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`available under
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`the
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`Patent Cooperation Treaty (PCT)
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`International application number: PCT/NO2007/000402
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`International filing date:
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`15 November 2007 (15.11.2007)
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`Document type:
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`Certified copy of priority document
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`Document details:
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`Country/Office: US
`Number:
`60/859,289
`Filing date:
`16 November 2006 (16.11.2006)
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`Date of receipt at the International Bureau:
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`11 December 2007 (11.12.2007)
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`Remark:
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`Priority document submitted or transmitted to the International Bureau in
`compliance with Rule 17.1(a) or (b)
`
`
`
`World Intellectual Property Organization (WIPO) - Geneva, Switzerland
`Organisation Mondiale de la Propriété Intellectuelle (OMPI) - Geneve. Suisse
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`RIMFROST EXHIBIT 1036
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`RIMFROST EXHIBIT 1036 page 0001
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`UNITED STATES DEPARTMENT OF COMMERCE
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`United States Patent and Trademark Office
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`November 13, 2007
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`RIMFROST EXHIBIT 1036 page 0002
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`Given Name (first and middle [I1 anyl)
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`and either State or Forei 11 Count
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`RIMFROST EXHIBIT 1036 page 0003
`RIMFROST EXHIBIT 1036
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`separately numbered sheets attached hereto
`D Additional inventors are being named on the
`TITLE OF THE INVENTION (280 characters max)
`PROCESSES FOR PRODUCTION OF OMEGA-3 RICH MARINE PHOSPHOLIPIDS FROM KRILL
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`CORRESPONDENCE ADDRESS
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`—-———+
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`Type Customer Number here
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`AMOUNT (S)
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`200.00
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`I
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`rli
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`15.1 1.2006
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`P60602925USOO
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`krill
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`Pronova Biocare AS
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`PO. Box 420
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`N-1327 Lysaker
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`NORWAY
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`Inventor:
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`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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`RIMFROST EXHIBIT 1036 page 0004
`Copy provided by USPTO from the IFW Image Database on 11/08/2007
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`RIMFROST EXHIBIT 1036
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`page 0004
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`Field of the invention
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`The present invention relates to a process for preparing a substantially total lipid
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`fraction from fresh krill, and a process for separating phospholipids from the other
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`5
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`lipids.
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`Background of the invention
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`Marine phospholipids are useful in medical products, health food and human nutrition,
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`l0
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`as well as in fish feed and means for increasing the rate of survival of fish larval and fry
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`of marine species like cod, halibut and turbot.
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`Phospholipids from marine organisms comprise omega-3 fatty acids. Omega-3 fatty
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`acids bound to marine phospholipids are assumed to have particularly useful properties.
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`IS
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`Products such as fish milt and roe are traditional raw materials for marine
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`phospholipids. However, these raw materials are available in limited volumes and the
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`price of said raw materials is high.
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`20 Krill are small, shrimp-like animals, containing relatively high concentrations of
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`phospholipids. In the group Euphasiids, there is more than 80 species, of which the
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`Antarctic krill is one of these. The current greatest potential for commercial utilisation is
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`the Antarctic Euphausia superba. E. superba has a length of 2-6 cm. Another krill
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`species is E. crystallorphias.
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`n
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`Fresh krill contains up to around 10 % of lipids, of that approximately 50 of %
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`phospholipids. Phospholipids from krill comprise a very high level of omega—3 fatty
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`acids, whereof the content of eicosapentaenoic acid (EPA) and docosahexaenoic acid
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`(DHA) is above 40 %. The approximate composition of lipids from the two main
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`30
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`species of Antarctic krill is given in Table 1.
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`35
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`RIMFROST EXHIBIT 1036 page 0005
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`RIMFRO ST EXHIBIT 1036
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`page 0005
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`-----EPA/DHA
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`Table l: ComIosition o krill IiIids. LiIid classes, aI Iroximate sum EPA + DHA
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`su Ierba
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`c stallo Ihias
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`Furthermore, Antarctic krill has lower level of environmental pollutants than traditional
`fish oils.
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`5
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`The krill has a digestive system with enzymes, including lipases, that are very active
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`around 0° C. The lipases stay active after the krill is dead, hydrolysing part of the krill
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`lipids. An unwanted effect of this is that krill oil normally contains several percents of
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`lo
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`free fatty acids. If the krill has to be cut into smaller fragments before being processed,
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`the person skilled in the art will immediately realise that this will increase the degree of
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`hydrolysis. Thus, it is a desire to find a process that can utilise whole, fresh krill, or
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`whole body parts from krill, as such a process will provide a product with improved
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`quality and low degree of hydrolysis of lipids. This improved quality Will affect all
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`15
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`groups of krill lipids, including phospholipids, triglycerides and astaxanthin esters.
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`Krill lipids are to a large extent located in the animals’ head. A process that can utilise
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`fresh krill is therefore also well suited for immediate processing of the by-products from
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`krill wherefrom the head is peeled off, a product that can be produced onboard the
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`20
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`fishing vessel.
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`From US Patent No. 6,800,299 of Beaudion et al. it is disclosed a method for extracting
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`total lipid fractions from krill by successive extraction at low temperatures using
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`organic solvents like acetone and ethanol. This process involves extraction with large
`amounts of organic solvents which is unfavourable.
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`25
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`K. Yamaguchi et al. (J. Agric. Food Chem. 1986 34, 904-907) showed that
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`supercritical fluid extraction with carbon dioxide, which is the most common solvent for
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`supercritical fluid extraction, of Antarctic krill resulted in a product mainly consisting of
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`30
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`unpolar lipids (mostly triglycerides), and no phospholipids.
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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 0006
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`RIMFRO ST EXHIBIT 1036
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`page 0006
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`Y. Tanaka and T. Ohkubo (J. Oleo. Sci. (2003), 52, 295-301) quotes the work of
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`Yamaguci et al. in relation to their own work on extraction of lipids from salmon roe. In
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`a more recent publication (Y. Tanaka et al. (2004), J. Oleo. Sci, 53, 417-424) the same
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`authors try to solve this problem by using a mixture of ethanol and C02 for extracting
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`the phospholipids. By using C02 with 5 % ethanol no phospholipids were removed
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`from freeze dried salmon roe, while by adding 10 % ethanol, 30 % of the phospholipids
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`were removed, and by adding as much as 30 % ethanol, more than 80 % of the
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`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
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`available by commercial krill fisheries.
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`Tanaka et a1. tried to optimise the process by varying the temperature of the extraction,
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`and found that low temperatures gave the best results. 33°C, a temperature just above
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`the critical temperature for C02, was chosen as giving best results.
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`Contrary to these findings, we have surprisingly found a process for extraction of a
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`substantially total lipid fraction fi'om fresh krill, without the need for complicated and
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`costly pre-treatment like freeze drying of large volumes. The lipid fraction contained
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`triglycerides, astaxanthin and phospholipids. We did not have to dry or deoil the raw
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`material before processing. Contrary to Tanaka et al. we have found that a short heating
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`of the marine raw material was positive for the extraction yield. It was also shown that
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`pre—treatment like a short-time heating to moderate temperatures, or contact with a solid
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`drying agent like molecular sieve, of the krill can make ethanol wash alone efficient in
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`removing phospholipids from fresh krill.
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`Summary of the invention
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`10
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`IS
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`20
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`25
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`It is a main object of the present invention to provide a process for preparing a
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`substantially total lipid fraction from fresh krill without using organic solvents like
`acetone.
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`30
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`The exposure to the supercritical fluid will prevent oxidation from taking place, and the
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`combined carbon dioxide/ethanol is expected to deactivate any enzymatic hydrolysis of
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`the krill lipids. As the process according to the invention requires a minimum of
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`35
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`handling of the raw materials, and is well suited to be used on fresh krill, for example
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`onboard the fishing vessel, the product according to the invention is expected to contain
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`substantially less hydrolysed and/or oxidised lipids than lipid produced by conventional
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`RIMFROST EXHIBIT 1036 page 0007
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`page 0007
`Copy provided by USPTO from the IFW Image Database on 11/08/2007
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`RIMFROST EXHIBIT 1036
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`4
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`processes. This also means that there is expected to be less deterioration of the krill lipid
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`antioxidants than from conventional processing. The optional pre-treatment involving
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`short-time heating of the fresh krill will also give an inactivation of enzymatic
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`decomposition of the lipids, thus ensuring a product with very low levels of free fatty
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`s
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`acids.
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`Another object of the present invention is to provide a process for preparing a
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`substantially total lipid fraction from other marine raw materials like fish gonads,
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`Calanus species, or high quality krill meal.
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`10
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`Another object of the present invention is to provide a substantially total lipid fraction
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`high in long chain polyunsaturated omega-3 fatty acids.
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`These and other objects are obtained by the process and lipid fraction as defined in the
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`Is
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`accompanying claims.
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`'
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`According to the invention it is provided a process for extracting a substantially total
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`lipid fraction from fresh krill, comprising the steps of:
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`a) reducing the water content of krill raw material; and
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`20
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`b) extracting the water reduced krill material from step a) with supercritical C02
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`containing ethanol, methanol, propanol and/or iso-propanol.
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`In a preferred embodiment the invention it is provided a process for extracting a
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`substantially total lipid fraction from fresh krill, comprising the steps of:
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`25
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`a) reducing the water content of krill raw material; and
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`V
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`b) extracting the water reduced krill material from step a) with supercritical C02
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`containing ethanol.
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`In a preferred embodiment of the invention, step a) comprises washing of the krill raw
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`3o material with ethanol, methanol, propanol and/or iso-propanol in a weight ratio 120.5 to
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`1:5. Preferably, the krill raw material is heated to 60-100 °C, more preferably 70-95 °C
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`before washing. Furthermore, the krill raw material is preferably heated for about 1 to
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`15 minutes, more preferably for about 1 to 5 minutes, before washing.
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`35
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`In another preferred embodiment of the invention, step a) comprises bringing the krill
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`raw material in contact with molecular sieve or an other form of membrane for removal
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`of water.
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`RIMFROST EXHIBIT 1036 page 0008
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`Copy provided by USPTO from the IFW Image Database on 11/08/2007
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`RIMFROST EXHIBIT 1036
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`page 0008
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`. Preferably, the amount of ethanol, methanol, propanol and/or iso—propanol in step b) is
`5-20 % by weight, more preferably 10-15 % by weight.
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`5
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`In addition to producing a product containing the total lipids of krill, the invention also
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`can be used for separating phospholipids from the other lipids. To make separate the
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`total lipids obtained by the supercritical extraction of the present invention into the
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`different lipid classes, extraction of the said total lipids with pure carbon dioxide will
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`remove the nonpolar lipids from the omega-3 rich phospholipids. Extraction of the total
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`10
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`lipids with carbon dioxide containing less than 5 % ethanol or methanol is another
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`option.
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`As the phospholipids are much richer in the valuable omega-3 fatty acids than the other
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`lipid classes, this makes the invention useful for producing high concentrates of omega-
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`15
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`3 fatty acids. While commercially available fish oils contain 11-33% total omega-3
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`.'
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`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
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`Publishing Ltd, Cambridge, pp. 56-79), the phospholipids of krill contain much higher
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`levels (Ellingsen, TE (1982) Biokjemiske studier over antarktisk krill, PhD thesis,
`20 Norges tekniske hoyskole, Trondheim. English summary in Publication no. 52 of the
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`Norwegian Antarctic Research Expeditions (1976/77 and 1978/79)), see also Table 1.
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`The omega-3 rich phospholipids can be used as they are, giving the various positive
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`biological effects that are attributed to omega-3 containing phospholipids.
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`Alternatively, the phospholipids can be transesterified or hydrolysed in order to give
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`25
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`esters (typically ethyl esters) or free fatty acids or other derivatives that are suitable for
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`further concentration of the omega-3 fatty acids. As examples, the ethyl esters of krill
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`phospholipids will be valuable as an intermediate product for producing concentrates
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`that comply with the European Pharmacopoeia monographs no. 1250 (Omega—3-acid
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`ethyl ester 90), 2062 (Omega -3-acid ethyl esters 60) and 1352 (Omega-3—acid
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`30
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`triglycerides). At the same time, the remaining lipids (astaxanthin, antioxidants,
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`triglycerides, wax esters) can be used as they are for various applications, including feed
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`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
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`35
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`phospholipids from the other lipids as described above.
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`Detailed description of the invention.
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`RIMFROST EXHIBIT 1036 page 0009
` RIMFROST EXHIBIT 1036
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`Copy provided by USPTO from the IFW Image Database on 11/08/2007
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`page 0009
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`
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`The process can be performed with a wide variety of processing conditions, some of
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`which are exemplified below.
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`5
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`In the following “fresh” krill is defined as krill that is treated immediately alter
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`harvesting, or sufficiently short time after harvesting to avoid quality deterioration like
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`hydrolysis or oxidation of lipids, or krill that is frozen immediately after harvesting.
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`Fresh krill can be the whole krill, or by—products from fresh krill (i.e. after peeling).
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`10 Moreover “krill” also includes krill meal.
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`Examples
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`Example 1
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`15
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`Processing of freeze dried krill
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`Freeze dried krill was extracted with supercritical C02. This gave a product of 90 g/kg.
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`Analysis showed that the extract contained a sum of EPA plus DHA of only 5.4%,
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`showing that this did not contain a significant amount of the omega-3 rich
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`phospholipids. A second extraction with C02 containing 10 % ethanol resulted in an
`extract of lOOg/kg (calculated from starting sample weight). 3'P NMR showed that the
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`20
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`product contained phospholipids. The extract contained a sum of EPA plus DHA of
`33.5 %.
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`25
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`so
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`In both steps the extraction conditions were 300 bar, 50°C.
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`Thus, it is possible substantially to separate the omega-3 rich phospholipids from the
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`less omega-3 rich components of the krill lipids.
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`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 ‘3C and 3‘P NMR. The analyses showed that the product
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`contained triglycerides and phospholipids as major components. Like the previous
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`extracts the dark red colour showed that the extract contained astaxanthin.
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`We are not aware that a process according to Example 1 has been used for freeze dried
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`krill. It could be argued that this could be anticipated from Y. Tanaka et a1. (2004) .1.
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`
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`i
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`RIMFROST EXHIBIT 1036 page 0010
`RIMFROST EXHIBIT 1036
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`7
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`Oleo Sci. 53, 417-424. However, in this prior art CO2 with 10 % ethanol resulted in
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`only 30 % of the phospholipids being extracted. 20 % ethanol had to be used in order to
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`extract 80 % of the phospholipids.
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`'
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`Examples according to the invention:
`
`Example 2
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`Fresh E. superba (200 g) was washed with ethanol (1:1, 200 g) at around 0°C. The
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`10
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`ethanol extract (1.5 %) contained inorganic salts (mainly NaCl) and some organic
`material.
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`The ethanol washed krill was extracted with C02 containing 10 % ethanol. This gave an
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`extract of 12 g (6 % based on starting krill). Analysis (TLC and NMR) showed that the
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`15
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`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
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`solvent for ethanol. Thus, an alternative procedure for modifying the solvent power of
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`the C02 is to utilise pressure/temperature conditions so that ethanol is dissolve directly
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`20
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`from the ethanol containing krill raw material, without having to be added by a pre-
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`treatment of the C02. This also applies for the examples below.
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`Example 3
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`Fresh E. superba (200 g) was washed with ethanol (1:3, 600 g) at around 0°C. The
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`25
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`ethanol extract (7.2 %) contained phospholipids, triglycerides and astaxanthin , and
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`some inorganic salts. The extract contained 26.3 % (EPA + DHA), showing that the
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`relative content of phospholipids was high.
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`The ethanol washed krill was extracted with CO2 containing 10 % ethanol. This gave an
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`30
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`extract of 2.2 % based on starting krill. Analysis (TLC and NMR) showed that the
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`extract contained phospholipids, triglycerides and astaxanthin. However, as the extract
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`contained only 8.1 % (EPA + DHA) it was concluded that the phospholipids content
`was low.
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`35
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`RIMFROST EXHIBIT 1036 page 0011
`RIMFROST EXHIBIT 1036
`page 0011
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`Example 4
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`Fresh E. superba was treated with the same two-step process as above, except that the
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`ethanol amount in the washing step was increased to 4:1. The ethanol extract was 7.2 %
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`compared to the starting material, while the supercritical fluid extract was 2.6 %.
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`Example 5
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`Fresh E. superba (200 g) was put in contact with molecular sieve (A3, 280 g) in order to
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`remove water from the krill raw material. Extraction with C02 containing 10 % ethanol
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`gave an extract of 5.2 % calculated from the starting weight of krill. Analyses showed
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`that the extract contained triglycerides, phospholipids and astaxanthin. The extracted,
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`whole krill, was completely white, except for the black eyes.
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`Example 5 shows the effect of removing water. Molecular sieve was chosen as an
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`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
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`mild and cost effective alternatives to freeze drying.
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`Example 6
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`Fresh E. superba (200 g) was washed with ethanol (1:1) as in example 2, but with the
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`difference that the raw material had been pre-treated at 80°C for 5 minutes. This gave
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`an ethanol extract of 7.3 %. Supercritical fluid extraction with C02 containing 10 %
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`ethanol gave an additional extract of 2.6 % calculated from the fresh raw material. The
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`total extract was 9.9%, and analyses (TLC, NMR) showed that the extract was rich in
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`phospholipids, and also contained triglycerides and astaxanthin. The remaining, whole
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`krill was completely white, except for the black eyes.
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`The results show that heat-treatment gives an increased yield of lipids compared to
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`the same treatment with no heating. After heat-treatment of the raw material, one part
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`(weight) of ethanol gave the same result as four parts of ethanol without heat treatment.
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`so -
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`The heat treatment gives as additional result that the highly active krill digestive
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`enzymes are inactivated, reducing the potential lipid hydrolysis.
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`The person skilled in the art will realise that the processes described above also can be
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`used for other raw materials than krill, for example the isolation of omega—3 rich
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`phospholipids from fish gonads, or from Calanus species. Some krill species are rich in
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`wax esters (example: E. crystallorphias), and the same will be the case for Calanus
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`RIMFROST EXHIBIT 1036 page 0012
`RIMFROST EXHIBIT 1036
`page 0012
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`Copy provided by USPTO from the IFW Image Database on 11/08/2007
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`species. The person skilled in the art will realiSe that by processing as described above,
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`the wax esters will be concentrated in the unpolar lipid fractions.
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`Furthermore, the person skilled in the art will realise that combination of process steps
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`as given above can be used for separating the polar (i.e. phospholipids) and unpolar
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`lipids of krill. It will also be possible to make an extract of the total lipids of krill
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`according to one of the examples above, and then make a second extraction of this
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`intermediary product in order to separate the lipid classes. For example, an extraction
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`with pure carbon dioxide would remove the nonpolar lipids from the omega-3 rich
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`phospholipids.
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`In another embodiment, the process according to the invention is used to extract krill
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`meal, wherein provided the krill meal has been produced in a sufficiently mild way to
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`avoid deterioration of the krill lipids.
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`The person skilled in the art will also realise that a process as described above can be
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`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
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`may have some additional advantages related to quality compared to known krill oil
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`products (produced by conventional processes), such as for instance a krill oil from
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`Neptune Biotechnologies & Bioresources extracted from a Japanese krill source
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`(species not specified) with the following composition:
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`Total Phospholipids
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`Esterified astaxanthin
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`Vitamin A
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`Vitamin E
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`Vitamin D
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`30
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`Total Omega-3
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`EPA
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`DHA
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`2 40.0 %
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`2 1.0 mg/g
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`2 1.0 IU/g
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`.
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`2 0.005 IU/g
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`2 0,1 IU/g
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`2 30.0 %
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`2 15.0 %
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`2 9.0 %
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`IS
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`A lipid product or fraction according to the invention is expected to;
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` Copy provided by USPTO trom the IFW Image Database on 11/08/2007
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`RIMFROST EXHIBIT 1036
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`RIMFROST EXHIBIT 1036 page 0013
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`page 0013
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`0
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`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
`processing,
`contain very low levels of free fatty acids, and/or
`be substantially free from trace of organic solvents.
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`5
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`1
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`By “oxidised” lipids is meant both primary oxidation products (typically measured as
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`peroxide value), secondary oxidation products (typically carbonyl products, often
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`analysed as anisidine value) and teritiary oxidation products (oligomers and polymers).
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`Thus, the invention includes commercial lipid or krill oil products produced by the
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`process according to the invention.
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`15 Moreover, examples of a lipid compositions obtained by the process according to the
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`invention are presented in the tables below, and also included herein.
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`Table 2
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`>3o-40%b weiht
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`20 According to the invention, the extract can be concentrated with respect to the content
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`of phospholipids. Some typical lipid compositions are illustrated by table 3-5, and
`included herein:
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`Table 3
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`Table 4
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`RIMFROST EXHIBIT 1036 page 0014
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`Copy provided by USPTO from the IFW Image Database on 11/08/2007
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`RIMFRO ST EXHIBIT 1036
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`page 0014
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`11
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`Table 5
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`290m weuu
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`The invention shall not be limited to the shown embodiments and examples.
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` RIMFRO ST EXHIBIT 1036
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`RIMFROST EXHIBIT 1036 page 0015
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`page 0015
`Copy provided by USPTO from the IFW Image Database on 11/08/2007
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`Patent
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`claims
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`12
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`1.
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`A process for extracting a substantially total lipid fraction from fresh krill,
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`comprising the steps of:
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`a) reducing the water content of krill raw material; and
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`b) extracting the water reduced kn'll material from step a) with supercritical C02
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`containing ethanol, methanol, propanol or iso-propanol.
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`A process of claim 1, wherein step a) comprises washing of the krill raw
`2.
`IO material with ethanol.
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`3.
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`A process of claim 1, wherein step a) comprises washing of the krill raw
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`.
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`material with ethanol, methanol, propanol or iso-propanol in a weight ratio 1:05 to 1:5.
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`4.
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`A process of claim 1, wherein the krill raw material was heated to 60-100 °C
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`before washing.
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`5.
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`A process of claim 4, wherein the krill raw material was heated to 70-95 °C
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`before washing.
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`20
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`6.
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`A process of claims 4 or 5, wherein the krill raw material was heated for about 1
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`to 15 minutes before washing.
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`7.
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`A process of claim 6, wherein the krill raw material was heated for about 1 to 5 '
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`25 minutes before washing.
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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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`9.
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`A process of claim 1, wherein the amount of ethanol, methanol, propanol or iso-
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`propanol in step b) is 5-20 % by weight.
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`10.
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`A process of claim 9, wherein the amount of ethanol, methanol, propanol or iso-
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`propanol in step b) is 10-15 % by weight.
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`11.
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`A substantially total lipid fraction comprising triglycerides, astaxanthin and
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`phospholipids obtainable by the process of claim 1.
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`RIMFROST EXHIBIT 1036 page 0016
`Copy provided by USPTO from the IFW Image Database on 11/08/2007
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`RIMFROST EXHIBIT 1036
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`page 0016
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`13
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`12.
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`A lipid fraction of claim 11, being substantially free from oxidised lipids.
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`13.
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`A total lipid fraction according to any one of claims 11 or 12, for use as a
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`s medicament and/or as a food supplement.
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`'-
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`14.
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`A process for separating phospholipids from the other lipids, comprising
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`extracting the total lipid fraction obtained by the process of claim 1, with pure carbon
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`dioxide, or carbon dioxide containing less than 5 % ethanol, methanol, propanol or iso-
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`l0
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`propanol.
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`15.
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`A phospholipids fraction obtainable by the process of claim 14.
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`16.
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`The phospholipids of claim 15, wherein the phospholipids are filrther
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`transesterified or hydrolysed.
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`17.
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`The phospholipids of claim 14, wherein the concentration of omega-3 fatty acids
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`is at least 40 % by weight.
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`RIMFROST EXHIBIT 1036 page 0017
` RIMFROST EXHIBIT 1036
`Copy provided by USPTO trom the IFW Image Database on 11/08/2007
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`page 0017
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`Abstract
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`0. nr. P60601677n000 _
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`The present invention relates to a process for preparing a substantially total lipid
`fraction from fresh hill, and a process for separating phospholipids from the other
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`lipids.
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`RIMFRO ST EXHIBIT 1036
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`page 0018
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`RIMFROST EXHIBIT 1036 page 0018
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`Copy provided by USPTO from the IFW Image Database on 11/08/2007
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`My Mt-
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