Report Confidential K- 313  Published September 2006
`
`
`
`Extraction of krill meal with ethanol
`
`
`Eyolf Langmyhr
`
`AKER EXHIBIT 2013 PAGE 0001
`
`

`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Norut Group Ltd. consists of six research
`institutes located in Tromsø, Narvik and Alta.
`The Norut Group has 220 employees whose
`applied research and development
`encompasses a wide variety of interdisciplinary
`fields. Each subsidiary institute has a specific
`research emphasis, but common to all is
`activity centered around the polar and Barents
`regions.
`
`Norut Group LTD consist of:
`
`Fiskeriforskning (Norwegian Institute of
`Fisheries and Aquaculture Research), Tromsø
`and Bergen
`
`Norut Information Technology Ltd, Tromsø
`
`Norut Social Science Researc Ltd, Tromsø
`
`Norut Technology Ltd, Narvik
`
`Norut Medicine and Health Ltd, Tromsø
`
`Norut NIBR Finnmark AS, Alta
`
`
`
`
`Fiskeriforskning (Norwegian Institute of
`Fisheries and Aquaculture Research) conducts
`research and development for the fisheries and
`aquaculture industry. The Institute covers
`virtually all links in the value chain – “from sea
`bottom to tabletop”. Fiskeriforskning is a national
`research institute – owned by the Norut Group
`Ltd. (51 %) and the Norwegian Ministry of
`Fisheries (49 %). Located in Tromsø (head
`office) and Bergen, the facilities at
`Fiskeriforskning are an important part of the
`national infrastructure for fisheries and
`aquaculture research.
`
`Fiskeriforskning have five main areas of research:
` Seafood and industrial processing
` Marine biotechnology and fish health
` Aquaculture
` Aquafeed and marine processing
` Economics and marketing
`
`Tromsø (head office)
`Muninbakken 9-13, Breivika
`P.O.box 6122
`NO-9291 Tromsø
`Norway
`Tel.: +47 77 62 90 00
`Fax: +47 77 62 91 00
`E-mail:post@fiskeriforskning.no
`
`Bergen
`Kjerreidviken 16
`NO-5141 Fyllingsdalen
`Norway
`Tel.: +47 55 50 12 00
`Fax: +47 55 50 12 99
`E-mail: office@fiskeriforskning.no
`
`Internet: www.fiskeriforskning.no
`
`AKER EXHIBIT 2013 PAGE 0002
`
`

`

`
`
`Norwegian Institute of Fisheries and
`Aquaculture Research
`Main office: P.O.box 6122, NO-9291 Tromsø
`Visiting address: Muninbakken 9-13,
`Tlf.: +47 77 62 90 00, fax: +47 77 62 91 00
`E-mail: post@fiskeriforskning.no
`
`Dept. Bergen: Kjerreidviken 16, NO-5141 Fyllingsdalen
`Tlf.: +47 55 50 12 00, fax: +47 55 50 12 99
`E-mail: office@fiskeriforskning.no
`
`Internet: www.fiskeriforskning.no
`
`Organisation no.: NO 964 441 898 MVA
`
`Accessibility:
`Confidential
`
`K - 313
`
`Date:
`14.09.2006
`Number of pages and appendixes:
`6 p
`Director of Research:
`Ivan Burkow
`Project no:
`20217
`Employers ref.:
`Snorre Tilseth
`
`
`
`
`
`
`
`
`
`
`
`
`REPORT
`Tittel:
`Extraction of krill meal with ethanol
`
`
`
`Author (s):
`Eyolf Langmyhr
`By agreement with:
`Aker Seafoods AS
`3 keywords:
`Krill meal, Extraction, Phospholipids
`Summary:
`Krill meal has been extracted with ethanol at Fresenius Kabi, Sweden.
`
`94 % of the lipids in the krill meal are extracted in the process.
`
`The protein quality of the krill meal has not changed in the extraction process.
`
`The flow of the krill meal is greatly improved by the extraction.
`
`Only 23 % of the astaxanthin is recovered. The greatest loss seems to be during the removal of
`solvent from the extracted lipid.
`
`Krill meal treated with ethoxyquin should not be used for extraction as the antioxidant may
`accumulate in the solvent.
`
`AKER EXHIBIT 2013 PAGE 0003
`
`

`

`TABLE OF CONTENTS
`
`1 MATERIAL AND METHODS ........................................................................................ 1
`
`2
`
`3
`
`EXTRACTION ................................................................................................................. 1
`
`ANALYTICAL RESULTS .............................................................................................. 1
`
`4 MASS BALANCE OF THE EXTRACTION .................................................................. 6
`
`
`AKER EXHIBIT 2013 PAGE 0004
`
`

`

`1 MATERIAL AND METHODS
`
`Krill meal, 3210 kg (107 bags á 30 kg) produced on board F/V Atlantic Navigator 06.05.05,
`was extracted with ethanol at Fresenius Kabi, Kungsängen, Sweden.
`
`The analytical methods is described in Fiskeriforskning Report K-300 “F/T Atlantic
`Navigator 2004-2005” and in Fiskeriforskning Report K-308 “Extraction of krill meal at
`Extractis”.
`
`2
`
`EXTRACTION
`
` The aim of the extraction was to test ethanol as extraction medium in Fresenius Kabi’s
`extraction unit for the production of marine phospholipids.
`
`From 3200 kg krill meal they got 660 kg crude krill lipid and 2500 kg delipidated krill
`powder. The krill lipid was dark red liquid with high viscosity. The colour of the krill powder
`was light red and it smelled of ethanol. The time and temperature of the extraction are not
`given.
`
`
`Figure 1 Krill meal before and after extraction
`
`
`
`3
`
`ANALYTICAL RESULTS
`
`By using ethanol to extract lipids from krill meal, only a small part of the lipid is left in the
`extracted krill meal (Table 1).
`
`The delipidated krill powder smelled of ethanol, so it is likely that the weight loss at drying is
`mostly ethanol.
`
`AKER EXHIBIT 2013 PAGE 0005
`
`

`

`The water and ethanol was removed from the Crude Krill Lipid by evaporation in a Büchi
`Rotavapor. The temperature at the end of the evaporation was 80 °C, and the weight reduction
`was 85 g/kg (Table 1). The water content of the Crude Krill Lipid was determined to 54 g/kg
`by Karl Fischer titration, and the difference, 31 g/kg, is the same as the ethanol content
`reported by Fresenius Kabi.
`
`After removal of water and ethanol from the Crude Krill Lipid the viscosity of the lipid
`increased considerably. The Crude Krill Lipid could be poured from the container at room
`temperature, but after removal of water/ethanol the lipid will move slowly in an inverted
`beaker. The viscosity of the dewatered lipid was determined to assure that there will be no
`problem in the molecular distillation unit (Table 2).
`
`Table 1 Composition and properties of the krill meal and products after extraction
`
`
`
`
`
`Crude Krill Lipid
`
`Delipidated
`
`Krill Powder
`
`Krill meal *
`
`Crude protein (Nx6.25)
`
`Fat (Bligh & Dyer)
`
`Fat (Soxhlet)
`
`Moisture/Ethanol
`
`Water (Karl Fischer)
`
`Ethanol (Fresenius Kabi)
`
`Insoluble impurities
`
`Free fatty acid
`
`Astaxanthin Free
`
`Asthaxanthin esters
`
` Diesters
`
`g/kg
`
`g/kg
`
`g/kg
`
`g/kg
`
`g/kg
`
`g/kg
`
`g/kg
`
`g/kg
`
`mg/kg
`
`mg/kg
`
`mg/kg
`
`
`
`
`
`
`
`85
`
`54
`
`31
`
`9
`
`99
`
`<1
`
`117
`
`37
`
`80
`
`735
`
`18
`
`2
`
`134
`
`
`
`
`
`
`
`
`
`<1
`
`10
`
`8.5
`
`1.8
`
`586
`
`223
`
`
`
`71
`
`
`
`
`
`
`
`
`
`<1
`
`144
`
`110
`
`33
`
` Monoesters
`
` Diesters
`
` Monoesters
`
`Asthaxanthin esters
`
` Diesters
`
` Monoesters
`
`Ethoxyquin
`
`Ethoxyquin in fat
`
`mg/kg
`
`
`
`
`
`mg/kg lipid
`
`mg/kg lipid
`
`mg/kg lipid
`
`mg/kg
`
`mg/kg lipid
`
`Biological digestible protein
`
`g/kg protein
`
`Flow number
`
`Density 30 °C
`
`Density 50 °C
`
`
`
`g/cm²
`
`g/cm²
`
`32 %
`
`68 %
`
`117
`
`37
`
`80
`
`760
`
`831
`
`
`
`
`
`0.996
`
`0.979
`
`85 %
`
`18 %
`
`572
`
`472
`
`100
`
`0.3
`
`17
`
`870
`
`1.9
`
`
`
`
`
`*) Data from Fiskeriforskning report K-300 ”F/T Atlantic Navigator 2004-2005”
`
`
`Table 2 Viscosity in Crude Krill Lipid after removal of water and ethanol
`
`Viscosity 60 °C
`
`Viscosity 80 °C
`
`
`
`cP
`
`cP
`
`1275
`
`409
`
`76 %
`
`23 %
`
`646
`
`493
`
`148
`
`191
`
`857
`
`854
`
`4.8
`
`
`
`
`
`AKER EXHIBIT 2013 PAGE 0006
`
`

`

`The concentration of astaxanthin in the Crude Krill Lipid is lower than in the lipid phase in
`the krill meal. The colour of the extract was dark red, but a considerable part of the
`astaxanthin is lost during the extraction and isolation process.
`
`Monoesters of astaxanthin are more polar than the diesters, so by ethanol extraction the
`proportion of astaxanthin monoester increases in the extracted lipid.
`
`Ethoxyquin is soluble in ethanol and is extracted together with the lipids. There are only
`traces of ethoxyquin left in the delipidated krill powder.
`
`The biological digestible protein of the krill meal did not change during the extraction process
`at Fresenius Kabi. Due to the ethanol in the extracted krill meal, the krill meal was mixed with
`cod fillet in the feed preparation.
`
`The flow of the krill meal is greatly improved by removing the lipids. A flow number below 2
`signify a powder with good flow properties, while a flow number above 5 signify a powder
`with poor flow properties.
`
`The density of the krill lipid is near 1 g/cm³ at 30 °C with a slight decrease at higher
`temperatures.
`
`The lipid class distribution in the products was analysed at NIFES, Bergen, by thin layer
`chromatography (HPTLC) and some compounds were also analysed at Fresenius Kabi (Table
`3). The lipid class distribution in the krill meal was analysed at Havforskningsinstituttet,
`Matre. The laboratories may disagree in the identification of some of minor compounds.
`
`The results show that the proportion of neutral lipids increased in the lipid extract. This is a
`result of increased amount of free fatty acids. This increase may have happened during
`storage of the krill meal or during the extraction process. The amount of free fatty acid in the
`krill lipid is higher when analysed by thin layer chromatography (Table 3) than by titration
`(Table 1). It is an effect we usually observe, but the reason is unknown.
`
`
`
`Table 3 Lipid class distribution
`
`
`
`
`
`Cholesterol esters
`
`Triacylglyserol
`
`Free fatty acid
`
`Cholesterol
`
`Diacylglyserol
`
`Monoacylglyserol
`
`Sphingolipid
`
`Phosphatidylethanolamin
`
`Cardiolipin
`
`Phosphatidylinositol
`
`Phosphatidylserin
`
`Phosphatidylcholin
`
`Lysophosphatidylcholin
`
`Crude Krill Lipid
`
`Delipidated Krill Powder
`
`Krill meal 1)
`
`NIFES
`
`Fresenius
`
`
`
`31.1
`
`16.0
`
`12.6
`
`3.3
`
`
`
`2.8*
`
`2.7
`
`
`
`
`
`
`
`25.3
`
`6.2
`
`
`
`
`
`
`
`
`
`
`
`
`
`0.5
`
`0
`
`
`
`
`
`
`
`24.3
`
`4.8
`
`
`
`
`
`37.4
`
`14.1
`
`8.0
`
`
`
`
`
`
`
`2.5
`
`4.2
`
`11.0
`
`
`
`20.2
`
`2.6
`
`
`
`3.5
`
`32.7
`
`7.8
`
`9.1
`
`1.1
`
`3.7
`
`
`
`6.5
`
`
`
`1.1
`
`1.4
`
`28.6
`
`2.9
`
`AKER EXHIBIT 2013 PAGE 0007
`
`

`

`
`
`Total polar lipids
`
`Total neutral lipids
`
`
`
`36.9
`
`63.1
`
`
`
`
`
`
`
`
`
`40.5
`
`59.5
`
`1) Data from F/T Atlantic Navigator 2005-2005, Fiskeriforskning report K300
`*) Identification not comfirmed
`
`
`
`40.6
`
`54.2
`
`AKER EXHIBIT 2013 PAGE 0008
`
`

`

`There is little change in the fatty acid composition from krill meal to lipid extract (Table 4).
`Some of the fatty acids were determined by Fresenius Kabi. These results are higher because
`they are calculated as percentage of total fatty acids. Recalculated on the basis of lipid there is
`good agreement with the results from Fiskeriforskning.
`
`Table 4 Fatty acid composition
`
`Crude Krill Lipid
`
`Krill meal
`
`Fiskeriforskning
`
`Fresenius
`
`6.4
`
`14.7
`
`0.7
`
`0.1
`
`
`
`
`
`14:0
`
`16:0
`
`18:0
`
`20:0
`
`22:0
`
`
`
`
`
`g/100g lipid
`
`g/100g lipid
`
`g/100g lipid
`
`g/100g lipid
`
`g/100g lipid
`
`<0,1
`
`16:1 n-7
`
`g/100g lipid
`
`18:1 (n-9)+(n-7)+(n-5)
`
`g/100g lipid
`
`20:1 (n-9)+(n-7)
`
`g/100g lipid
`
`22:1 (n-11)+(n-9)+(n-7)
`
`g/100g lipid
`
`4.2
`
`11.8
`
`0.6
`
`0.4
`
`24:1 n-9
`
`16:2 n-4
`
`16:3 n-4
`
`16:4 n-1
`
`18:2 n-6
`
`18:3 n-6
`
`20:2 n-6
`
`g/100g lipid
`
`<0,1
`
`g/100g lipid
`
`g/100g lipid
`
`g/100g lipid
`
`g/100g lipid
`
`g/100g lipid
`
`g/100g lipid
`
`0.5
`
`0.1
`
`0.4
`
`1.2
`
`<0,1
`
`<0,1
`
`10.2
`
`23.2
`
`1.4
`
`
`
`
`
`7.6
`
`19.7
`
`0.8
`
`
`
`
`
`
`
`
`
`
`
`2.4
`
`0.7
`
`
`
`
`
`
`
`7.8
`
`15.8
`
`0.9
`
`0.1
`
`<0,1
`
`5.1
`
`13.4
`
`0.8
`
`0.5
`
`0.1
`
`0.7
`
`0.1
`
`0.5
`
`1.1
`
`0.1
`
`<0,1
`
`0.1
`
`g/100g lipid
`
`<0,1
`
`g/100g lipid
`
`0.2
`
`0.4
`
`
`
`
`
`
`
`
`
`
`
`16.2
`
`
`
`
`
`6.6
`
`
`
`
`
`
`
`
`
`
`
`
`
`89.2
`
`2.45
`
`0.2
`
`<0,1
`
`0.4
`
`1.1
`
`<0,1
`
`0.2
`
`10.5
`
`0.3
`
`0.2
`
`5.4
`
`
`
`24.6
`
`19.9
`
`1.4
`
`18.2
`
`21.0
`
`65.5
`
`1.94
`
`20:3 n-6
`
`20:4 n-6
`
`22:4 n-6
`
`18:3 n-3
`
`18:4 n-3
`
`20:3 n-3
`
`20:4 n-3
`
`20:5 n-3
`
`21:5 n-3
`
`22:5 n-3
`
`22:6 n-3
`
`
`
`g/100g lipid
`
`<0,1
`
`g/100g lipid
`
`g/100g lipid
`
`0.4
`
`1.0
`
`g/100g lipid
`
`<0,1
`
`g/100g lipid
`
`g/100g lipid
`
`0.2
`
`10.4
`
`g/100g lipid
`
`<0,1
`
`g/100g lipid
`
`g/100g lipid
`
`
`
`0.2
`
`4.8
`
`
`
`21.9
`
`17.0
`
`1.4
`
`17.0
`
`19.4
`
`58.2
`
`Total saturated fatty acids
`
`g/100g lipid
`
`Total monoene fatty acids
`
`g/100g lipid
`
`Total PUFA (n-6) fatty acids g/100g lipid
`
`Total PUFA (n-3) fatty acids g/100g lipid
`
`Total PUFA fatty acids
`
`g/100g lipid
`
`Total fatty acids
`
`g/100g lipid
`
`EPA/DHA
`
`
`
`2.17
`
`
`
`
`
`AKER EXHIBIT 2013 PAGE 0009
`
`

`

`4 MASS BALANCE OF THE EXTRACTION
`
`The amount of products and various compounds by extraction of 1000 g krill meal with
`ethanol is given in Table 5. Some of the discrepancies in Table 5 of the amount before and
`after extraction may be due to variation within the batch of krill meal, and some to the
`reporting of the amount of products in round numbers (2 Extraction).
`
`In the extraction process at Fresenius Kabi 94 % of the lipids in the krill meal are extracted.
`
`Of the 144 mg astaxanthin which goes into the process, only 33 mg (23 %) is recovered.
`Since the loss seems to be in the lipid fraction (Table 1), it may be that the conditions during
`removal of ethanol are unfavourable for astaxanthin.
`
`Some ethoxyquin (17 %) is lost and probably ended up in the recovered ethanol.
`
`
`Table 5 Yields by extraction of 1000 g krill meal
`
`
`
` Crude Krill Lipid
`
`Delipidated Krill
`Powder
`
`Total after
`extractinon
`
`Total
`
`
`
`Protein (N x 6,25)
`
`Fat
`
`Dry matter
`
`
`
`Astaxanthin
`
` Diester
`
` Monoester
`
`
`
`Ethoxyquin
`
`
`
`Polar lipids
`
`Neutral lipids
`
`
`
`g
`
`
`
`g
`
`g
`
`g
`
`
`
`mg
`
`mg
`
`mg
`
`
`
`mg
`
`
`
`g
`
`g
`
`209
`
`
`
`
`
`191
`
`191
`
`
`
`24
`
`8
`
`17
`
`
`
`159
`
`
`
`71
`
`121
`
`791
`
`
`
`581
`
`14
`
`685
`
`
`
`8
`
`7
`
`1
`
`
`
`0.2
`
`
`
`6
`
`8
`
`1000
`
`
`
`581
`
`205
`
`876
`
`
`
`33
`
`14
`
`18
`
`
`
`159
`
`
`
`77
`
`129
`
`Krill meal
`
`1000
`
`
`
`586
`
`223
`
`929
`
`
`
`144
`
`110
`
`33
`
`
`
`191
`
`
`
`91
`
`129
`
`AKER EXHIBIT 2013 PAGE 0010
`
`

`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Tromsø (head office)
`Muninbakken 9-13, Breivika
`P.O.box 6122
`NO-9291 Tromsø
`Norway
`Tel.: +47 77 62 90 00
`Fax: +47 77 62 91 00
`E-mail:post@fiskeriforskning.no
`
`Bergen
`Kjerreidviken 16
`NO-5141 Fyllingsdalen
`Norway
`Tel.: +47 55 50 12 00
`Fax: +47 55 50 12 99
`E-mail: office@fiskeriforskning.no
`
`Internet: www.fiskeriforskning.no
`
`
`
`
`
`
`AKER EXHIBIT 2013 PAGE 0011
`
`