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`International application number: PCT/NO2007/000402
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`International filing date:
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`Documenttype:
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`60/859,289
`Filing date:
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`Date of receipt at the International Bureau:
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`Remark:
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`compliance with Rule 17.1(a) or (b)
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`
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`World Intellectual Property Organization (WIPO) - Geneva, Switzerland
`Organisation Mondiale de la Propriété Intellectuelle (OMPI) - Genéve. Suisse
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`RIMFROST EXHIBIT 1036
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`RIMFROST EXHIBIT 1036 page 0001
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`DOALT,7WHOMTHESE; PRESENTS; SHAG, COMES
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`PROCESSES FOR PRODUCTION OF OMEGA-3 RICH MARINE PHOSPHOLIPIDS FROM KRILL
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`rli
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`15.11.2006
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`P6060292S5US00
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`krill
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`Pronova Biocare AS
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`P.O. Box 420
`
`N-1327 Lysaker
`NORWAY
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`Inventor:
`
`Harald Breivik
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`Uranusvn. 22
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`N-3942 Porsgrunn
`NORWAY
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`Title: "Processes for production of omega-3 rich marine phospholipids from krill”
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`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.
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`Background of the invention
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`Marine phospholipids are useful in medical products, health food and humannutrition,
`as well as in fish feed and meansfor increasing the rate of survivalof 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.
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`Products such asfish milt and roe are traditional raw materials for marine
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`phospholipids. However, these raw materials are available in limited volumesand the
`price of said raw materials is high.
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`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. Anotherkrill
`species is E. crystallorphias.
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`Fresh krill contains up to around 10 % oflipids, of that approximately 50 of %
`phospholipids. Phospholipids from krill comprise a very high level of omega-3 fatty
`acids, whereofthe content of eicosapentaenoic acid (EPA) and docosahexaenoic acid
`(DHA)is above 40 %. The approximate composition oflipids from the two main
`species of Antarctic krill is given in Table 1.
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`Table 1: Composition of krill lipids. Lipid classes,
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`superba
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`crystallorphias
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`(approximate sum EPA + DHA
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`EPA/DHA
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`Furthermore, Antarctic krill has lower level of environmental pollutants than traditional
`fish oils.
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`s
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`The krill has a digestive system with enzymes, including lipases, that are very active
`around 0° C. Thelipases 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 bodyparts 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
`groupsofkrill lipids, including phospholipids, triglycerides and astaxanthin esters.
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`Krill lipids are to a large extent located in the animals’ head. A processthat 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 onboardthe
`fishing vessel.
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`From USPatent No. 6,800,299 of Beaudionetal. 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
`amountsof organic solvents which is unfavourable.
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`K. Yamaguchiet al. (J. Agric. Food Chem. 1986 34, 904-907) showedthat
`supercritical fluid extraction with carbon dioxide, which is the most commonsolvent for
`supercritical fluid extraction, of Antarctic krill resulted in a product mainly consisting of
`~=unpolar lipids (mostly triglycerides), and no phospholipids.
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`3
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`Y. Tanaka and T. Ohkubo (J. Oleo. Sci.(2003), 52, 295-301) quotes the work of
`Yamagucief al. in relation to their own work on extraction oflipids from salmonroe. In
`a more recent publication (Y. Tanakaet al. (2004), J. Oleo. Sci., 53, 417-424) the same
`authorstry to solve this problem by using a mixture of ethanol and CO) for extracting
`the phospholipids. By using CO, 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 treatmentof the very large volumesof raw materials that will become
`available by commercial krill fisheries.
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`Tanakaet al. tried to optimise the process by varying the temperatureof the extraction,
`and found that low temperatures gavethe best results. 33°C, a temperature just above
`the critical temperature for CO2, was chosenas giving best results.
`
`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-treatmentlike freeze drying of large volumes.Thelipid fraction contained
`triglycerides, astaxanthin and phospholipids. We did not have to dry or deoil the raw
`material before processing. Contrary to Tanakaet al. we have foundthat a short heating
`of the marine raw material was positive for the extraction yield. It was also shown that
`pre-treatmentlike a short-time heating to moderate temperatures, or contact with a solid
`drying agent like molecular sieve, of the krill can make ethanol wash aloneefficient in
`removing phospholipids from fresh krill.
`
`Summary of the invention
`
`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 solventslike
`acetone.
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`The exposure to the supercritical fluid will prevent oxidation from taking place, and the
`combined carbon dioxide/ethanolis 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 onfresh krill, 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
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`processes. This also meansthat 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 ofthe lipids, thus ensuring a product with very low levels of free fatty
`acids.
`
`Anotherobject 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.
`
`Another object of the present invention is to provide a substantially total lipid fraction
`high in long chain polyunsaturated omega-3 fatty acids.
`
`These andother objects are obtained by the processandlipid fraction as defined in the
`accompanying claims.
`.
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`According to the inventionit is provided a process for extracting a substantially total
`lipid fraction from fresh krill, comprising the steps of:
`a) reducing the water contentof krill raw material; and
`b) extracting the water reduced kmill material from step a) with supercritical CO,
`containing ethanol, methanol, propanol and/or iso-propanol.
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`In a preferred embodimentthe 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 ofkrill raw material; and
`b) extracting the water reducedkrill material from step a) with supercritical CO?
`containing ethanol.
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`In a preferred embodimentof the invention, step a) comprises washing ofthe krill raw
`material with ethanol, methanol, propanol and/or iso-propanolin a weight ratio 1:0.5 to
`1:5. Preferably, the krill raw material is heated to 60-100 °C, more preferably 70-95 °C
`before washing. Furthermore,the krill raw material is preferably heated for about 1 to
`15 minutes, more preferably for about 1 to 5 minutes, before washing.
`
`In another preferred embodimentofthe invention, step a) comprises bringingthe krill
`raw material in contact with molecular sieve or an other form of membrane for removal
`
`of water.
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`_ Preferably, the amount of ethanol, methanol, propanol and/or iso-propanolin step b) is
`5-20 % by weight, more preferably 10-15 % by weight.
`
`In addition to producing a product containingthe total lipids of krill, the invention also
`can be used for separating phospholipids from the other lipids. To make separate the
`total lipids obtained by the supercritical extraction of the present invention into the
`different lipid classes, extraction of the said total lipids with pure carbon dioxide will
`remove the nonpolar lipids from the omega-3 rich phospholipids. Extraction of the total
`lipids with carbon dioxide containing less than 5 % ethanol or methanolis another
`option.
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`Asthe phospholipids are muchricher 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, B and Haraldsson, GG (2006)Fish oils and lipids from marine
`sources, In: Modifying Lipidsfor Use in Food (FD Gunstone, ed), Woodhead
`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 hoyskole, 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 usedastheyare, giving the various positive
`biological effects that are attributed to omega-3 containing phospholipids.
`Alternatively, the phospholipids can be transesterified or hydrolysed in order to give
`esters (typically ethy! 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 monographsno. 1250 (Omega-3-acid
`ethyl ester 90), 2062 (Omega -3-acid ethyl esters 60) and 1352 (Omega-3-acid
`triglycerides). At the sametime, the remaininglipids (astaxanthin, antioxidants,
`triglycerides, wax esters) can be used as they are for various applications, including feed
`in aquaculture, or the lipid classes can be further separated.
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`Thus,still another object of the present invention is to provide a process for separating
`phospholipids from the other lipids as described above.
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`35
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`Detailed description of the invention.
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`RIMFROST EXHIBIT 1036 page 0009
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`The process can be performed with a wide variety of processing conditions, some of
`which are exemplified below.
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`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 oflipids, or krill that is frozen immediately after harvesting.
`Fresh krill can be the wholekrill, or by-products from fresh krill (i.e. after peeling).
`
`Moreover“krill” also includeskrill meal.
`
`Examples
`
`Example 1
`Processing of freeze dried krill
`Freeze dried krill was extracted with supercritical CO2. This gave a product of 90 g/kg.
`Analysis showedthat the extract contained a sum of EPA plus DHA of only 5.4%,
`showingthat this did not contain a significant amount of the omega-3 rich
`phospholipids. A second extraction with CO, containing 10 % ethanolresulted in an
`extract of 100g/kg (calculated from starting sample weight). 3>'P NMR showedthatthe
`product contained phospholipids. The extract contained a sum of EPA plus DHA of
`33.5%.
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`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 componentsofthe krill lipids.
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`In a second experimentthe freeze dried krill was extracted twice with the same pressure
`and temperature as above,first with 167 parts (weight) of pure CO2, and then with 167
`part (weight) of CO2 containing 10 % ethanol. The combined extract (280 g/kg raw
`material) was analysed by °C and *'P NMR. Theanalyses showedthatthe product
`contained triglycerides and phospholipids as major components. Like the previous
`extracts the dark red colour showedthat the extract contained astaxanthin.
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`Weare not awarethat a process according to Example 1 has been used for freeze dried
`krill. It could be argued that this could be anticipated from Y. Tanakaet al. (2004) J.
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`7
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`Oleo Sci. 53, 417-424. However,in this prior art CO) with 10 % ethanol resulted in
`only 30 % of the phospholipids being extracted. 20 % ethanol had to be usedin orderto
`extract 80 % of the phospholipids.
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`Examples accordingto 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.
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`The ethanol washedkrill was extracted with CO, containing 10 % ethanol. This gave an
`extract of 12 g (6 % basedonstarting krill). Analysis (TLC and NMR) showedthat the
`extract contained phospholipids, triglycerides and astaxanthin.
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`The person skilled in the art will realise that supercritical carbon dioxide can act as a
`solvent for ethanol. Thus, an alternative procedure for modifying the solvent power of
`the CO}is to utilise pressure/temperature conditions so that ethanolis dissolve directly
`from the ethanol containing krill raw material, without having to be addedbya pre-
`treatment of the CO. This also applies for the examples below.
`
`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 of phospholipids was high.
`
`The ethanol washedkrill was extracted with CO2 containing 10 % ethanol. This gave an
`extract of 2.2 % based onstarting krill. Analysis (TLC and NMR)showedthat the
`extract contained phospholipids,triglycerides and astaxanthin. However,as the extract
`contained only 8.1 % (EPA + DHA)it was concluded that the phospholipids content
`waslow.
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`Example 4
`Fresh E. superba wastreated with the same two-step process as above, exceptthat the
`ethanol amountin the washingstep was increased to 4:1. The ethanol extract was 7.2 %
`compared to the starting material, while the supercritical fluid extract was 2.6 %.
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`Example 5
`Fresh E. superba (200 g) was put in contact with molecular sieve (A3, 280 g) in orderto
`remove water from the krill raw material. Extraction with COcontaining 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.
`
`Example 5 showsthe effect of removing water. Molecular sieve was chosen as an
`alternative to ethanol. These examplesare 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.
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`Example 6
`Fresh E. superba (200 g) was washedwith 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 ethanolextract of 7.3 %. Supercritical fluid extraction with CO, 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) showedthat the extract wasrich in
`phospholipids, and also contained triglycerides and astaxanthin. The remaining, whole
`krill was completely white, except for the black eyes.
`
`Theresults show that heat-treatmentgives 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 sameresult as four parts of ethanol without heat treatment.
`
`Theheat treatment givesas additional result that the highly active krill digestive
`enzymesare inactivated, reducing the potential lipid hydrolysis.
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`The personskilled 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. Somekrill species are rich in
`wax esters (example: E. crystallorphias), and the same will be the case for Calanus
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`RIMFROST EXHIBIT 1036
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`RIMFROST EXHIBIT 1036 page 0012
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`species. The personskilled in the art will realise that by processing as described above,
`the wax esters will be concentrated in the unpolar lipid fractions.
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`9
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`Furthermore, the person skilled in the art will realise that combination of process steps
`as given abovecan be usedfor separating the polar (i.e. phospholipids) and unpolar
`lipids of krill. It will also be possible to make an extract ofthe total lipids of krill
`according to one of the examples above, and then make a secondextraction of this
`intermediary product in orderto separate the lipid classes. For example, an extraction
`with pure carbon dioxide would removethe nonpolar lipids from the omega-3 rich
`phospholipids.
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`5
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`10
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`In another embodiment, the process accordingto the invention is used to extractkrill
`meal, wherein provided the krill meal has been produced in a sufficiently mild way to
`avoid deterioration of the krill lipids.
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`The personskilled 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.
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`A lipid fraction, or lipid product, derived from the process according to the invention
`20 may have some additional advantagesrelated to quality compared to known krill oil
`products (produced by conventional processes), such as for instancea krill oi] from
`Neptune Biotechnologies & Bioresources extracted from a Japanese krill source
`(species not specified) with thefollowing composition:
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`25
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`Total Phospholipids
`Esterified astaxanthin
`Vitamin A
`Vitamin E
`Vitamin D
`30 Total Omega-3
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`EPA
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`DHA
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`> 40.0 %
`2 1.0 mg/g
`= 1.0 IU/g
`> 0.005 IU/g
`> 0,1 IU/g
`> 30.0%
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`> 15.0%
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`=9.0%
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`A lipid product or fraction according to the invention is expected to;
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`Copy provided by USPTO from the IFW Image Database on 11/08/2007
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`RIMFROST EXHIBIT 1036 page 0013
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`contain substantially less hydrolysed and/or oxidised lipids than lipid produced
`by conventional processes,
`beless deterioration of the krill lipid antioxidants than from conventional
`processing,
`contain very low levels of free fatty acids, and/or
`be substantially free from trace of organic solvents.
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`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) andteritiary oxidation products (oligomers and polymers).
`
`Thus, the invention includes commerciallipid or krill 01] products produced by the
`process accordingto the invention.
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`5
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`-
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`15. Moreover, examples ofa lipid compositions obtained by the process according to the
`invention are presented in the tables below, and also includedherein.
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`Table 2
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`[Lipidcomposition|
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`> 30 - 40 % by weight
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`20 According to the invention, the extract can be concentrated with respect to the content
`of phospholipids. Sometypical lipid compositionsare illustrated by table 3-5, and
`includedherein:
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`[Lipidcomposition=|sd
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`Phospholipids
`> 50 % by weight
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`EPA
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`[Lipidcomposition|
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`Copyprovided by USPTO from the IFW {mage Database on 11/08/2007
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`RIMFROST EXHIBIT 1036 page 0014
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`RIMFROST EXHIBIT 1036
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`11
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`Table 5
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`[Lipidcomposition|
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`> 90 % by weight
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`The invention shall not be limited to the shown embodiments and examples.
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`RIMFROST EXHIBIT 1036 page 0015
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`RIMFROST EXHIBIT 1036
`Copy provided by USPTO from the IFW Image Database on 11/08/2007
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`page 0015
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`
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`Patent
`
`claims
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`12
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`A processfor extracting a substantially total lipid fraction from freshkrill,
`1.
`comprising the steps of:
`a) reducing the water content of krill raw material; and
`b) extracting the water reducedkrill material from step a) with supercritical CO,
`containing ethanol, methanol, propanol or iso-propanol.
`
`A process of claim 1, wherein step a) comprises washing ofthe krill raw
`2.
`material with ethanol.
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`A processof claim 1, wherein step a) comprises washingofthe krill raw
`3.
`material with ethanol, methanol, propanol or iso-propanolin a weightratio 1:0.5 to 1:5.
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`A processof claim 1, wherein the krill raw material was heated to 60-100 °C
`4.
`before washing.
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`A process of claim 4, wherein the krill raw material was heated to 70-95 °C
`5.
`before washing.
`
`20
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`A process of claims 4 or 5, wherein the krill raw material was heated for about 1
`6.
`to 15 minutes before washing.
`
`A processof claim 6, wherein the krill raw material was heated for about 1 to 5 ©
`7.
`minutes before washing.
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`25
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`A process of claim 1, wherein step a) comprises bringing the krill raw material
`8.
`in contact with molecular sieve.
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`A process of claim 1, wherein the amountof ethanol, methanol, propanolor iso-
`9.
`propanolin step b) is 5-20 % by weight.
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`A process of claim 9, wherein the amountof ethanol, methanol, propanoloriso-
`10.
`propanolin step b) is 10-15 % by weight.
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`30
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`35
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`A substantially total lipid fraction comprising triglycerides, astaxanthin and
`11.
`phospholipids obtainable by the process of claim 1.
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`page 0016
`RIMFROST EXHIBIT 1036
`RIMFROST EXHIBIT 1036 page 0016
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`The phospholipids of claim 15, wherein the phospholipids are further
`16.
`1s_transesterified or hydrolysed.
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`The phospholipids of claim 14, wherein the concentration of omega-3 fatty acids
`17.
`is at least 40 % by weight.
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`20
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`12._Alipid fraction of claim 11, being substantially free from oxidisedlipids.
`
`13
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`A total lipid fraction according to any one of claims 11 or 12, for use as a
`13.
`5 medicament and/or as a food supplement.
`
`*
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`A process for separating phospholipids from the other lipids, comprising
`14.
`extracting the total lipid fraction obtained by the process of claim 1, with pure carbon
`dioxide, or carbon dioxide containing less than 5 % ethanol, methanol, propanoloriso-
`propanol.
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`10
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`15.
`
`A phospholipids fraction obtainable by the process of claim 14.
`
`
` RIMFROST EXHIBIT 1036
`page 0017
`RIMFROST EXHIBIT 1036 page 0017
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`Copy provided by USPTOfrom the IFW Image Database on 11/08/2007
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`

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`
`
`Abstract
`
`O. nr. P60601677n000 |
`
`The presentinvention relates to a process for preparing a substantially total lipid
`fraction from fresh krill, and a process for separating phospholipids from the other
`lipids.
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`page 0018
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`RIMFROST EXHIBIT 1036
`RIMFROST EXHIBIT 1036 page 0018
`Copy provided by USPTOfrom the IFW Image Database on 11/08/2007
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