ARA.

Non-marine lipid material: Lipid material not comprising the highly unsaturated fatty acids DHA, EPA and ARA. Depending on the source the material may comprise to a very low degree other highly unsaturated fatty acids such as C22:5n3.