111111
`
`1111111111111111111111111111111111111111111111111111111111111111111111111111
`US 20100143571Al
`
`(19) United States
`c12) Patent Application Publication
`Breivik
`
`(10) Pub. No.: US 2010/0143571 A1
`Jun. 10, 2010
`(43) Pub. Date:
`
`(54) PROCESS FOR PRODUCTION OF OMEGA-3
`RICH MARINE PHOSPHOLIPIDS FROM
`KRILL
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/859,289, filed on Nov.
`16,2006.
`
`(76)
`
`Inventor:
`
`Harald Breivik, Porsgrunn (NO)
`
`Publication Classification
`
`Correspondence Address:
`FINNEGAN, HENDERSON, FARABOW, GAR(cid:173)
`RETT & DUNNER
`LLP
`901 NEW YORK AVENUE, NW
`WASHINGTON, DC 20001-4413 (US)
`
`(21)
`
`Appl. No.:
`
`12/515,098
`
`(22)
`
`PCTFiled:
`
`Nov.15, 2007
`
`(86)
`
`PCTNo.:
`
`PCT/N007/00402
`
`§ 371 (c)(l),
`(2), ( 4) Date:
`
`Feb.17,2010
`
`(51)
`
`Int. Cl.
`(2006.01)
`A23L 11325
`(2006.01)
`A23K 1110
`(2006.01)
`A23K 1118
`(2006.01)
`CllB 1110
`(2006.01)
`C07C 45178
`(2006.01)
`COBB 37108
`(52) U.S. Cl. ........... 426/643; 426/417; 554/21; 568/366;
`536/20
`
`(57)
`
`ABSTRACT
`
`The present disclosure relates to a process for preparing a
`substantially total lipid fraction from fresh krill, a process for
`separating phospholipids from the other lipids, and a process
`for producing krill meal.
`
`RIMFROST EXHIBIT 1035 page 0001
`
`

`

`Patent Application Publication
`
`Jun. 10, 2010 Sheet 1 of 2
`
`US 2010/0143571 A1
`
`Fig. 1
`
`RIMFROST EXHIBIT 1035 page 0002
`
`

`

`Patent Application Publication
`
`Jun. 10, 2010 Sheet 2 of 2
`
`US 2010/0143571 A1
`
`Fig. 2
`
`RIMFROST EXHIBIT 1035 page 0003
`
`

`

`US 2010/0143571 AI
`
`Jun. 10,2010
`
`1
`
`PROCESS FOR PRODUCTION OF OMEGA-3
`RICH MARINE PHOSPHOLIPIDS FROM
`KRILL
`
`FIELD OF THE INVENTION
`
`[0001] The present invention relates to a process for pre(cid:173)
`paring a substantially total lipid fraction from fresh hill, and a
`process for separating phospholipids from the other lipids.
`The invention also relates to a process for production of high
`quality krill meal.
`
`BACKGROUND OF THE INVENTION
`
`[0002] Marine phospholipids are useful in medical prod(cid:173)
`ucts, health food and human nutrition, as well as in fish feed
`and means for increasing the rate of survival of fish larval and
`fry of marine species like cod, halibut and turbot.
`[0003] Phospholipids from marine organisms comprise
`omega-3 fatty acids. Omega-3 fatty acids bound to marine
`phospholipids are assumed to have particularly useful prop(cid:173)
`erties.
`[0004] Products such as fish milt and roe are traditional raw
`materials for marine phospholipids. However, these raw
`materials are available in limited volumes and the price of
`said raw materials is high.
`[0005] Krill are small, shrimp-like animals, containing
`relatively high concentrations of phospholipids. In the group
`Euphasiids, there is more than 80 species, of which the Ant(cid:173)
`arctic krill is one of these. The current greatest potential for
`commercial utilisation is the Antarctic Euphausia superba. E.
`superba has a length of 2-6 em. Another Antarctic krill spe(cid:173)
`cies is E. crystallorphias. Meganyctiphanes norvegica,
`Thysanoessa inermis and T. raschii are examples of northern
`krill.
`[0006] Fresh hill contains up to around 10% of lipids, of
`that approximately 50 of % phospholipids in Euphausia
`superba. Phospholipids from krill comprise a very high level
`of omega-3 fatty acids, whereof the content of eicosapen(cid:173)
`taenoic acid (EPA) anddocosahexaenoic acid (DHA) is above
`40%. The approximate composition of lipids from the two
`main species of Antarctic krill is given in Table 1.
`
`TABLE 1
`
`Composition of krill lipids. Lipid classes,
`(approximate sum EPA+ DHA)
`
`Ratio
`Wax esters Glycerides Phospholipids EPA/DHA
`
`50 (7)
`
`50 (40-45)
`
`1.4-1.5
`
`40
`
`20 (4)
`
`40 (30-33)
`
`1.3
`
`Euphausia
`superba
`Euphausia
`crystallorphias
`
`[0007] Furthermore, Antarctic krill has lower level of envi(cid:173)
`ronmental pollutants than traditional fish oils.
`[0008] The krill has a digestive system with enzymes,
`including lipases that are very active around oo C. The lipases
`stay active after the krill is dead, hydrolysing part of the krill
`lipids. An unwanted effect of this is that krill oil normally
`contains several percents offree fatty acids. If the krill has to
`be cut into smaller fragments before being processed, the
`person skilled in the art will immediately realise that this will
`increase the degree ofhydrolysis. Thus, it is a desire to find a
`process that can utilise whole, fresh krill, or whole body parts
`
`from krill, as such a process will provide a product with
`improved quality and low degree ofhydrolysis oflipids. This
`improved quality will affect all groups of krill lipids, includ(cid:173)
`ing phospholipids, triglycerides and astaxanthin esters.
`[0009] Krill lipids are to a large extent located in the ani(cid:173)
`mals' head. A process that can utilise fresh krill is therefore
`also well suited for immediate processing of the by-products
`from krill wherefrom the head is peeled off, a product that can
`be produced onboard the fishing vessel.
`[0010] From U.S. Pat. No. 6,800,299 ofBeaudionetal. it is
`disclosed a method for extracting total lipid fractions from
`krill by successive extraction at low temperatures using
`organic solvents like acetone and ethanol. This process
`involves extraction with large amounts of organic solvents
`which is unfavourable.
`[0011] K. Yamaguchi eta!. (J. Agric. Food Chern. 1986 34,
`904-907) showed that supercritical fluid extraction with car(cid:173)
`bon dioxide, which is the most common solvent for super(cid:173)
`critical fluid extraction, of freeze dried Antarctic krill resulted
`in a product mainly consisting ofunpolar lipids (mostly trig(cid:173)
`lycerides), and no phospholipids. Yamaguchi eta!. reported
`that oil in krill meal was deteriorated by oxidation or poly(cid:173)
`merisation to such an extent that only limited extraction
`occurred with supercritical C02 . Y. Tanaka and T. Ohkubo (J.
`Oleo. Sci. (2003), 52, 295-301) quotes the work ofYamaguci
`eta!. in relation to their own work on extraction oflipids from
`salmon roe. In a more recent publication (Y. Tanaka et a!.
`(2004), J. Oleo. Sci., 53, 417-424) the same authors try to
`solve this problem by using a mixture of ethanol and C02 for
`extracting the phospholipids. By using C02 with 5% ethanol
`no phospholipids were removed from freeze dried salmon
`roe, while by adding 10% ethanol, 30% of the phospholipids
`were removed, and by adding as much as 30% ethanol, more
`than 80% of the phospholipids were removed. Freeze drying
`is a costly and energy consuming process, and not suited for
`treatment of the very large volumes of raw materials that will
`become available by commercial krill fisheries.
`[0012] Tanaka eta!. tried to optimise the process by varying
`the temperature of the extraction, and found that low tempera(cid:173)
`tures gave the best results. 33 o C., a temperature just above the
`critical temperature for C02 , was chosen as giving best
`results.
`[0013] Contrary to these findings, we have surprisingly
`found a process for extraction of a substantially total lipid
`fraction from fresh krill, without the need for complicated and
`costly pre-treatment like freeze drying oflarge volumes. The
`lipid fraction contained triglycerides, astaxanthin and phos(cid:173)
`pholipids. We did not have to dry or deoil the raw material
`before processing. Contrary to Tanaka et a!. we have found
`that a short heating of the marine raw material was positive for
`the extraction yield. It was also shown that pre-treatment like
`a short-time heating to moderate temperatures, or contact
`with a solid drying agent like molecular sieve, of the krill can
`make ethanol wash alone efficient in removing phospholipids
`from fresh krill.
`
`SUMMARY OF THE INVENTION
`
`[0014]
`It is a main object of the present invention to provide
`a process for preparing a substantially total lipid fraction from
`fresh krill without using organic solvents like acetone.
`[0015] The exposure to the fluid under supercritical pres(cid:173)
`sure will prevent oxidation from taking place, and the com(cid:173)
`bined carbon dioxide/ethanol is expected to deactivate any
`enzymatic hydrolysis of the krill lipids. As the process
`
`RIMFROST EXHIBIT 1035 page 0004
`
`

`

`US 2010/0143571 AI
`
`Jun. 10,2010
`
`2
`
`according to the invention requires a minimum ofhandling of
`the raw materials, and is well suited to be used on fresh hill,
`for example onboard the fishing vessel, the product according
`to the invention is expected to contain substantially less
`hydrolysed and/or oxidised lipids than lipid produced by
`conventional processes. This also means that there is
`expected to be less deterioration of the krill lipid antioxidants
`than from conventional processing. The optional pre-treat(cid:173)
`ment involving short-time heating of the fresh krill will also
`give an inactivation of enzymatic decomposition of the lipids,
`thus ensuring a product with very low levels of free fatty
`acids.
`[0016] Another object of the present invention is to provide
`a process for preparing a substantially total lipid fraction from
`other marine raw materials like fish gonads, Calanus species,
`or high quality krill meal.
`[0017] Another object of the present invention is to provide
`a substantially total lipid fraction high in long chain polyun(cid:173)
`saturated omega-3 fatty acids.
`[0018] These and other objects are obtained by the process
`and lipid fraction as defined in the accompanying claims.
`[0019] According to the invention it is provided a process
`for extracting a substantially total lipid fraction from fresh
`krill, comprising the steps of:
`a) reducing the water content of krill raw material; and
`b) isolating the lipid fraction.
`[0020] Optionally, the above-mentioned process compris(cid:173)
`ing a further step of:
`a-1) extracting the water reduced krill material from step a)
`with C02 at supercritical pressure containing ethanol, metha(cid:173)
`nol, propanol or iso-propanol. This step, a-1 ), is performed
`directly after step a).
`[0021]
`In a preferred embodiment of the invention it is
`provided a process for extracting a substantially total lipid
`fraction from fresh krill, comprising the steps of:
`a) reducing the water content of krill raw material;
`a-1) extracting the water reduced krill material from step a)
`with C02 containing ethanol, the extraction taking place at
`supercritical pressure; and
`b) isolating the lipid fraction from the ethanol.
`[0022]
`In a preferred embodiment of the invention, step a)
`comprises washing of the krill raw material with ethanol,
`methanol, propanol and/or iso-propanol in a weight ratio
`1:0.5 to 1:5. Preferably, the krill raw material is heated to
`60-100° C., more preferred to 70-100° C., and most preferred
`to 80-95° C., before washing. Furthermore, the krill raw
`material is preferably heated for about 1 to 40 minutes, more
`preferred about 1 to 15 minutes, and most preferred for about
`1 to 5 minutes, before washing.
`[0023]
`In another preferred embodiment of the invention,
`step a) comprises bringing the krill raw material in contact
`with molecular sieve or another form of membrane, such as a
`water absorbing membrane, for removal of water.
`[0024] Preferably, the amount of ethanol, methanol, pro(cid:173)
`panol and/or iso-propanol in step a-1) is 5-20% by weight,
`more preferably 10-15% by weight.
`[0025]
`In addition to producing a product containing the
`total lipids of krill, the invention also can be used for sepa(cid:173)
`rating phospholipids from the other lipids. To separate the
`total lipids obtained by extraction at supercritical pressure,
`according to the present invention into the different lipid
`classes, extraction of the said total lipids with pure carbon
`dioxide can remove the non-polar lipids from the omega-3
`
`rich phospholipids. Extraction of the total lipids with carbon
`dioxide containing less than 5% ethanol or methanol is
`another option.
`[0026] As the phospholipids are much richer in the valuable
`omega-3 fatty acids than the other lipid classes, this makes the
`invention useful for producing high concentrates of omega-3
`fatty acids. While commercially available fish oils contain
`11-33% total omega-3 fatty acids (Hjaltason, Band Haralds(cid:173)
`son, G G (2006) Fish oils and lipids from marine sources, In:
`ModifYing Lipids for Use in Food (FD Gunstone, ed), Wood(cid:173)
`head Publishing Ltd, Cambridge, pp. 56-79), the phospholip(cid:173)
`ids of krill contain much higher levels (Ellingsen, T E (1982)
`Biokjemiske studier over antarktisk krill, PhD thesis, Norges
`tekniske hoyskole, Trondheim. English summary in Publica(cid:173)
`tion no. 52 of the Norwegian Antarctic Research Expeditions
`(1976/77 and 1978/79)), see also Table 1. The omega-3 rich
`phospholipids can be used as they are, giving the various
`positive biological effects that are attributed to omega-3 con(cid:173)
`taining phospholipids. Alternatively, the phospholipids can
`be transesterified or hydrolysed in order to give esters (typi(cid:173)
`cally ethyl esters) or free fatty acids or other derivatives that
`are suitable for further concentration of the omega-3 fatty
`acids.As examples, the ethyl esters ofkrill phospholipids will
`be valuable as an intermediate product for producing concen(cid:173)
`trates that comply with the European Pharmacopoeia mono(cid:173)
`graphs no. 1250 (Omega-3-acid ethyl ester 90), 2062
`(Omega-3-acid ethyl esters 60) and 1352 (Omega-3-acid trig(cid:173)
`lycerides). At the same time, the remaining lipids (astaxan(cid:173)
`thin, antioxidants, triglycerides, wax esters) can be used as
`they are for various applications, including feed in aquacul(cid:173)
`ture, or the lipid classes can be further separated.
`[0027] Thus, still another object of the present invention is
`to provide a process for separating phospholipids from the
`other lipids as described above.
`[0028] Another object of the invention is to produce a high
`quality krill meal. As the lipids are removed at an initial step
`of the process, the meal will be substantially free of oxidised
`and polymerised lipids. This will make the meal very well
`suited for applications where it is important to avoid oxidative
`stress, i.e. for use in aquaculture feed, especially starting feed
`for marine fish species. The krill meal of the present invention
`is thus well suited for feeding fish larvae and fry, as well as
`fish and crustaceans. Furthermore, the krill meal of the inven(cid:173)
`tion may be used as a source for production of high quality
`chi to san.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0029] The process can be performed with a wide variety of
`processing conditions, some of which are exemplified below.
`[0030]
`In the following "fresh" krill is defined as krill that is
`treated immediately after harvesting, or sufficiently short
`time after harvesting to avoid quality deterioration like
`hydrolysis or oxidation of lipids, or krill that is frozen imme(cid:173)
`diately after harvesting. Fresh krill can be the whole krill, or
`by-products from fresh krill (i.e. after peeling). Fresh krill can
`also be hill, or by-products from krill, that have been frozen
`shortly after harvesting.
`[0031] Moreover "krill" also includes krill meal.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`[0032] FIG. 1 shows a picture of E. superba used as raw
`material for extraction.
`
`RIMFROST EXHIBIT 1035 page 0005
`
`

`

`US 2010/0143571 AI
`
`Jun. 10,2010
`
`3
`
`[0033] FIG. 2 shows the material after extraction as
`described in Example 7 below.
`
`EXAMPLES
`
`Example 1
`
`Processing of Freeze Dried Krill
`
`[0034] Freeze dried krill was extracted with C02 at super(cid:173)
`critical pressure. This gave a product of 90 g/kg. Analysis
`showed that the extract contained a sum of EPA plus DHA of
`only 5.4%, showing that this did not contain a significant
`amount of the omega-3 rich phospholipids. A second extrac(cid:173)
`tion with C02 containing 10% ethanol resulted in an extract of
`100 g/kg (calculated from starting sample weight). 31 P NMR
`showed that the product contained phospholipids. The extract
`contained a sum of EPA plus DHA of33.5%.
`[0035]
`In both steps the extraction conditions were 300 bar,
`50° C.
`[0036] Thus, it is possible substantially to separate the
`omega-3 rich phospholipids from the less omega-3 rich com(cid:173)
`ponents of the krill lipids.
`[0037]
`In a second experiment the freeze dried krill was
`extracted twice with the same pressure and temperature as
`above, first with 167 parts (weight) of pure C02 , and then with
`167 part (weight) of C02 containing 10% ethanol. The com(cid:173)
`bined extract (280 g/kg raw material) was analysed by 13C
`and 31P NMR. The analyses showed that the product con(cid:173)
`tained triglycerides and phospholipids as major components.
`Like the previous extracts the dark red colour showed that the
`extract contained astaxanthin.
`[0038] We are not aware that a process according to
`Example 1 has been used for freeze dried hill. It could be
`argued that this could be anticipated from Y. Tanaka et a!.
`(2004) J. Oleo Sci. 53, 417-424. However, in this prior art
`C02 with 10% ethanol resulted in only 30% of the phospho(cid:173)
`lipids being extracted. 20% ethanol had to be used in order to
`extract 80% of the phospholipids.
`
`Examples According to the Invention
`
`Example 2
`
`[0039] Fresh E. superba (200 g) was washed with ethanol
`(1:1, 200 g) at around oo C. The ethanol extract (1.5%) con(cid:173)
`tained inorganic salts (mainly NaCl) and some organic mate(cid:173)
`rial.
`[0040] The ethanol washed krill was extracted with C02
`containing 10% ethanol. This gave an extract of 12 g (6%
`based on starting krill). Analysis (TLC and NMR) showed
`that the extract contained phospholipids, triglycerides and
`astaxanthin.
`[0041] The person skilled in the art will realise that carbon
`dioxide at supercritical pressure can act as a solvent for etha(cid:173)
`nol. Thus, an alternative procedure for modifying the solvent
`power of the C02 is to utilise pressure/temperature conditions
`so that ethanol is dissolve directly from the ethanol containing
`krill raw material, without having to be added by a pre(cid:173)
`treatment of the C02 . This also applies for the examples
`below.
`
`Example 3
`
`[0042] Fresh E. superba (200 g) was washed with ethanol
`(1:3, 600 g) at around oo C. The ethanol extract (7.2%) con(cid:173)
`tained phospholipids, triglycerides and astaxanthin, and
`
`some inorganic salts. The extract contained 26.3% (EPA+
`DHA), showing that the relative content of phospholipids was
`high.
`[0043] The ethanol washed krill was extracted with C02
`containing 10% ethanol. This gave an extract of 2.2% based
`on starting krill. Analysis (TLC and NMR) showed that the
`extract contained phospholipids, triglycerides and astaxan(cid:173)
`thin. However, as the extract contained only 8.1% (EPA+
`DHA) it was concluded that the phospholipids content was
`low.
`
`Example 4
`
`[0044] Fresh E. superba was treated with the same two-step
`process as above, except that the ethanol amount in the wash(cid:173)
`ing step was increased to 4:1. The ethanol extract was 7.2%
`compared to the starting material, while the supercritical fluid
`extract was 2.6%.
`
`Example 5
`
`[0045] Fresh E. superba (200 g) was put in contact with
`molecular sieve (A3, 280 g) in order to remove water from the
`krill raw material. Extraction with C02 containing 10% etha(cid:173)
`nol gave an extract of 5.2% calculated from the starting
`weight of krill. Analyses showed that the extract contained
`triglycerides, phospholipids and astaxanthin. The extracted
`whole krill was completely white, except for the black eyes.
`[0046] Example 5 shows the effect of removing water.
`Molecular sieve was chosen as an alternative to ethanol.
`These examples are not intended to be limiting with regard to
`potential agents for removal of water. Molecular sieve and
`other drying agents can be mild and cost effective alternatives
`to freeze drying.
`
`Example 6
`
`[0047] Fresh E. superba (200 g) was washed with ethanol
`(1: 1) as in example 2, but with the difference that the raw
`material had been pre-treated at 80° C. for 5 minutes. This
`gave an ethanol extract of7 .3%. Supercritical fluid extraction
`with C02 containing 10% ethanol gave an additional extract
`of 2.6% calculated from the fresh raw material. The total
`extract was 9.9%, and analyses (TLC, NMR) showed that the
`extract was rich in phospholipids, and also contained triglyc(cid:173)
`erides and astaxanthin. The remaining, whole krill was com(cid:173)
`pletely white, except for the black eyes.
`
`Example 7
`
`[0048] Fresh E. superba (12 kg) was heated to 80° C. for a
`few minutes and thereafter extracted with ethanol (26 kg).
`This gave an ethanol extract of0.82 kg (7% ). Analysis oflipid
`classes (HPLC; Colunm: Alltima HP silica 3 f.tm; detector:
`DEDL Sedere; Solvents: Chloroform/methanol) showed a
`content of 58% phospholipids. Analysis by GC (area %)
`showed a content of 24.0% EPA and 11.4% DHA, sum EPA+
`DHA=35.4%.
`[0049] The remaining krill was extracted at 280 bar and 50°
`C. with C02 (156kg) containing ethanol (15 kg). This gave an
`extract of0.24 kg (2% ). The remaining krill was white, except
`for the dark eyes. Analysis oflipid classes showed a content of
`19% phospholipids. The extract contained 8.9% EPA and
`4.8% DHA (sum 13.7%). Extraction of the remaining krill
`material (Folch method) showed a content of only 0.08 kg
`
`RIMFROST EXHIBIT 1035 page 0006
`
`

`

`US 2010/0143571 AI
`
`Jun. 10,2010
`
`4
`
`lipids (0.7% compared to initial krill weight). This means that
`substantially all lipids had been extracted.
`
`Example 8
`
`[0050] Fresh E. superba (12 kg) was extracted with ethanol
`(33 kg) without heat treatment. This gave an extract of0.29 kg
`(2.4% ). Analysis oflipid classes as above showed a content of
`28.5% phospholipids.
`[0051] The results show that heat-treatment gives an
`increased yield oflipids compared to the same treatment with
`no heating. After heat-treatment of the raw material, one part
`(weight) of ethanol gave the same result as four parts of
`ethanol without heat treatment. Also, heating gave an ethanol
`extract that was more rich in phospholipids and omega-3 fatty
`acids than when the ethanol treatment was performed without
`heating.
`[0052] The heating times in the examples should not be
`limiting for the invention. The person known in the art will
`realise that exact heating times are difficult to monitor for
`large volumes of biological material. Thus, the heating time
`may vary depending of the amount of krill that is to be
`processed at a specific time. Also, the temperature used for
`pre-heating is not limited to the temperature given in the
`examples. Experiments showed that pre-heating to 95° C.
`tended to increase the yield of lipids in step a) even higher
`than pre-heating to 80° C. Also, for large volumes of krill it
`may be difficult to obtain exactly the same temperature in all
`the krill material.
`[0053] The heat treatment gives as additional result that the
`highly active krill digestive enzymes are inactivated, reducing
`the potential lipid hydrolysis.
`
`Example 9
`
`[0054] FIG. 1 shows a picture of E. superba used as raw
`material for extraction. FIG. 2 shows the material after extrac(cid:173)
`tion as described in Example 7. The other examples gave very
`similar material after extraction. The extracted krill is dry, and
`can easily be made into a powder, even manually by pressing
`between the fingers. The de-fatted powder contains proteins
`as well as chitosan and other non-lipid components from the
`krill. The powders smell similar to dry cod. As this powder is
`substantially free of lipids, it will give a meal substantially
`without oxidised polyunsaturated fatty acids. This is very
`different from krill meal produced according to traditional
`processes, where substantially all of the phospholipid frac(cid:173)
`tion will be remain in the meal, giving rise to oxidised and
`polymerised material. Krill meal produced according to the
`present process will thus give much reduced oxidative stress
`compared to traditional krill meal or fish meal when used in
`feed for aquaculture. The krill meal will also be very suitable
`in feed for crustaceans, including lobster, and for feeding
`wild-caught King Crabs (Paralithodes camtschatica) in order
`to increase the quality and volume of the crab meat. As the
`meal is substantially free of polymerised lipids, it will also be
`beneficial for production of high quality chi to san, and for
`other processed where a high quality meal is needed.
`[0055] Because the krill lipids oxidises very rapidly, and
`become less soluble in common solvents, the person skilled in
`the art will realise that a similar high quality krill meal could
`not be obtained by de-fatting of traditional krill meal, for
`example by use of organic solvents.
`[0056] The person skilled in the art will realise that the
`processes described above also can be used for other raw
`
`materials than krill, for example the isolation of omega-3 rich
`phospholipids from fish gonads, or from Calanus species.
`Some krill species are rich in wax esters (example: E. crys(cid:173)
`tallorphias), and the same will be the case for Calanus spe(cid:173)
`cies. The person skilled in the art will realise that by process(cid:173)
`ing as described above, the wax esters will be concentrated in
`the unpolar lipid fractions.
`[0057] Furthermore, the person skilled in the art will realise
`that combination of process steps as given above can be used
`for separating the polar (i.e. phospholipids) and unpolar lipids
`of krill. It will also be possible to make an extract of the total
`lipids of krill according to one of the examples above, and
`then make a second extraction of this intermediary product in
`order to separate the lipid classes. For example, an extraction
`with pure carbon dioxide would remove the non-polar lipids
`from the omega-3 rich phospholipids.
`[0058]
`In another embodiment, the process according to the
`invention is used to extract krill meal, wherein provided the
`krill meal has been produced in a sufficiently mild way to
`avoid deterioration of the krill lipids.
`[0059] The person skilled in the art will also realise that a
`process as described above can be used to extract other marine
`raw materials like fish gonads and Calanus species.
`[0060] A lipid fraction, or lipid product, derived from the
`process according to the invention may have some additional
`advantages related to quality compared to known hill oil
`products (produced by conventional processes), such as for
`instance a krill oil from Neptune Biotechnologies & Biore(cid:173)
`sources extracted from a Japanese krill source (species not
`specified) with the following composition:
`
`Total Phospholipids
`Esterified astaxanthin
`Vitamin A
`VitaminE
`VitaminD
`Total Omega-3
`EPA
`DHA
`
`"':40.0%
`"':1.0 mg/g
`"':1.0 IU/g
`"':0.005 IU/g
`"':0.1 IU/g
`"':30.0%
`"':15.0%
`~9.0%
`
`[0061] A lipid product or fraction according to the inven(cid:173)
`tion is expected to;
`[0062]
`contain substantially less hydrolysed and/or oxi(cid:173)
`dised lipids than lipid produced by conventional pro(cid:173)
`cesses,
`[0063] be less deterioration of the krill lipid antioxidants
`than from conventional processing,
`[0064]
`contain very low levels offree fatty acids, and/or
`[0065] be substantially free from trace of organic sol-
`vents.
`[0066] By "oxidised" lipids is meant both primary oxida(cid:173)
`tion products (typically measured as peroxide value), second(cid:173)
`ary oxidation products (typically carbonyl products, often
`analysed as anisidine value) and tertiary oxidation products
`(oligomers and polymers).
`[0067] Thus, the invention includes commercial lipid or
`krill oil products produced by one of the processes according
`to the invention.
`[0068] Products like, for instance, the dietary supplement,
`Superba ™ (Aker BioMarine, Norway), might be produced by
`a process according to the present invention.
`[0069] The person skilled in the art will realise that the
`quality of a product produced by a process of the present
`
`RIMFROST EXHIBIT 1035 page 0007
`
`

`

`US 2010/0143571 AI
`
`Jun. 10,2010
`
`5
`
`invention will be improved compared to a product produced
`by traditional extraction of krill meal.
`lipid compositions
`[0070] Moreover, examples of a
`obtained by the process according to the invention are pre(cid:173)
`sented in the tables below, and also included herein.
`
`TABLE2
`
`Lipid composition
`
`Phospholipids
`EPA
`DHA
`
`> 30-40% by weight
`>5-15% by weight
`>5-15% by weight
`
`[0071] According to the invention, the extract can be con(cid:173)
`centrated with respect to the content of phospholipids. Some
`typical lipid compositions are illustrated by table 3-5, and
`included herein:
`
`TABLE3
`
`Lipid composition
`
`Phospholipids
`EPA
`DHA
`
`"':50% by weight
`~15%
`~10%
`
`[0072] As can be seen from Example 7, a lipid composition
`as described in Table 3 can also be obtained by only applying
`extraction according to step a) of the invention.
`
`TABLE4
`
`Lipid composition
`
`Phospholipids
`EPA
`DHA
`
`"':80% by weight
`"':20%
`"':13%
`
`TABLES
`
`Lipid composition
`
`Phospholipids
`EPA
`DHA
`
`"':90% by weight
`~23%
`~15%
`
`[0073] The invention shall not be limited to the shown
`embodiments and examples.
`1. A process for extracting a substantially total lipid frac(cid:173)
`tion from fresh krill, comprising the steps of:
`a) reducing the water content ofkrill raw material by wash(cid:173)
`ing with at least one alcohol chosen from ethanol,
`methanol, propanol, and iso-propanol in a weight ratio
`of krill raw material:at least one alcohol ranging from
`1:0.5 to 1:5; and
`b) isolating the lipid fraction from the at least one alcohol.
`2. (canceled)
`3. The process of claim 1, wherein at least one alcohol is
`ethanol.
`4. The process of claim 1, further comprising a step:
`a-1) extracting the water-reducedkrill material from step a)
`with C02 at supercritical pressure comprising at least
`one alcohol chosen from ethanol, methanol, propanol
`and iso-propanol;
`wherein step a-1) occurs immediately after step a).
`
`5. The process of claim 1, wherein the krill raw material is
`heated at a temperature ranging from 60-1 00° C. before wash(cid:173)
`in g.
`6. The process of claim 5, wherein the krill raw material is
`heated at a temperature ranging from 70-100° C. before wash(cid:173)
`ing.
`7. The process of claim 6, wherein the krill raw material is
`heated at a temperature ranging from 80-95° C. before wash(cid:173)
`ing.
`8. The process of claim 5, wherein the krill raw material is
`heated for about 1 to 40 minutes before washing.
`9. The process of claim 8, wherein the krill raw material is
`heated for about 1 to 15 minutes before washing.
`10. The process of claim 8, wherein the krill raw material is
`heated for about 1 to 5 minutes before washing.
`11. (canceled)
`12. (canceled)
`13. The process of claim 4, wherein the amount of the at
`least one alcohol in step a-1) is 5-20% by weight.
`14. The process of claim 13, wherein the amount of alcohol
`in step a-1) is 10-15% by weight.
`15. A substantially total lipid fraction according to claim 1,
`wherein the lipid fraction comprises at least one oftriglycer(cid:173)
`ides, astaxanthin, and phospholipids, and is substantially free
`from oxidized lipids.
`16. (canceled)
`17. A medicament or food supplement comprising the sub(cid:173)
`stantially total lipid fraction according to claim 15.
`18. A process for separating phospholipids from other lip(cid:173)
`ids, comprising extracting the total lipid fraction obtained by
`the process of claim 1 with pure carbon dioxide, or carbon
`dioxide comprising less than 5% alcohol chosen from etha(cid:173)
`nol, methanol, propanol and iso-propanol.
`19. The phospholipids fraction obtainable by the process of
`claim 18.
`20. The phospholipids fraction of claim 19, wherein the
`phospholipids are further transesterified or hydrolysed.
`21. The phospholipids fraction of claim 19, wherein the
`concentration of omega-3 fatty acids is at least 40% by
`weight.
`22. A process for producing krill meal, comprising extract(cid:173)
`ing a substantially total lipid fraction according to the process
`of; claim 1, and isolating the remaining krill raw material.
`23. A krill meal substantially free of oxidised polyunsatu(cid:173)
`rated fatty acids and other lipids according to claim 22.
`24. An animal feed comprising the meal of claim 23.
`25. An aquaculture feed comprising the krill meal of claim
`23.
`26. The aquaculture feed of claim 25, suitable for feeding at
`least one marine