(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`I lllll llllllll II llllll lllll lllll lllll llll I II Ill lllll lllll lllll 111111111111111111111111111111111
`
`( 43) International Publication Date
`22 May 2008 (22.05.2008)
`
`(51) International Patent Classification:
`CllB 1110 (2006.01)
`CllB 3114 (2006.01)
`A23K 1110 (2006.01)
`
`PCT
`
`(10) International Publication Number
`WO 2008/060163 Al
`(74) Agent: LILLEGRAVEN, Rita; Zacco Norway AS, P.O.
`Box 2003, N-0125 Oslo (NO).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH,
`CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG,
`ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL,
`IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK,
`LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW,
`MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL,
`PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY,
`TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA,
`ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL, PL,
`PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`with international search report
`
`(21) International Application Number:
`PCT/N02007 /000402
`
`(22) International Filing Date:
`15 November 2007 ( 15 .11.2007)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/859,289
`
`16 November 2006 (16.11.2006) US
`
`(71) Applicant
`(for all designated States except US):
`PRONOVA BIOPHARMA NORGE AS [NO/NO];
`Lysaker Torg 8, N-1327 Lysaker (NO).
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): BREIVIK, Harald
`[NO/NO]; Uranusveien 22, N-3942 Porsgrunn (NO).
`
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`Q
`M (54) Title: PROCESS FOR PRODUCTION OF OMEGA-3 RICH MARINE PHOSPHOLIPIDS FROM KRILL
`~ (57) Abstract: The present invention 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 1037 page 0000
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`WO 2008/060163
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`1
`
`PROCESS FOR PRODUCTION OF OMEGA-3 RICH MARINE
`PHOSPHOLIPIDS FROM KRILL
`
`5
`
`Field of the invention
`
`The present invention relates to a process for preparing a substantially total lipid
`:fraction from fresh krill, and a process for separating phospholipids from the other
`lipids. The invention also relates to a process for production of high quality krill meal.
`
`10
`
`Background of the invention
`
`15
`
`Marine phospholipids are useful in medical products, 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.
`
`Phospholipids from marine organisms comprise omega-3 fatty acids. Omega-3 fatty
`acids bound to marine phospholipids are assumed to have particularly useful properties.
`
`20
`
`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.
`
`25 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
`Antarctic 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 cm. Another
`Antarctic krill species is E. crystallorphias. Meganyctiphanes norvegica, Thysanoessa
`inermis and T. raschii are examples of northern krill.
`
`30
`
`Fresh krill 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 eicosapentaenoic acid (EPA) and
`docosahexaenoic acid (DHA) is above 40 %. The approximate composition of lipids
`from the two main species of Antarctic krill is given in Table 1.
`
`35
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`RIMFROST EXHIBIT 1037 page 0001
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`WO 2008/060163
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`2
`
`Table 1: Composition of krill lipids. Lipid classes, (ar, oroximate sum EPA + DHA)
`Wax esters
`Glycerides
`Phospholipids
`Ratio
`EPA/DHA
`1.4-1.5
`
`1
`
`50 (7)
`
`50 (40-45)
`
`Euphausia
`superba
`Euphausia
`crystallorphias
`
`40
`
`20 (4)
`
`40 (30-33)
`
`1.3
`
`Furthermore, Antarctic krill has lower level of environmental pollutants than traditional
`fish oils.
`
`The krill has a digestive system with enzymes, including lipases that are very active
`around 0 °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 of
`free 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 of
`hydrolysis. 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 of hydrolysis of lipids. This improved quality will affect all
`groups of krill lipids, including phospholipids, triglycerides and astaxanthin esters.·
`
`Krill lipids are to a large extent located in the animals' 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.
`
`From US Patent No. 6,800,299 of Beaudion et al. 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.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`K. Yamaguchi et al. (J Agric. Food Chem. 1986 34, 904-907) showed that
`supercritical fluid extraction with carbon dioxide, which is the most common solvent for
`supercritical fluid extraction, of freeze dried Antarctic krill resulted in a product mainly
`consisting ofunpolar lipids (mostly triglycerides), and no phospholipids. Yamaguchi et
`30 al. reported that oil in krill meal was deteriorated by oxidation or polymerisation to such
`an extent that only limited extraction occurred with supercritical C02•
`
`RIMFROST EXHIBIT 1037 page 0002
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`WO 2008/060163
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`3
`
`Y. Tanaka and T. Ohkubo (J. Oleo. Sci. (2003), 52, 295-301) quotes the work of
`Y amaguci et al. in relation to their own work on extraction of lipids from salmon roe. In
`a more recent publication (Y. Tanaka et al. (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.
`
`5
`
`10
`
`Tanaka et al. tried to optimise the process by varying the temperature of the extraction,
`and found that low temperatures gave the best results. 33°C, a temperature just above
`the critical temperature for C02, was chosen as giving best results.
`
`15
`
`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 of large volumes. The lipid fraction contained
`triglycerides, astaxanthin and phospholipids. We did not have to dry or deoil the raw
`material before processing. Contrary to Tanaka et al. 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.
`
`20
`
`25
`
`Summary of the invention
`
`30
`
`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.
`
`35
`
`The exposure to the fluid under supercritical pressure will prevent oxidation from taking
`place, and the combined carbon dioxide/ethanol is expected to deactivate any enzymatic
`hydrolysis of the krill lipids. As the process according to the invention requires a
`minimum of handling of the raw materials, and is well suited to be used on fresh krill,
`for example onboard the fishing vessel, the product according to the invention is
`
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`4
`
`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-treatment 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.
`
`5
`
`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,
`10 Ca/anus species, or high quality krill meal.
`
`Another object of the present invention is to provide a substantially total lipid fraction
`high in long chain polyunsaturated omega-3 fatty acids.
`
`15 These and other objects are obtained by the process and lipid fraction as defined in the
`accompanying claims.
`
`20
`
`25
`
`30
`
`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.
`
`Optionally, the above-mentioned process comprising a further step of:
`a-1) extracting the water reduced krill material from step a) with C02 at supercritical
`pressure containing ethanol, methanol, propanol or iso-prop_anol. This step, a-1 ), is
`performed directly after step a).
`
`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.
`
`35
`
`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
`
`RIMFROST EXHIBIT 1037 page 0004
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`WO 2008/060163
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`5
`
`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.
`
`5
`
`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.
`
`Preferably, the amount of ethanol, methanol, propanol and/or iso-propanol in step a-1)
`is 5-20 % by weight, more preferably 10-15 % by weight.
`
`10
`
`15
`
`In addition to producing a product containing the total lipids of krill, the invention also
`can be used for separating 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.
`
`20 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 Haraldsson, GG (2006) Fish oils and lipids from marine
`sources, In: Modifying Lipids for Use in Food (FD Gunstone, ed), Woodhead
`25 Publishing Ltd, Cambridge, pp. 56-79), the phospholipids of krill contain much higher
`levels (Ellingsen, TE (1982) Biokjemiske studier over antarktisk krill, PhD thesis,
`Norges tekniske lwyskole, Trondheim. English summary in Publication 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
`30 biological effects that are attributed to omega-3 containing phospholipids.
`Alternatively, the phospholipids can be transesterified or hydrolysed in order to give
`esters (typically 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 of krill
`phospholipids will be valuable as an intermediate product for producing concentrates
`that comply with the European Pharmacopoeia monographs no. 1250 (Omega-3-acid
`ethyl ester 90), 2062 (Omega -3-acid ethyl esters 60) and 1352 (Omega-3-acid
`triglycerides). At the same time, the remaining lipids (astaxanthin, antioxidants,
`
`35
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`RIMFROST EXHIBIT 1037 page 0005
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`

`WO 2008/060163
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`PCT /N02007 /000402
`
`triglycerides, wax esters) can be used as they are for various applications, including feed
`in aquaculture, or the lipid classes can be further separated.
`
`6
`
`Thus, still another object of the present invention is to provide a process for separating
`phospholipids from the other lipids as described above.
`
`5
`
`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 invention may be used as a source for production of high quality
`chitosan.
`
`Detailed description of the invention.
`
`The process can be performed with a wide variety of processing conditions, some of
`which are exemplified below.
`
`10
`
`15
`
`20
`
`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 immediately after har\resting.
`Fresh krill can be the whole krill, or by-products from fresh krill (i.e. after peeling).
`25 Fresh krill can also be krill, or by-products from krill, that have been frozen shortly after
`harvesting.
`
`Moreover "krill" also includes krill meal.
`
`30 Brief description of the figures.
`
`Figure 1 shows a picture of E. superba used as raw material for extraction.
`Figure 2 shows the material after extraction as described in Example 7 below.
`
`35 Examples
`
`Example 1
`
`RIMFROST EXHIBIT 1037 page 0006
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`WO 2008/060163
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`7
`
`Processing of freeze dried krill
`Freeze dried krill was extracted with C02 at supercritical 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
`5 phospholipids. A second extraction with C02 containing 10 % ethanol resulted in an
`extract of lOOg/kg (calculated from starting sample weight). 31P NMR showed that the
`product contained phospholipids. The extract contained a sum of EPA plus DHA of
`33.5 %.
`
`10
`
`In both steps the extraction conditions were 300 bar, 50°C.
`
`Thus, it is possible substantially to separate the omega-3 rich phospholipids from the
`less omega-3 rich components of the krill lipids.
`
`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 combined extract (280 g/kg raw
`material) was analysed by 13C and 31P NMR. The analyses showed that the product
`contained triglycerides and phospholipids as major components. Like the previous
`extracts the dark red colour showed that the extract contained astaxanthin.
`
`We are not aware that a process according to Example 1 has been used for freeze dried
`krill. It could be argued that this could be anticipated from Y. Tanaka et al. (2004) J.
`Oleo Sci. 53, 417-424. However, in this prior art C02 with 10 % ethanol resulted in
`only 30 % of the phospholipids being extracted. 20 % ethanol had to be used in order to
`extract 80 % of the phospholipids.
`
`Examples according to the invention:
`
`Example 2
`Fresh E. superba (200 g) was washed with ethanol (1: 1, 200 g) at around 0°C. The
`ethanol extract (1.5 %) contained inorganic salts (mainly NaCl) and some organic
`material.
`
`15
`
`20
`
`25
`
`30
`
`35
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`WO 2008/060163
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`8
`
`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.
`
`5 The person skilled in the art will realise that carbon dioxide at supercritical pressure can
`act as a solvent for ethanol. 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-treatment of the C02. This also applies for the examples below.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Example 3
`Fresh E. superba (200 g) was washed with ethanol (1 :3, 600 g) at around 0°C. The
`ethanol extract (7.2 %) contained phospholipids, triglycerides and astaxanthin, and
`some inorganic salts. The extract contained 26.3 % (EPA + DHA), showing that the
`relative content ofphospholipids was high.
`
`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 astaxanthin. However, as the extract
`contained only 8.1 % (EPA+ DHA) it was concluded that the phospholipids content
`was low.
`
`Example 4
`Fresh E. superba was treated with the same two-step process as above, except that the
`ethanol amount in the washing 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
`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 % ethanol
`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.
`
`35 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
`
`RIMFROST EXHIBIT 1037 page 0008
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`

`WO 2008/060163
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`PCT /N02007 /000402
`
`potential agents for removal of water. Molecular sieve and other drying agents can be
`mild and cost effective alternatives to freeze drying.
`
`9
`
`Example 6
`5 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 of 7.3 %. 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
`10 phospholipids, and also contained triglycerides and astaxanthin. The remaining, whole
`krill was completely white, except for the black eyes.
`
`Example 7
`Fresh E. superba (12 kg) was heated to 80°C for a few minutes and thereafter extracted
`15 with ethanol (26 kg). This gave an ethanol extract of 0.82 kg (7 %). Analysis oflipid
`classes (HPLC; Column: Alltima HP silica 3µm; detector: DEDL Sedere; Solvents:
`Chloroform/methanol) showed a content of 58 % phospholipids. Analysis by GC (area
`%) showed a content of24.0 % EPA and 11.4 % DHA, sum EPA+DHA = 35.4 %.
`
`20 The remaining krill was extracted at 280 bar and 50°C with C02 (156 kg) containing
`ethanol (15 kg). This gave an extract of 0.24 kg (2%). The remaining krill was white,
`except for the dark eyes. Analysis of lipid 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 lipids (0. 7 % compared to initial krill weight). This means that substantially all lipids
`had been extracted.
`
`25
`
`Example 8
`Fresh E. superba (12 kg) was extracted with ethanol (33 kg) without heat treatment.
`30 This gave an extract of 0.29 kg (2.4 %). Analysis oflipid classes as above showed a
`content of 2S.5 % phospholipids.
`
`35
`
`The results show that heat-treatment gives an increased yield of lipids 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.
`
`RIMFROST EXHIBIT 1037 page 0009
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`WO 2008/060163
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`10
`
`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 oflipids 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.
`
`5
`
`10
`
`The heat treatment gives as additional result that the highly active krill digestive
`enzymes are inactivated, reducing the potential lipid hydrolysis.
`
`Example 9
`15 Figure 1 shows a picture of E. superba used as raw material for extraction. Figure 2
`shows the material after extraction 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
`20 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 fraction 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 chitosan, and for other processed where a high quality
`meal is needed.
`
`30
`
`25
`
`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.
`
`35
`
`RIMFROST EXHIBIT 1037 page 0010
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`WO 2008/060163
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`PCT /N02007 /000402
`
`11
`
`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 Ca/anus species. Some krill species are rich in
`wax esters (example: E. crystallorphias), and the same will be the case for Ca/anus
`species. The person skilled in the art will realise that by processing as described above,
`the wax esters will be concentrated in the unpolar lipid fractions.
`
`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.
`
`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.
`
`5
`
`10
`
`15
`
`20 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 Ca/anus species.
`
`25
`
`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 krill oil
`products (produced by conventional processes), such as for instance a krill oil from
`Neptune Biotechnologies & Bioresources extracted from a Japanese krill source
`(species not specified) with the following composition:
`
`Total Phospholipids
`
`30 Esterified astaxanthin
`
`Vitamin A
`
`Vitamin E
`
`VitaminD
`
`Total Omega-3
`
`35 EPA
`
`DHA
`
`2: 40.0 %
`
`2: 1.0 mg/g
`
`2: 1.0 IU/g
`
`2: 0.005 IU/g
`
`2: 0.1 IU/g
`
`2: 30.0 %
`
`2: 15.0 %
`
`2: 9.0 %
`
`RIMFROST EXHIBIT 1037 page 0011
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`WO 2008/060163
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`PCT /N02007 /000402
`
`12
`
`A lipid product or fraction according to the invention is expected to;
`
`•
`
`contain substantially less hydrolysed and/or oxidised lipids than lipid produced
`by conventional processes,
`• be less deterioration of the krill lipid antioxidants than from conventional
`processmg,
`contain very low levels of free fatty acids, and/or
`•
`• be substantially free from trace of organic solvents.
`
`5
`
`10
`
`By "oxidised" lipids is meant both primary oxidation products (typically measured as
`peroxide value), secondary oxidation products (typically carbonyl products, often
`analysed as anisidine value) and tertiary oxidation products (oligomers and polymers).
`
`15 Thus, the invention includes commercial lipid or krill oil products produced by one of
`the processes according to the invention.
`
`Products like, for instance, the dietary supplement, Superba ™ (Aker BioMarine,
`Norway), might be produced by a process according to the present invention.
`
`20
`
`The person skilled in the art will realise that the quality of a product produced by a
`process of the present invention will be improved compared to a product produced by
`traditional extraction of krill meal.
`
`25 Moreover, examples of a lipid compositions obtained by the process according to the
`invention are presented in the tables below, and also included herein.
`
`Table 2
`
`Lipid composition
`Phospholipids
`EPA
`DHA
`
`> 30 - 40 % by weight
`> 5 -15 % by weight
`> 5-15 % by weight
`
`30 According to the invention, the extract can be concentrated with respect to the content
`of phospholipids. Some typical lipid compositions are illustrated by table 3-5, and
`included herein:
`
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`WO 2008/060163
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`PCT /N02007 /000402
`
`Table 3
`Lipid composition
`Phospholipids
`EPA
`DHA
`
`13
`
`?: 50 % by weight
`?: 15 %
`?:10 %
`
`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.
`
`5
`
`Table 4
`Lipid composition
`Phospholipids
`EPA
`DHA
`
`Table 5
`Lipid composition
`Phospholipids
`EPA
`DHA
`
`?: 80 % by weight
`?:20 %
`?:13 %
`
`?: 90 % by weight
`?:23 %
`?:15 %
`
`10 The invention shall not be limited to the shown embodiments and examples.
`
`RIMFROST EXHIBIT 1037 page 0013
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`WO 2008/060163
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`PCT /N02007 /000402
`
`14
`
`P A T E N T CLAIMS
`
`1.
`5 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.
`
`10 2.
`A process of claim 1, wherein step a) comprises washing of the krill raw material with
`ethanol, methanol, propanol or iso-propanol in a weight ratio 1 :0.5 to 1: 5; and
`step b) comprises isolating the lipid fraction from the alcohol.
`
`15
`
`3.
`A process of claim 1or2, wherein step a) comprises washing of the krill raw material
`with ethanol; and
`step b) comprises isolating the lipid fraction from the ethanol.
`
`20 4.
`A process of any one of claims 1-3, comprising a further process step of:
`a-1) extracting the water reduced krill material from step a) with C02 at supercritical
`pressure containing ethanol, methanol, propanol or iso-propanol.
`
`25
`
`5.
`A process of any one of the preceding claims, wherein the krill raw material was heated
`to 60-100 °C before washing.
`
`6.
`30 A process of claim 5, wherein the krill raw material was heated to 70-100 °C before
`washing.
`
`7.
`A process of claims 5 or 6, wherein the krill raw material was heated to 80-95 °C before
`35 washing.
`
`RIMFROST EXHIBIT 1037 page 0014
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`WO 2008/060163
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`PCT /N02007 /000402
`
`15
`
`8.
`A process of any one of claims 5 to 7, wherein the krill raw material was heated for
`about 1 to 40 minutes before washing.
`
`5 9.
`A process of claim 8, wherein the krill raw material was heated for about 1 to 15
`minutes before washing.
`
`10.
`10 A process of claims 8 or 9, wherein the krill raw material was heated for about 1 to 5
`minutes before washing.
`
`11.
`A process of claim 1, wherein step a) comprises bringing the krill raw material in
`contact with molecular sieve.
`
`15
`
`12.
`A process claim 1, wherein step a) comprises bringing the krill material in contact with
`water absorbing membranes.
`
`20
`
`13.
`A process of claim 1, wherein the amount of ethanol, methanol, propanol or iso(cid:173)
`propanol in step a-1) is 5-20 % by weight.
`
`25
`
`14.
`A process of claim 13, wherein the amount of ethanol, met