CX—1274C
`CX Page 1
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`Weight of krill lipids in filter agitation acetone teclmique
`
`"
`
`Cup 7: l.0548g — O.9864g = 0.0684g
`Cup 8: 1.0561g — O.9860g = 0.701g
`Cup 9: 1.0566g O.9886g = 0.068g
`
`X = 0.0688g
`
`% of krill lipids of the dry polytron weight
`
`acetone technique
`
`0.799g/2ml = x/10ml x = 0.3995 g
`
`0.3995g/5g = x/100g x= 7.99%
`
`% of krill lipids of dry weight agitation
`0.1973g / 2 ml = x/10ml x = O.9865g
`29/7/98: 7 mL instead of IO mL? I3.8l% rather than 19.73%. Where did the 3mls that S]
`
`acetone technique
`
`pipetted go?
`
`10 ml acetone. X = 19.73%
`
`O.9865g/5g = x/ 100g
`
`% of krill lipids of the dry polytron filter weight acetone technique
`0.0760g/2 mL = x/ ml x = 0.3800g
`
`0.3800/5g = x/100g x=7.6%
`% of krill lipids of the dry filter weight agitation acetone technique
`
`x = 0.3440g
`0.0688g/ 2 mL = x/10 mL
`0.3440g/5g = x/100g x=6.88%
`
`6/3/98 Results: acetone technique yield
`Krill homogenized to polytron: 7.99 g lipids/ 100g dry weight
`Krill homogenized by agitation: 19.73g lipids/ 100g dry weight
`Results: acetone and contents technical yicld
`filtered in ethanol re-suspension MeOH
`Krill homogenized polytron: 7.99 lipids /100g dry weight + 7.60g lipids/ 100g dw = 15.59g/
`100g
`See 2"d and 3"‘ trials -'> Krill homogenized agitation: l9.73g lipids/100g dw + 6.88g lipids 100g
`dw = 26.61g/100g dw
`
`Page 1
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`N EP877|TC-00679899
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`NEPN Ex. 2031
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`Aker v. Neptune
`[PR2014-00003
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`CX Page 1
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`Weight of krill lipids in filter agitation acetone teclmique
`
`"
`
`Cup 7: l.0548g — O.9864g = 0.0684g
`Cup 8: 1.0561g — O.9860g = 0.701g
`Cup 9: 1.0566g O.9886g = 0.068g
`
`X = 0.0688g
`
`% of krill lipids of the dry polytron weight
`
`acetone technique
`
`0.799g/2ml = x/10ml x = 0.3995 g
`
`0.3995g/5g = x/100g x= 7.99%
`
`% of krill lipids of dry weight agitation
`0.1973g / 2 ml = x/10ml x = O.9865g
`29/7/98: 7 mL instead of IO mL? I3.8l% rather than 19.73%. Where did the 3mls that S]
`
`acetone technique
`
`pipetted go?
`
`10 ml acetone. X = 19.73%
`
`O.9865g/5g = x/ 100g
`
`% of krill lipids of the dry polytron filter weight acetone technique
`0.0760g/2 mL = x/ ml x = 0.3800g
`
`0.3800/5g = x/100g x=7.6%
`% of krill lipids of the dry filter weight agitation acetone technique
`
`x = 0.3440g
`0.0688g/ 2 mL = x/10 mL
`0.3440g/5g = x/100g x=6.88%
`
`6/3/98 Results: acetone technique yield
`Krill homogenized to polytron: 7.99 g lipids/ 100g dry weight
`Krill homogenized by agitation: 19.73g lipids/ 100g dry weight
`Results: acetone and contents technical yicld
`filtered in ethanol re-suspension MeOH
`Krill homogenized polytron: 7.99 lipids /100g dry weight + 7.60g lipids/ 100g dw = 15.59g/
`100g
`See 2"d and 3"‘ trials -'> Krill homogenized agitation: l9.73g lipids/100g dw + 6.88g lipids 100g
`dw = 26.61g/100g dw
`
`Page 1
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`N EP877|TC-00679899
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`NEPN Ex. 2031
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`Aker v. Neptune
`[PR2014-00003
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`CX Page 2
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`
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`OK 9/3/98 re-done
`
`Experiments to be done: re-do extraction with 5 g acetone dry krill agitation (given the 19.73%
`result) 3/10/98 recovery ethanol filter deposit OK 3/9/98 re-done
`Re-do extraction OK
`
`Chloromethanol (to validate the results) 9/9/98 re-done
`D0 acetone extraction
`
`[notes in the side margins; cm oflso it Zsjust the translation of the visible words: And to verify
`the impact of the solvent volume on the efficacy of the extraction]
`Ethanol l :1 (50 mL total) 10/3/98 re-done rinse with 25 1nL total
`Change the solvent volumes (double 100 mL)
`
`6/3/98
`
`After evaporation, re-suspend in l0mL acetone-ethanol l:l
`l2/3/98 done fresh krill acetone technique
`Procedure ( 15' trial): weigh 25g fresh krill 25.08g
`225 mL pure cold (freezer) acetone
`agitation 20 min (27 min)
`
`*(- vacuum filtration
`
` (not the first time) rinse cold acetone
`/illegible English addition/
`Up to 85 C'°
`Evaporation acetone (revolving evapo) pay attention to bubbles
`Vacuum = ? No bubbles
`l l/3/98
`
`Cold the H20 lipid mixture on ice
`Add hexane or pentane 25 mL
`To H20 — lipids mixture
`Decantation ampulla, mix
`See p. 15
`Collect lipids and hexane
`Evaporation (rotating evapo)
`Re-suspend lOmL hexane
`Vacuum evaporation
`
`'
`
`'
`
`
`
`7-5.»
`
`C!
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`Page 2
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`NEP877|TC-00679900
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`CX Page 3
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`Filter deposit re-suspend mix 100 mL EtOH
`2 papers Whatman 12.5cm 202
`Ordinary filtration keep powder good for fish
`1 1/3/98 Vacuum =? Evaporation
`We had to heat a lot to compensate for the mediocre vacuum
`85°C bath water
`
`Re-suspension ethanol 10 mL
`F.vapora.ti on
`Weighing lipids, average
`Chloro technique MeOH
`6/3/98: Preparation of the egg lipids (standard)
`1 egg yolk transfer pipette
`Chloro 50 mL
`
`Methanol 25 mL
`15 mL saline
`
`mix decantation ampulla
`let it separate | one weekend
`9/3/98 Evaporation (rotating evaporation)
`Re-suspension 10 mL chloroforme — methanol 1 :1
`3 samples of 2mL each
`Transfer each 2 mL in aluminum cups
`Evaporation without N
`Egg (- Weighing lipids, average
`Tech. chloro — MeOH Egg
`Cup 1 1.0l50g 1.1548g 1.1777 with lipids 1.1667g l.l670g
`Cup 2 1.0l54g1.l642gl.1737g
`l.1644g l.l659g
`Cup 3 l.0134g l.l62lg1.1758g
`1.16520 l.1623g
`
`Page 3
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`NEP877ITC-00679901
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`CX Page 4
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`6/3/98 Look for Medline acetone
`
`Acetone toxicity
`Lipids classes
`Calamus
`
`6/3/98 Calculations: krill lipids weight technique chloroforme methanol)
`Cup 1; l.l470g — 0.9927g = 0. 1 543g
`Cup 2: l.1447g — O.989lg = O.l556g
`Cup 3: l.1709g—1.0l55g = 0. l554g
`x = 0. 155 lg
`
`% of lipids in dry weight chloro-MeOH technique
`0. l551g/2mL = x/ 10 mL x= O.7755g O.7755g/5g = x/100g x= 15.51%
`Result: chloroforme-methanol technical yield
`15.51 g lipids/ 100g krill dry weight
`
`9/3/98 Notes: possibility of water in the egg lipids, chloroform, methanol, saline solution since
`the mixture was boiling at evaporation under rotating evaporator (perhaps because I washed the
`bubble with water)
`Empty I l/3/98 with lipids 10/3/98
`Cup 4 l.Ol60g
`1.0976g
`1.0971g
`Cup 5
`l.Ol§31g
`1.0956g
`l.O938g
`Cup 6 l.0l90g
`l.O977g
`l.O997g
`
`1.0958g
`l.0965g
`l.0995g
`
`l.O959g
`l.0973g
`1.0993,;
`
`Note: instead of letting it rest for one night, the ampulla solution (methanol chlorofonn recovery
`method from 5/3/98 and 6/3/98) rested 5 and a half hours.
`
`Cup7 l.02l8g
`Cup8 l.0l88g
`Cup9 1.0227g
`
`1.l778g
`l.l788g
`1.l826g
`
`l.1687g
`l.1679g
`l.1729g
`
`1.l654g
`l.l668g
`l.l7l9g
`
`Page 4
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`NEP877lTC-00679902
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`CX Page 5
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`10/3/98
`
`Calculations: egg lipids weight chloroform methanol technique
`cup 1; 1.1670-1.0l50g = 0.1520g
`cup 2: 1.1659g —1.0154g = 0.1505g
`cup 3: 1.1623g —1.0134g = O.1489g
`
`[-w5 5,.)
`
`x= 0. 1SO5g
`
`% of egg lipids of dry weight with chloro-MeOH technique
`
`0. l505gZ mL = x/10mL X=0.7525g
`
`0.7525g/5g = x/100g x = 15.05%
`
`(transfer pipette)
`
`10/3/98 Calculations: Krill lipids weight chloro-MeOH technique 2”‘ trial
`Cup 7; l.1687g- 1.02l8g = O.1469g
`Cup 8: l.1679g—1.0l88g= 0.l49lg
`Cup 9: 1.1729g — 1.0227g = 0.1502g
`X = 0. l487g
`
`% krill lipids dry weight chloro-MeOH technique
`0.1487g/2 mL = X/' 10 mL
`x= 0.7435g
`
`0.74325g / Sg = x/100g
`
`x= 14.87%
`
`ids
`with li
`empty 12/3/98
`10/3/98
`filter deposit agitation acetone technique 2" trial 10/3/98
`
`11/3/98
`
`l.0268g
`Cup 1:
`Cup 2: l.0275g
`Cup 3: 1.0250g
`
`1.l348g
`1.l342g
`1.1316g
`
`l.1679g
`l.15l8g
`1.1525g
`
`1.1388g
`l.l390g
`1 1370g
`
`Calculations: krill lipids weight agitation acetone technique
`
`Cup 4: l.0959g — l.Ol60g = 0.0799g
`Cup 5; l.O973g — l.Ol3lg — 0.0842g
`Cup 6: l.O993g— 1.o19og= 0.0803g
`
`X=0.0815g
`
`Krill lipids weight in filter agitation acetone technique 2"" trial
`Cup 1: l.1388g— l.O268g= O.1120g
`Cup 2: l.1390g— 1.0275g=0.11lSg
`Cup 3:1.1370g—1.0250g= O.1120g X=0.1118g
`% of krill lipids of dry weight agitation acetone technique 2'” trial
`0.0815g/2mL = X/10 mL x= 0_4075g
`0.4075g/5g = x/100g x=8. 1 5%
`
`(next page)
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`Page 5
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`N EP877lTC-00679903
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`CX Page 6
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`(continued)
`0.2835g/25g = x/100g X=1.13%
`
`X=1.70% for 15 mL instead of 10 mL
`
`ha
`
`13/3/98 Result: acetone technical yield + second decantation + contents of 1st filtration in ETOH
`(lg trial)
`
`2.14glipids/100g fresh weight (fw) krill + 0.12g lipids/100g fw
`Krill + 2 Mg lipidsl 100g fw Krill = 4.40g lipids/' 100g fw krill
`
`Note: Result of 2.14% of fresh weight of the contents. 1”‘ filtration of EtOII being questionable
`(by comparing the cups) and very poor vacuum during evaporation, do 2"“ trial. With, per the 15‘
`remark on p. 16, double acetone extraction (see protocol pp. 18-19)
`
`Result: Technical acetone yield (double acetone extraction) + filter contents 3” filtration in EtoH
`
`2.25g lipids/100g krill fresh weight + l. l3g lipids/100g fw krill = 3.38g lipids/100g krill fresh
`weight
`
`Result; precipitation test with TCA if proteins are present
`Aqueous solution 2"" decantation 11/3/98: no precipitate = no proteins
`Aqueous solution 2'“ decantation I2/3/98: no precipitate = lack of protein
`
`:0,
`.
`J
`Eeésgulgi, ‘I? W‘: 6’ '
`
`.
`.
`.
`.
`'.' )e:gl" gene "img. E: el5l'.lll:“ ill” 5 ' 5.‘ll’
`
`Result: dry weight krill compared with fresh weight krill
`20.26% of dry weight by relation to fresh weight
`
`Result: levels of H20 of the fresh krill
`
`79.74g water per 100g fresh krill
`Calculation: 3.38g lipids -> 100g fresh krill
`20,26g dry Krill -> 100g fresh Krill
`3.38g lipids = X
`X= -1-6.-68% acetone technique
`Summary P. 14 dry Krill
`
`Page 6
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`NEP877|TC-00679904
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`CX Page 7
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`Recapitulation Flesh Krill 13/3/98
`1“ trial ll/3/98 pages 8 and 15
`25g fresh Krill 225 lnL acctonc
`agitation 20 min
`
`Deposit
`l0()mL Et0H
`
`deposit 1'lltratio1l(2)
`2 filters 1"i ltrate
`
`evaporation
`10 mL EIOH
`evapo N
`weighing cups
`7-8-9
`
`2.14%
`(Doubtful)
`
`Filtration (1)
`Filtiate
`
`e\ aporation
`25 lnL hexane
`
`rejected version
`
`decant anlpullas (1)
`H30 (lower phase)
`hcxanc lipids (llppcr phase)
`evaporation
`10 mL hexane
`10 lnL hexane
`decant ampulla (2)
`evapo N
`hexane lipids
`H30 keep for TCA
`
`weighing cups weighing cups
`1-2-3
`2.14%
`
`4-5-6
`0. 12%
`
`12/3/98 pages 18 and 19
`25 g fresh Krill 225 lnL acetone
`agitation 20 llllll
`deposit filtration
`50 mL acetone
`
`filtrate
`
`Deposit filtration filtrate
`100 tnL EtOH
`
`evaporation
`25 mL hexane
`
`2"“ trial
`
`Deposit filtration (3)
`Filtiate
`Evaporation
`
`docant ampulla
`hexane lipids
`H20 (lower phase)
`(upper phase)
`10 ml. hexane
`Evaporation
`dccant amplllla (2)
`l0mL hexane
`hexane lipids
`10 mL EIOH
`evapo
`5lnL hexane
`centnftlgation /illegible/ Weighing cups 1-2-3
`3000
`NO
`2 25%
`15 min
`
`modified version
`
`H20 keep for TC A
`
`Supernatant
`Residue
`(soluble composition)
`Evapo
`
`For each of two groups
`
`Weighing cups 4-5-6
`1.13%
`
`Page 7
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`NEP877lTC-00679905
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`
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`CX Page 8
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`For each of two groups of filtersl3/3/98
`Page 21
`40mL saline 9%
`
`agitation
`ccntrifugation 3000 rpm 15 min
`polytron
`centrifugalion 3000 rpm 15 min
`supernatant
`residue
`combine and
`20 mL saline
`
`to improve: reduce the Krill to powder before
`suspension in 40mL saline
`
`put at 4 degrees C
`
`Centrifugation 3000 15 min
`snpematant
`residue
`20 mL acetone
`
`Cemrifugation 3000 rpm 5 n1i11
`Remove acetone
`Residue 25 mL acetone
`
`Centrifugation 3000 rpm 5 min
`Remove acetone
`
`Residue in ampulla
`Heating plate — oven one wcckcnd- weighing
`
`Page 8
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`NEP877|TC-00679906
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`CX Page 9
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`16/3/98
`
`3"‘ trial fresh Krill acetone technique
`protocol see p 24 / 12/3/98
`place on hot plate cups which spent the weekend in the oven because of defective
`
`oven
`
`Note: Krill solution boiled during the 15' evaporation (it was very hot in the room = sun)
`
`Aqueous phase 15' decant: 45 mL
`Aqueous phase 2“ decant: 40 mL
`
`Calculations: weight of filter contents filtration at EtOH (insol. residue)
`Cup 1: 3. l955g— l.O255g = 2. l700g
`Cup 2: 3 l900g— l.Ol9lg= 2.l709g
`
`16/3/98
`17/3/98
`
`empty
`18/3/98
`
`with lipids
`
`Krill lipids
`Fresh extraction
`
`Acetone after
`Decantation
`(3"‘ trial)
`
`cup 1: 1.0267g
`
`1.l278g 1.1272g
`
`1.1279g
`
`cup 2: 1.0237gl.1273g l.l252g
`cup 3: l.0275gl.l3l2g l.l29lg
`
`l.1271g
`l.l297g
`
`Lowry protein assay to be done
`
`- Done 17/3/98
`
`I;.”l..i
`
`; 21:;
`
`Et0H{3"' trial
`3/16/98
`Notes: During centrifugation of the re-suspended solution
`EtOH, the tube split and the liquid was lost with insoluble dry proteins
`
`Filter Cup 1 l.0367g3.7808g
`5'.
`go
`E
`L
`‘E
`
`3.8344g 18/3/98
`
`17/3/98 Note: forgot to reduce the deposit to powder, thus took the supernatant (the carcasses)
`and reduced them to powder using a mortar, and put back into solution and recentrifuged.
`Instead of using polytron
`
`Page 9
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`NEP877|TC-00679907
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`CX Page 10
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`Note: perfect: obtained no layer on top after the second Centrifugation, and the supernatant was
`very clear, thus no need for a re-suspension 20 mL saline /illegible/, immediately re-suspend 20
`mL acetone.
`
`One step less
`
`Acetone re-suspension 10 mL 20 mL
`
`X redo protocol with fresh from A to Z to validate the results and with vacuum filtration
`17/3/98
`Lowry protein (fraction P) assay (p. 11 black binder)
`lmL sample 9 200 mL
`5 ml. reactive 91ml. 49 ml, A +1 ml,B +1 mI,C
`No SDS added
`
`No Na2I-IP04 = no buffer
`
`CUSO4 SHQO
`
`500 mg/ml. for std
`OK albumen solution preparation
`Stock solution 2-5-ml:-H90-m-I: 75 mL total: 0.0375g 75 mL
`500 mg 9 1 mL
`0105-1-ag
`50
`
`+00
`
`-50-mL
`2-5 mg
`Do not stir hard
`
`12,500 mg (-25 mL
`(=12.5 mg)
`Aliquot 1.5 1 mL
`so (0.0l25g)
`20 frozen aliquots, keep in bottles GIBCO refrigerator
`preparation of Folin reactive (Phenol IN)
`Phenol C6 ZN -> IN
`
`25 mL H20 + 2.5 mL Folin = IN 5 mL
`
`Selutien—A
`preparation Na2CO3 2%
`Solution A
`2g 9 100 mL
`(4g) (- 200 mL
`OK preparation NaOH 0.] N Sela!-ien—Ar
`MW = 40g 9 lmole/L 9 IN
`(4g) (-0.1 mole/L (-0.1 N
`4g 9 1000 1nL
`(0.8g) (- 200 mL
`
`Solution B OK — preparation NaK tartrate 0.02%
`(002g) 9 100 mL
`(-0:09-l-g-)(—5mL
`we
`
`Page 10
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`NEP877ITC-00679908
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`CX Page 11
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`A; -:f‘:
`I
`-"'0
`
`on C OK
`
`17/3/98
`
`X=0.l012g
`
`CUSO4 05%
`Preparation
`0.5g-)100mL
`(0.025g) (-5 10 mL
`
`18/3/98
`Clsg-> l00mL
`(0.25g) (- 50 mL
`
`Calculations: Fresh Krill weight acetone technique 3“ trial
`Cup 1: l.l272g —1.0267g = O.1005gl
`Cup 2; l.l252g—1.0237g—O.lOl5g
`Cup 3; l.1291g —1.0275g= 0_1016g
`
`% fresh Krill lipids weights acetone technique 3"‘ trial
`O.l0l2g/2mL= X/IO mL
`X: O.5060g
`0.5060g/25g = X/100g
`x=2.024%
`
`Calculations: weight of filter contents filtration EtOH (insoluble proteins) album 500 mg
`Cup 1: 3.7808g— l.O367g= 2.7441g
`
`iilllllllllli
`
`IiiiillllllllfiIIIIIIIIIIIIIE
`
`50ul-> lmL
`
`10ug/mL
`
`18/3/98 S - ectro 0 hotometer
`
`solution
`
`I
`
`8 9
`
`-2
`
`10
`
`11 blank
`12
`
`(supernatant
`
`1 1
`
`3
`
`(supernatant
`2)
`14
`
`(supernatant
`3
`
`Page 11
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`N EP877|TC-00679909
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`CX Page 12
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`
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`Separation of neutral lipids and phospholipids by
`20/3/98
`TLC: place 20 uL by “spot” white tips
`1.5 cm bottom of plate
`2 same plates -) l phospholipids
`-) 1 neutral lipids
`
`On the plate (each)
`
`Egg | Chloro-MeOH | Dry Krill Acetone | Fresh Kn'll Acetone | Dry Krill MeOH | Fresh Krill
`EtOH l Eggl
`
`lipids
`I
`I
`BCM I
`Phospho Chloro 30: EtOH 34: H20 8: Tiimethylamine 35 Photo 9-10-11
`Neutrals Hexane 90 — etherdiethyl:20 = acetic acidzl
`Photo 9-1-0-1-1-6-7-8
`
`Notes: phospholipids not well separated, thus redo — develop with iodine
`
`SBCL5 for TLC or SBCI3 development in cliloro
`very toxic chemical
`20-40%
`
`20-40%
`
`m%mwfp
`antimony pentachloride
`
`or SBCI3 in acetic acid
`50%
`80-96 C° 3-5 min
`
`Take cholesterol as std 3°/o solution
`
`See Boywer
`
`Chloroform MeOH
`20 mg/mL
`
`20 mL for each spot
`
`Page 12
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`NEP877ITC-00679910
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`CX Page 13
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`20/3/98 calculations: (continuation of page 40) concentration in soluble proteins from D.O based
`on standard curve of albumin 500 mg/mL at 500 nm
`Supernatant 1 1/50 y =0.0009x + 0.0076
`0.126= 0.0009x + 0.0076
`
`0.126 — 0.0076 / 0.0009 = 0.0009x/0.0009
`
`131.5556 mg/mL X
`
`131.5556 mg/mL x 50 = 6577.78 mg/mL
`= 6.5778 mg/mL
`
`6.5778 mg/mL x 40 mL = 263.1120 mg in supernatant
`
`263.1120 mg/20.26% x 25g = 263.1120 mg/5.0650g = X/100g
`
`X = 51947088 mg/100 g dry weight
`X= 5.19g/100g dry weight
`
`Supernatant 2 1,10 Y=0.0009 x + 0.0076
`0.472= 0.0009x + 0.0076
`0.472-0.0076/0.0009 = 0.0009x/0.0009
`
`516 mg/mL = X
`516 mg/mL x 10 = 5160 mg/mL
`= 5.1600 mg/mL
`5.1600 mg/'mL x 40 mL = 206.4 mg supematam
`
`206.4 mg/ 20.26% 25g = 206.4 mg/5.0650g = X/100g
`x=4075.o247 mg/100g dw
`X=4.07g/100g dw
`
`Page 13
`
`NEP877|TC-00679911
`
`
`
`CX Page 14
`
`-..>..5
`
`20/3/98 Supernatant 2 1/50 Y=0.0009x + 0.0076
`0.108 = 0.0009); + 0.0076
`
`9,108-0.0076/0.0009 = 0.0009x/0.0009
`
`111.5556 mg/mL = X
`111.5556 mg/mL x 50 = 5577.78 mg/mL
`= 5.5778 mg/mL
`5.5778 mg/mLx40 mL
`—223.1l2O mg on 2
`223.1120 mg / 20.26%x25g = 223.1 120mg/5.0650g = X/100g
`x = 4404.9753 mg/100g dw
`= 4.4050g/100g dw
`
`Supernatant 3 1/10
`
`Y—0.0009x+0.0076
`0.3335=0.0009x+0.0076
`
`0.335-0.0076/0.0009 = 0.0009x/0.0009
`
`363.7778 mg/mL = X
`363.7778 mg/mL x 10 = 3637.7778O mg/mL
`= 3.6378 mg/mL
`3.6378 mg/mL x 33 mL = 120.0474 mg
`
`120.0474mg/20.26°/ox25g = 120.0474mg/5.0650g = x/100g
`X=2370.1362 mg/100g dw
`X=2.3701g/100g dw
`
`Supernatant 3 1/50 Y=0.0009x+0.0076
`0.074=0.0009x+0.0076
`
`0.074-0.0076/0.0009 = 0.0009x/0.0009
`
`73.7778 mg/mL = X
`73.7778 mg/mL x 50 = 3688.89 mg/mL
`= 3.6889 mg/mL
`(continues on page 44)
`
`Page 14
`
`NEP877|TC-00679912
`
`
`
`Unédenti.
`
`(200prlgfmi}ugfmlg}18.15
`
`
`18.72 2589 21,72'16,.t3§(500
`PolyH-Poly4:3?
`30,242,7211,112,48fit1,18
`26,344,6724,5241,79
`
`Di 1,91 175 32,03 3,23 1,24
`
`CX Page 15
`
`26/3/98 3.6889 mg/mL x 33 mL = 121.7337 mg in on 3
`121.7337 mg/20.26% x 25g = 121.7337 mg/5.065g = x/100g
`. x= 2403.4294mg
`= 2.4034g/100g p.s.
`
`identification of methyl esters (methylated esters) derivatives
`19/3/98
`by GLC (gas liquid chromatography)
`methylation:
`l l, 15, 26 and 28 separately
`200 mL Biovials 7,
`glass tubes with glass cap 15 mL
`Evaporation of solvents under N
`lillegiblel Antioxidant 10mg/100 mL
`[-1380 l0% in MeOHeaeh—talae
`In each tube
`
`In 65C° bath 2h glass caps
`Cool the ice
`nano
`
`H20 in tubes en-iee (attention)
`Hexane
`
`/illegible upper phase Pasteur pipette
`Hexane in lower phase tube
`Collect upper phase
`Hexane of lower phase
`Collect upper phase
`Combine upper phases of each tube of
`BIOVIAL and evaporate hexane under N
`
`Page 15
`
`NEP877lTC-00679913
`
`
`
`
`
`SaturatedUnsatura. Mono‘(7)GM25,18Fattyacidscomposétéon(‘#3)TubeNo.
`
`
`22,54(11}sum21,422,18
`
`
`
`(26)srott19,39‘22.11
`
`
`
`
`
`22.98
`
`
`
`
`
`
`
`(28)301245,9322,9645,96
`
`
`
`CX Page 16
`
`A‘
`_z.
`.‘ .,.
`
`20/3/98 3.6889 mg/mL x 33 mL = 121.7337 mg on 3
`121.733 7mg/20.26%x25g = 121 .7337mg/5.065g = x/100g
`x=24o3.4294mg
`= 2.4034g 1/100g dw
`
`Identification of methyl esters (methylated esters) derivatives by GLC (gas-liquid
`19/3/98
`chromatography)
`Methylation
`200 mL BIOVIALS 7, ll, 15, 26 and 28 separately
`Glass tubes with glass cap 15 mL
`Evapo of solvents under N
`l0mg/I 0OmL
`Hydroquinone Antioxidant
`2-5-ml: H2804 10% in MeOH in-eaeh—t~ube
`2.5 mL in each tube
`
`Heat a bath at 650C 2h glass caps
`Cool the ice
`nano
`
`2.5 mL H20 in tubes en-iee
`
`(caution, may produce heat)
`1 mL hexane
`
`collect upper phase Pasteur pipette
`1mL hexane in lower phase tube
`collect upper sup
`combine upper phases of each tube of BIOVIAL and evaporate hexane under Njust before GLC.
`
`Page 16
`
`NEP877|TC-00679914
`
`
`
`CX Page 17
`
`19/3/98
`
`Chromatogram TLC 20/3/98 (phospholipids)
`1.
`egg 2. BIOVIAL 7 3: BIOVIAL ll 4: BIOVIAL 26 5: BIOVIAL 15 6: BIOVIAL 28 7:
`93%
`
`development with iodine
`
`Migration solvent: 30, 34, 8, 35
`chloroform — EtOH — H20 — triethylamine
`Chromatogram TLC 20/3/98 (neutral lipids)
`1.
`egg 2. BIOVIAL 7 3: BIOVIAL ll 4 BIOVIAL 26 5: BIOVIAL l5 6: BIOVIAL 28 7:
`988
`Migration solvent: 90: IO: 1
`hexane — diethyl__e_:t_l_ier - acetic acid development with iodine.
`f‘;-
`"Ia.
`l-.-“”)
`
`'._J
`
`\/4
`
`i ..
`
`/\
`
`/
`
`‘I1
`I"
`
`:
`i
`M.
`.1
`1;,
`
`I
`
`«
`
`[As
`” 3
`—-
`
`.‘,;.:—a,3}_
`’-""5
`--
`
`‘,'\“
`/,a\
`
`'-V.’
`l
`l
`\.__J
`
`, --~.
`KN»);
`
`3?:
`3
`3'
`.1:
`
`I
`
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`3/"
`;
`
`.
`/3
`'\
`I
`I.
`A,
`\\/V,
`
`c
`T
`'9“!
`'3.’
`
`_..\
`L;
`5
`
`I
`
`_
`/"\l
`I
`.
`'
`
`“\
`!_‘~,|
`
`IA
`
`1
`
`~
`
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`I
`
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`U
`
`2
`
`y\
`
`-"\
`LI’
`
`-[A
`
`,-
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`‘\
`1
`
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`}
`7’
`
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`-
`]
`\...—r'
`
`/'-\
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`._./
`
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`2‘
`t
`<./
`
`I/*\_‘
`I,
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`I
`I
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`M‘
`ilfliz
`M
`
`\
`
`{
`
`/N‘
`I -
`
`I x
`‘
`’
`K)
`
`Volume of20 pL-5 11L to be deposited by spots
`
`Page 17
`
`N EP877|TC-00679915
`
`
`
`CX Page 18
`
`Methylated: fat hydrolysis + BF3 MeOl-1 to esterify (saponification)
`transestedfication H2804 + MeOH
`
`boiling point
`this
`Methylated esters have a lot of affinity for organic solvents and not much for H20
`
`20/3/98 Calculations: RF from TLC chromatogram
`R, = D substitute/D solvent
`
`1. neutral lipids from bottom to top 2. Dry Krill chloro MeOH
`
`Rf= 0.7 cm/13.4cm = 0.05
`
`Rf=1.0/13.4 = 0.07
`
`Rf = 3.6/l3.4 = 0.27
`
`RF=5.0/I 3.4=0.37
`
`Rf=6.0/l3.4=0.45
`
`Rf=12.7/13.4=0.95
`
`Rf: 12.7/13.4=O.95
`
`Volume of 20 uL-5 uL to be deposited by spots.
`
`Page 18
`
`NEP877|TC-00679916
`
`
`
`CX Page 19
`
`Page 19
`
`N EP877|TC-00679917
`
`
`
`CX Page 20
`
`Page 20
`
`NEP877|TC-00679918
`
`
`
`CX Page 21
`
`Methylated: fat hydrolysis + BF 3 MeOH for esterification
`(saponification)
`/illegible/
`
`Note: Perhaps I should reduce the volume to be deposited by spots from 20 pl to 5 pL.
`
`Page 21
`
`NEP877|TC-00679919
`
`
`
`CX Page 22
`
`23/5/98 and 25/3/98
`Redo fresh Krill acetone tech
`
`With very thin filaments (not a lot) -) weigh and reweigh after filtration with deposit
`
`Polytron to obtain insoluble residue
`
`Done on 14/4/98 redo TLC with standard cholesterol
`
`10 mL
`
`Not immediately
`65
`25
`
`3
`
`4
`
`chloroform methanol H20
`
`acetic acid
`
`Theory: PC not soluble in acetone
`Cholesterol solubility 1.29% in EtOH 20 C°
`Put cholesterol in hexane
`
`Purple cholesterol with SBCI3
`
`Done 3/4/98
`
`Prepare charts Title: extraction of lipids from Krill
`
`Suspension (9 vol solvent 1)
`Filtration (metallic filter)
`(Vacuum)
`Suspension solvent 11
`
`Evaporation (solvent 1)
`
`Filtration
`
`Lipids fraction I
`
`evaporation (solvent II)
`Suspension
`lipids fraction II
`Saline
`Centrifugation or filtration
`Insoluble eesielue matter
`Fraction insoluble matter
`
`soluble proteins
`fraction P
`
`I.M.
`
`Page 22
`
`N EP877|TC-00679920
`
`
`
`CX Page 23
`
`1.13 mg,/mL=C;
`tube 28 5.67 mg/mL x10 mL = Czx 100 mL
`5.67 mg/ml, x 10 mL/100 mL = C;
`
`0.57 mg/mL = C2
`
`24/3/98
`
`Filter content weight before re-suspension Saline: l5.0089g, rest of acetone
`
`Note: 39 mL saline rather than 40 mL and solution in 2 tubes since 1 is too full
`
`Note: after the first centrifuge, no layer on top, thus no need for polytoner
`
`Note: 1000 rpm 5 min flask content
`10 mL EtOH and 5 mL MeOH in flask to remove content from the walls.
`
`Transfer MeOH flask to tube with residue (EtOH previously removed after centrifuge). 7 mL of
`MeOH supernatant
`
`GLC tubes 2"“ trial
`
`Note: take 200 uL rather than 10 uL
`2991+1T—>—1eL,117—>—1—.+r1, OK
`2011-I.->-(_-1—p-I7)->-1-9G0,—1—mL—9Ié
` OK
`and re-suspend (tube 15) in -1-00-p-1 20 uL C: 15 -2-00-p-I:->-1-G9-pi-1:
`-2eH¢1T—>+1o«1L9
`
`11.18 mg/100 pg (and less)x 20 uL = Cgx-10020 pL
`11.18 mg/100 pgx 20 uL/100 p.L = C2
`1l.18mg/ 100 ugx20mL=C2
`+99-1111720 pl
`11.18 mg/100 pg (and less)=C;
` =C2
` =C2
` =C2
`111.8 mg/mL (and less)= C;
`
`Page 23
`
`NEP877ITC-00679921
`
`
`
`CX Page 24
`
`.,v,
`.«-
`.A;-‘_’.
`
`Note: collected supernatant of both tubes in only 1 tube and put together both residues in 1 tube
`and added 10 mL saline to it and centrifuged at 3000 rpm
`
`24/3/98
`
`15 min
`
`with lipids
`empty
`l.2l68g
`l.2l65g
`l.0403g
`Cup 1
`Fresh Krill
`1 .2228g
`1.0371g
`Tech re-suspended hexane Cup 2
`l.2170g
`l.2168g
`Acetone 3
`1.037lg
`1.1299g
`1.1218g
`Double
`1.0324g
`5
`Extraction
`l.O350g
`l.l289g
`I .1273g
`EtOH re-suspended 6
`1.0319g
`l.1248g
`1.1206g
`With hydrosoluble subs
`
`4
`
`l.2l65g
`
`Re-suspended
`MeOH
`
`re-suspended
`saline
`
`residue
`
`7
`8
`9
`
`l.0263g
`l.0274g
`1.0276g
`
`1.0501 g
`l.0S89g
`1.0567g 0.-939-7—g
`l.0583g
`1.0584g
`l.0590g
`with insoluble residue
`
`10
`11
`
`2.67 10g
`l.0298g
`0.-93-081.0308g 2.567Sg
`
`4-94 2.0961g
`2.10l6g to be added since same
`
`l.8892g
`2. l370g
`l.0329g
`12
`Note: collected above—mentioned centnfugation supernatant for a supematant total of 44mL, then
`wash residue 20 mL acetone and centrifugation 3000 rpm 5 min. Re-wash with 20 mL acetone
`and re-centri 3000 rpm 5 min, residue in cups, place in the oven for 1 night, weighing.
`
`Note: take 1.5 mL instead of 2 for MeOH cups. Calculate with 7.5 mL rather than 10 mL of
`hexane.
`
`Krill 25g in 225 mL acetone incubation
`25/3/98
`1 piece 1 night agitation 20 min
`remains: see p. 24 modified version
`1 weight: 0_0778g
`0.1820g
`Very thin filaments
`
`Page 24
`
`NEP877lTC-00679922
`
`
`
`CX Page 25
`
`24/3/98
`
`25/3/98
`
`Cup 10
`Cup 11
`Cup 12
`
`7.3755g
`-3.7049g
`3.6706g
`
`6.0869g
`-3.7049g
`2.3820g
`
`‘
`J
`Difference of l.2886g
`.33..
`‘
`
`
`
`2 filaments’ weights: 0.0648gwith deposit 0.1385g
`
`Note: rinse the flask with 10 mL hexane after pouring 25 mL hexane and lipids into the ampulla.
`Good separation!
`Upon evaporation of the acetone, the solution was boiling very very easily and a lot even when
`acetone no longer evaporated: water? Especially as everything separated well by decantation in
`the ampulla without agitation.
`
`Note: there were debris between the aqueous phase and hexane lipids phase. I got rid of those
`debris by moving them to the aqueous phase but pigments followed, thus certainly small loss of
`lipids. For the second decantation also.
`
`Note: 3 filaments’ weights 0.0775g O.1728g
`Filter deposit was incubated for 1.5 hrs. in EtOH before evaporation of EtOH
`Note: Centrifuge 10 mL EtOH flask content 1000 rpm 5 min and 5 mL MeOH in flask to
`solubilize -) supernatant: 5 mL
`
`Page 25
`
`N EP877|TC-00679923
`
`
`
`CX Page 26
`
`
`
`Filter deposit weight before re-suspersion
`saline 12.6216 g (free fall)
`Note: Approx. same quantity of filter deposit in each tube + 20 1nL saline in each tube. agitation and centrifuge 3000
`rpm 15 min then 10 mL saline on each residue and re-centrifuge 3000 rpm 15 min
`Supernatant: 50.5 rnL total
`20 mL acetone per residue and centri 3000 rpm 5 min
`Re-wash with 20 mL acetone per residue and centri 3000 rpm 5 min
`Residue in cups . drying on heating plate. weighing
`
`Note: weight loss of 1 krill by removing acetone
`
`25/3/98
`1
`........
`
`2"“ wash
`
`‘
`’
`
`with iipidsyzg/3/93
`
`2 3
`
`Cup
`
`Cup
`
`enough 11/1e0H supernatarit for 3 cups) :
`
`..
`
`st
`
`“"°‘*7«;:'
`
`_
`_
`_
`To add since this is the
`V?-same residue
`;"
`
`
`
`_
`
`Re-suspended4
`hexane
`
`5 6
`
`Re-suspended7
`MeOH
`
`8 9
`
` Insoluble
`
`residue
`
`10
`11
`
`12
`
`V
`
`fresh Krill
`acetone tech
`double extraction
`
`‘
`
`Calculations:
`25/3/98 tube 7
`
`in tubes for GLC
`
`15.51 mg/mL x 0.2 mL = C3 x100 pL
`3.102mg/0.l00mL = C;
`-1—5:5-Hag
`31.02 mg/mL = C 3
`Tube 11 8.15 mg/mL X 0.2 mL = C‘; x 100 iiL
`1.630mg/0.1 n1L = C;
`
`Page 26
`
`NEP877ITC-00679924
`
`
`
`CX Page 27
`
`:'..§"§ s1.5
`
`«-
`
`
`Weight of fresh Krill éééféiié’{飧li}iiLi{ié'§}§}i£ii'}iiiE}3h1amems
`P. 48 (23/3/98) hexane
`
`Cup1
`
`°4’..,9,.f.._l$Fl_l.l..l.l.Ri9§_f!§§h ,w_ei r
`(v
`5 {av
`
`'73;\§
`
`ll’
`
`___:‘__'-:53_______________
`
`
`
`
`of krill lipids dry weight
`5.
`
`X‘: 13.4% dry weight 1
`»,:
`
`
`Page 27
`
`NEP877|TC-00679925
`
`I6.30mg/mL=C;
`
`flf§3,;.l:;:-,-‘.
`
`--35
`
`o
`
`... _
`‘
`\r‘1I
`;,_ H,,,,:__:
`
`1
`
`.
`
`‘$5:
`
`’(,2-1
`
`
`
`Calculations:
`Loss in thin filaments
`Those of 24/3/98
`‘If
`3
`
`
`
`_.r
`
`g
`
`
`1
`V
`5.: 2
`not very reliable because of oxidation
`% of dry weight which is loss infilters
`-
`. 1"“.
`.
`-.....»........,,¢....
`*-
`,<":3'.'.<.‘=».¢‘Q ....... ..
`
`
`
`
`
`CX Page 28
`
`Note: drop like oil in H20 in the 3.
`22/5/98 Note: N bottle emptied, then wait until Monday for EtOH and MeOH to evaporate
`under N.
`
`Calculations: Krill lipids weight acetone tech microfilaments
`hexane
`
`(.0 4;)“:
`
`
`
`
`
`-;
`3 974%
`:,,
`Krill weight acetone tech microfilaments MeOH
`....,.,~ .
`.1.-n.u..\~.y.-;r.... ..gr'<-W"-vzv,
`N"
`-
`.
`if
`
`.3
`
`
`
`
`
`~‘-"\,_.‘
`‘ "
`29/5-5/6 mission at sea Martha L Black
`
`9/6/98 absorption spectrum sun lotion done 12/5/98
`Against
`Against tap water
`
`Page 28
`
`NEP877|TC-00679926
`
`
`
`CX Page 29
`
`
`
`Aé£1i'1fst water _
`--3 <3
`1'}
`
`
` :
`
`=. '».~.~.;
`
`§%tfi?SR?
`
`
`
`
`
`Note: dilute 1/10 in oil and take water nzmo
`So sLm lotion with oil as blank
`'~gg:§'3L<R.«'.« 1,
`
`10/6/98
`
`Page 29
`
`NEP877|TC-00679927
`
`
`
`CX Page 30
`
`Harvested species Calamus hyperboreus
`Martha L. Black
`Meganyctiphanes norvegica
`Thysanoessa raschii
`Mysidicea
`Euchaeta
`
`10/6/98 Fresh Krill Meganyctiphanes norvegica
`stirred 20 min
`
`incubated 18h 4‘C
`
`vacuum filtration microfilaments
`flask rinsed 10 mL hexane
`
`flask on ice during evaporation
`Note: difficult evaporations (80 °C)
`
`Page 30
`
`N EP877|TC-00679928
`
`
`
`CX Page 31
`
`
`
`Note: incubation 10h50 — l3h50 4°C EtOH (45) 3h
`Note: 10 mL EtOH in flask5 min 1000 rpm flask content
`empty tube weight: 16.09g
`5 mL MeOH in flask
`
`Transfer MeOH to tube containing only centrifuged residue
`(EtOH removed)
`Note: During 2"" decantation, the upper portion is not colored contrary to the light yellow color
`with E. Pacifica
`
`Note: EtOH flask content as colored as the hexane flask content contrary to the EtOH flask
`which is usually more yellow (than orange) the hexane flask
`
`Note: the krill (Meganyctiphanes) remains pigmented after the EtOH step (EtOH filter deposit)
`
`Aqueous phase volume: 12.5 mL
`EtOH volume: 11.5 mL very colored
`MeOH volume: 5 mL colored
`
`Page 31
`
`NEP877|TC-00679929
`
`
`
`CX Page 32
`
`Note: MeOH eentei-it tube( residue safer)
`Centrifuge 5 min 1000 rpm
`Tube weight with MeOH residue: 16.07g
`(impossible empty = l6.09g!)
`For calculations: hexane 9 13.8 mL instead ofD
`EIOH 9 l 1.5 mL instead of
`
`ll/6/98
`
`MeOH 9 5 mL instead of
`Fresh Calanus: stirred for 20 min.
`
`Flask on ice
`
`incubated 18h 4°C
`
`Vacuum filtration microfilaments
`flask rinsed hexane
`
`During evaporation 10 mL EIOH in flask
`5 min 1000 rpm flask content
`5 mL MeOH in flask
`Transfer MeOH in tube
`
`containing only centrifuged residue (removed EtOH)
`Centrifuge 5 min 1000 rpm
`
`Note: flask content acetone + lipids
`Fluorescent orange Cal anus before evaporation
`
`Note: During first decantation, aqueous phase like cream soda not at all pigmented. Upper phase
`orange Mr. Freeze (fluo like in the flask before evaporation).
`
`Note: during 2"“ decantation, upper phase yellowish
`Lower phase like first decantation
`
`15.5905g tube + cap weight for MeOH
`
`Page 32
`
`NEP877ITC-00679930
`
`
`
`CX Page 33
`
`
`
`
`_
`__ V _!
`:
`Note: difficult evaporations! 80°C for hexane and 85°C for EtOH and very long. Not good
`vacuum, low H20 pressure
`
`_‘
`
`Aqueous phase volume: 20 mL
`EtOH volume: 14 mL
`MeOH volume: 5.5 mL MeOH
`
`For calculations: 17.5 mL hexane
`14 mL EtOH
`
`12/6/98
`5.5 mL MeOH5.5 cylinder 4.9 / pipette 5 mL!
`Note: Same orange Mr. Freeze coloration for hexane fraction and EtOH fraction
`
`Note: the Calanus remains pigmented
`Mr. Freeze orange after EtOH treatment (EtOH filter deposit)
`
`Tube weight MeOH 15.643 8g with deposit
`12/6/98 Fresh Krill Meganyctiphanes
`10 min stirred
`
`incubated 15h 4‘C
`
`l8h -)9h
`
`microfilaments filtration
`flask rinsed 10 mL hexane
`
`Flask on ice during evaporation
`
`Page 33
`
`NEP877|TC-00679931
`
`
`
`CX Page 34
`
`10 mL EtOH flask
`
`5 min 1000 rpm flask content
`5 mL MeOH in flask
`
`transfer MeOH to tube containing only centrifugated residue (EtOH removed)
`centrifuge 5 min 1000 rpm
`
`Note: acetone flask before evaporation is a lighter shade of orange than Mr. Freeze.
`
`Note: During 15' decantation, lower phase colored light kaki and the upper phase fluo orange
`
`Note: aqueous phase 12 mL greenish
`
`12/6/98
`cub
`
`__________
`
`‘I
`
`empty
`
`with lipids
`
`4/15/93
`
`«««««««-~
`
`
`
`
`
`
`
`
`
`
`Note: EtOH flask content as colored as hexane content
`Note: EtOH incubation 2 hours rather than 3
`
`tube weight for empty MeOH: 15.64g with residue: lS.64g
`
`Page 34
`
`N EP877ITC-00679932
`
`
`
`CX Page 35
`
`Note: Meganyctiphanes remains pigmented after washing with EtOH
`EtOH flask tube centrifuged very colored very red
`
`15/6/9
`Note: deposit stuck on the flask which cannot be removed with MeOH, perhaps because too
`much heat during evaporation
`
`EtOH volume: 11 mL
`MeOH volume: 5.5 mL
`
`Note: EtOH BIOVIAL tube (red) darker than hexane BIOVIAL tube (orange)
`
`15/6/98 Fresh Calanus: 20 min stirred
`
`incubated 15h 4°C 18h20 -) 9h20
`Microfilaments filtration
`1516/
`Rinsed flask lOmL hexane
`
`Flask on ice during evaporation
`
`10 mL EtOH flask
`
`5 min 1000 rpm flask content
`5 mL MeOH in flask
`
`Transfer MeOH to tube containing only centrifuged residue (EtOH removed)
`Centrifuge 5 min 1000 rpm
`
`Note: incubation in EtOH: 1h (10h2O -) 11h20)
`MeOH tube weight: 1S.34l8g empty
`15.3674g with residue
`
`MeOH
`
`Page 35
`
`NEP877ITC-00679933
`
`
`
`CX Page 36
`
`.’ I
`.*r%_:,:. "
`
`20/7/98
`
`Protein recovery a£ter—ene-weekend
`lg Krill insoluble substance in 10 mL NaOH 0, IN
`one weekend at 4 °C
`
` r..:“u
`*
`5
`é
`if
`,
`
`.5
`
`‘*5
`.1
`»
`" "
`.
`1%-p.‘
`
`_‘
`X».
`
`.
`fa
`
`v
`~
`
`:4.
`_
`.
`.3:
`'"
`5...:
`fig‘
`-f\:-
`“.
`g
`.
`4.
`A
`..
`Q“'~._
`'‘2;-'‘-
`8 J 5
`9 in Hi‘;
`‘A-‘
`5%
`,'.__ N .
`»
`:2
`51,
`.3
`
`-~
`£5
`
`.
`.v_y
`3‘
`,,
`..
`..'.*:*~
`2‘-‘
`1* g
`‘me
`
`ef
`;__
`st
`'-‘v
`
`.5
`,
`“Fe
`-- P
`0"
`‘.'.;‘§
`:3
`.1.
`(1,
`%>».
`1:‘
`J
`‘OUR.
`2?
`.33,
`
`0
`3;
`0
`M
`‘
`
`‘(T65
`
`1.65g -) 25g Krill
`(6.6) mag Krill
`therefore 6.6% of proteins
`
`Page 36
`
`NEP877|TC-00679934
`
`
`
`CX Page 37
`
`20/7/98 protein recovery aiftepeneaweekeaé
`lg Krill insoluble substance in 10 mL NaOH 0.1N
`-«om
`-
`_> H
`One ""°._"‘.?.$?.’__‘.‘.‘?‘. :17?
`
`—«..
`
`
`
`'23:‘
`
`-;:~
`
`:
`
`A 1- M”
`
`n¢'.;
`$32
`,«__h
`
`
`
`i'xI
`
`.fl.fl.
`
`1.65 —) 25g Krill
`(6.6) 100g Kn'll
`therefore 6.6% of proteins
`
`Page 37
`
`NEP877|TC-00679935
`
`
`
`CX Page 38
`
`¢._ .
`2- 2
`
`.;.
`:
`.’t‘§‘£2
`
`20/7/98
`
`Protein recovery afier-one-weekend
`lg Krill insoluble substance in 10 mL NaOH 0.1N
`one weekend at 4°C
`
`concentration in
`
`protein (mg/mL)
`
`4-?)
`
`3'3
`
`l.65g -) 25g Krill
`(6.6) 100g Krill
`therefore 6.6% of proteins
`
`Page 38
`
`NEP877|TC-00679936
`
`
`
`CX Page 39
`
` 3 7/20/98
`
`Protein recovery afi
`lg Krill insoluble substance in 10 mL NaOH 0 IN
`one weekend at 4°C
`
`centrifuge 4000 rpm 5 min
`samc aligmncm on plate as 17/7/98
`Note: bubbles. then reslan std curve
`
`NaOH blank
`
`.9-:
`
`
`
`.
`
`.
`
`:;..
`
`
`Based on standard curve (not very good, but to give an idea)
`1/10 D.O. 0.060 (removed blank)
`—) 5.5 mg/mL
`5.5 mg/mLx 10 = 55 mg/mL
`N
`
`
`
`Page 39
`
`NEP877|TC-00679937
`
`
`
`CX Page 40
`
`24/7/98 mackerel fish internal organs l6g in 144 mL
`I lh25 -) l0h30 I7 days 23h 4°C acetone
`
`24/7/98
`
`6/7/98
`slinecl 20 min
`microfilainents filtration
`flask rinsed with 10 mL hexane
`
`flask on ice evapo
`
`;....>.~ §\‘3 5.,»
`
`28/7/98
`
`pids
`
`
`« 4)
`
`
`
`Cup
`
`5'..................
`
`’
`
`
`
`
`
`Note: 2”“ decantation unusual: 3 distinct phases
`I only took the one on the top
`24/7/98 mackerel fish 4 tissues 25g in 225 mL
`111125 -) 131145
`18 days 2h25
`4'Cacetone
`
`6/7/98
`
`24/7/98
`
`-
`
`empty
`
`Notes: EIOI-l flask tissues filled with deposits on the wall contrary to the flask containing Etol-l internal organs
`27/7/98 seeding NA with Krill in acetone since 7/4/98
`
`25 1nL acetone orange flash Mr. Freeze
`Note: Krill 5g
`Krill 5g 10 ml.
`coral color
`5 inL
`salmon pink
`
`Smells of acetone and krill and not pulrefaction
`
`Page 40
`
`NEP877|TC-00679938
`
`
`
`CX Page 41
`
`J
`-~
`.-
`.
`‘
`'~’»‘=.’),’<‘~Z)1,.I§.:':27/7/98 extraction krill lipids in acetone since 7/4/98
`5g fresh krill in 10 mL acetone
`deposit 1“ filtration in 10 mL acetone
`deposit in 20 mL EtOH
`5 mL hexane and decantation
`rinse flask with 2ml hexane
`
`1
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