APPENDIX G

APPENDIX
G
NEPTUNE KRILL OILTM
&
AQUATEINETM (Krill Protein Concentrate)
EXTRACTION
PROCEDURES
WO 00/23546
PCTICA9910098;
PATENT COOPERATION
TREATY
From the INTERNATIONAL BUREAU
To:
PCT
DUBUC, Jean, H.
Goudreau Gage Dubuc & Martineau
Walker
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PCTlCA99100987
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I
/
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21 October 1998 (21.10.98)
ITE DE SHERBROOKE et al
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PCT/CA99/00987’
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PCT
INTERNATIONAL
WORLD
APPLICATION
51) International Patent Classitication 7 :
CllB
INlELLECl-UAL
COOPERATION
(11) International Publication Number:
I
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PCTlCA99tOO987
21 October 1999 (21.10.99)
21 October 1998 (21.10.98)
TREATY (PC-l-)
CA
71) Applicant fjbr all designated Sfarrs except VSj: UNIVERSITE
DE SHERBROOKE (CA/CA]; University Boulevard, Sherbrooke, Quebec J I K 2R I (CA).
‘72) Inventors; and
175)1nventorJApplicants (,for US only): BEAUDOIN. Adrien
[CA/CA]; 748. boulevard des V&&-am, Rock Forest, Quebec JIN I27 (CA). MARTIN. Genevitve [CA/CA]; 797,
McManamy. Sherbrooke, Quebec JIH 2N1 (CA).
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Es. m. GB. GD, GE. GH. GM, HR. Hu.ID.IL.IN. Is.JP,
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SD. SE. SG. Sl, SK, SL, TJ. TM. TR. IT, -l-Z, UA. UG,
US. UZ, VN, YU, ZA. ZW. ARIPO patent (GH, GM. KE,
LS. MW. SD, SL, SZ, Tz. UG, ZW). Eurasian patent (AM
AZ, BY, KG. KZ, MD, RU, TJ. TM), European patent (AT,
BE, CR CY. DE, DK. ES, FI, FR. GB. GR. IE. IT, LU
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Published
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4
:74) Agents: DUBUC. Jean. H. et al.; Goudreau Gage Dubuc &
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lE9 (CA).
(54) Title:
MElTlOD OF EXTRACllNG
LIPIDS FROM MARINE AND AQUATIC ANIMAL TISSUES
(57) Abstract
Provided herein is a method for extracting lipid fractions from marine and aquatic animal material by acetone extraction. The
resulting non-soluble and particulate fraction is preferably sub&ted to an additional solvent extraction with an alcohol, preferably ethanol
isopropanol or r-butanol or an esfer of acetic acid, preferably ethyl acetate to achieve extraction of the remaining soluble lipid fraction fron;
the marine and aquatic animal material. The remaining non-soluble particulate contents is also recovered since it is enriched in protein:
and contains a useful amount of active enzymes. Also provided herein is a krill extract.
-_-
FOR THE PURPOSES OF INFORMATION
ONLY
Codes used to identify States party to the PCT on the Front pages of pamphlets publishing international applications under the PCI’
AL
Ah1
AT
AU
AZ
BA
BB
BE
UF
BC
BJ
BR
BY
CA
CF
CC
CII
Cl
Chl
CN
cu
CZ
DE
DK
BE
Albania
Armenia
Amwir
Aurralia
Azerbaijan
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Belgium
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Benin
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Es
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FR
CA
CB
GE
CH
CN
CR
HU
IE
IL
IS
IT
JP
KE
KG
EP
CXlfIlCWOll
China
Cuba
Czech Republic
Germany
Denmark
Estomo
KR
Kz
LC
I.1
LK
LR
Spain
Finland
hlncc
GZhtl
Uniud Kingdom
Georgia
GhZl-la
Guinea
Greccc
Is
LT
LU
LV
MC
hlD
MC
MK
Huwar/
Ireland
lrncl
Iceland
_
ltaly
Japan
Kenya
Kyrgyrrrm
Dcmanlic
Pcoplc’s
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Republic of Korea
KZ?XkStUl
Saint Lucia
Licchtenstcin
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hlN
MR
MW
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NL
NO
NZ
PL
ET
RO
RU
SD
SE
SG
Lcrod!a
Lithuania
LuXCmbOUrg
Lalvu
hloruco
Republic of Moldova
Madagscu
The fomlcr Yugorlsv
Republic of Mlcedonia
Mali
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hlauimia
Malawi
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Niger
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Nowry
New Zwland
Poland
Pcflugal
Romania
Rursun Federation
St&n
SW&l
Singlporc
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SK
SN
sz
TD
TC
TJ
Thl
TR
l-r
UA
UC
US
uz
VN
YU
zw
Sbvenia
Sbvtia
Senegal
Swaziland
Chad
Tog0
TajikLcan
Turkmenistan
Turkey
Trinidad and Tobago
Ukraine
Ugacda
United Suwr of Amcr~ca
Uzbckiaan
Via Nam
Yugoslavia
Zimbabwe
wo
PCT/CA99/00987
QQr23546
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2
Fish farming
Among the lipids found in krill, Calanus and fish, high concentrations
. 205 (eicosapentaenoic
acid) and 22:6 (docosahexaenoic
of fatty acids
acid) are present. These
fatty acids are essential nutrients and are beneficial as fish feed. Furthermore, these
5
essential nutrients are carried over in human diet by eating the fish grown on such
diets.
Animal feed
Animal feed diets rich in omega-3 fatty acids may increase the level of unsaturated
fatty acids and decrease cholesterol levels of meat. This property is already exploited
10
in the poultry industry to improve the quality of eggs.
Various
methods
for extracting
marine and aquatic animal oils are known, For
example, it is known to extract fish oil using organic solvents such as hexane and
ethanol.
15
It is also known to measure the fat content in fish muscle tissue using
solvents such as acetone.
USP 4,331,695 describes a method using pressurized solvents which are gaseous
at room temperature,
such as propane, butane or hexane.
The extraction
is
performed at preferred temperatures of 15 to 80°C on shredded vegetable or finely
20
divided animal products.
The extracted oils are then made to precipitate under high
pressure and elevated temperatures
extraction
solvent
temperatures
25
for marine
of 50 to 2OO’C. However, hexane is a poor
animals
such as krill.
Furthermore,
the high
used in the precipitation step negatively alters the lipids.
Canadian Patent Application 2,115,571 describes a method for extracting oils from
various brown and read algae species. The method provides for example Soxhlet
extraction using nearly pure ethanol for 40 hours.
USP 5,006,281
30
animals
describes
such as fish.
a method for extracting oil from marine and aquatic
The marine and aquatic animal is first treated with an
antioxidant compound, finely divided and centrifuged to separate the oil phase from
PCTlCA9~~00987
WO OOt23546
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3
the aqueous
antioxidant
The oil phase is then further treated with
to remove undesirable odour or taste.
Canadian
5
phase and solid phase.
Patent 1,098,900 describes a method for extracting oils from krill. The
method involves emulsifying fresh or defrosted krill in an aqueous medium. The oil
fraction is recovered
by centrifugation.
Folch in the article published in the year 1957 in J. biol. Chem. 226: 497-509
simple method for the isolation and purification
10
of total /@ids from animal tissues”
proposes an extraction method using chloroform and methanol.
commercially
quantitative
improved
This method is not
feasible because of the toxicity of the solvents involved.
However, prior art processes are generally commercially
15
“A
yields.
unfeasible or provide low
Thus, it is an object of the present invention to provide an
marine and aquatic animal oil extraction
method allowing recovery of a
valuable lipid fraction and separate recovery of a valuable protein rich solid residue
that comprises
active enzymes.
Other objects and further scope of applicability of the present invention will become
20
apparent from the detailed description given hereinafter.
It should be understood,
however, that this detailed description, while indicating preferred embodiments
invention,
is given
modifications
by way of illustration
only,
since
various
changes
of the
and
within the spirit and scope of the invention will become apparent to
those skilled in the art.
25
BRIEF DESCRIPTION
Figure
1. Gas-liquid
OF THE DRAWINGS
chromalography
of fatty acids from dry krill (chloroform-
methanol)
30
Figure 2. Gas-liquid chromatography
of fatty acids from dry krill (acetone)
Figure 3. Gas-liquid chromatography
of fatty acids from frozen krill (acetone)
Figure 4. Gas-liquid chromatography
of fatty acids from frozen krill (ethanol)
Figure 5. Gas-liquid chromatography
of fatty acids from frozen krill (t-butanol)
PCT/CA99/00987
W O 00/23546
4
Figure 6. Gas-liquid chromatography
of fatty acids from frozen krill (ethyl
acetate)
Figure 7. Thin-layer chromatography
of neutral lipids of Caianus sp. and
M . nowegica
5
Figure 8. Thin-layer chromatography
of neutral lipids of E. pacifica
Figure 9. Thin-layer chromatography
of neutral lipids of M . schmitti
Figure 10. Thin-layer chromatography
of neutral lipids of G. galeus
Figure 11. Thin-layer chromatography
of neutral lipids of Angel Shark
Figure 12. Thin-layer chromatography of phospholipids of Calanus sp. and
10
M . norvegica
Figure 13. Thin-layer chromatography of phospholipids of E. pacifica
Figure 14. Thin-layer chromatography of phospholipids of M . schmiffi
Figure 15. Thin-layer chromatography of phospholipids of G. galeus
Figure 16. Thin-layer chromatography of phospholipids of Angel Shark
15
Figure 17. Influence of the volume of acetone on lipid extraction (E. pacifrca)
Figure 18. Influence of incubation time in acetone on lipid extraction
(E. pacifica)
Figure 19. Influence of the volume of ethanol on lipid extraction (E. pacifica)
Figure 20. Influence of incubation time in ethanol on lipid extraction
20
(T. raschii)
DETAILED DESCRIPTION
OF THE PREFERRED EMBODIMENT
Before describing the present invention in detail, it is to be understood that the
invention is not lim ited in its application to the process details described herein. The
25
invention is capable of other embodiments and of being practised in various ways.
It is also to be understood that the phraseology or terminology used herein is for the
purpose of description and not lim itation.
The method of the invention comprises suspending freshly collected marine and
30
aquatic material in acetone. Lipids are extracted with a ketone such as acetone.
This allows a rapid dehydration of animal tissue and a m igration of the lipid fraction
to the solvent. The dry residue is a valuable product rich in active enzymes.
PCTICA99(00987
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5
In a preferred embodiment, the extraction is carried out by successive acetone and
alcohol treatments.
Preferred alcohols are isopropanol, and t-butanol. The alcohol
’ may also be substituted with an ester of acetic acid such as ethyl acetate.
procedure
5
The
produces two successive lipid fractions and a dry residue enriched in
protein, including active enzymes. Recovery of total lipids is comparable to the Folch
et al. (1957) procedure reported in the background of the invention. It has been
tested with krill, Calanus, fish and shark tissues.
Surprisingly, it was found that successive extraction treatments as proposed by the
10
present invention has a better yield in lipid extraction that single solvent system
extractions. The extraction using two successive solvents which starts with a ketone
1’
such as acetone is especially advantageous since the acetone, in effect, dehydrates
the animal tissue. Having the animal tissue in dehydrated form greatly facilitates the
extraction process with the second solvent, alcohol or an ester of acetic acid such
15
as ethyl acetate.
In the case of zooplancton such as krill and Calanus and in the case of fish-filleting
by-products such as fish viscera, it is noted that extraction with acetone alone may
be sufficient to allow a cost-effective recovery of lipid fractions and separate recovery
20
of a dry solid product rich in proteins including active enzymes.
The general extraction method of the present invention will now be described. The
starting material consisting of freshly harvested and preferably finely divided marine’
and aquatic animal material is subjected to acetone extraction, for at about two hours
25
and preferably overnight. However extraction time is not critical to the yield of lipid
extraction.
To facilitate extraction, it is preferable to use particles of less than 5 m m
in diameter. Extraction is preferably conducted under inert atmosphere and at a
temperature in the order of about 5’C or less.
30
Preferably, the beginning of the extraction will be conducted under agitation for about
10 to 40 m inutes, preferably 20 m inutes. Although extraction time is not critical, it
1
PCT/C A99!00987
W O OOi23546
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6
was found that a 2 hour extraction with 6:l volume ratio of acetone to marine and
aquatic animal material is best.
The solubilized lipid fractions are separated from the solid material by standard
5
techniques
including,
for example,
filtration,
centrifugation
,
or sedimentation.
Filtration is preferably used.
After
separation by filtration on an organic solvent resistant filter (metal, glass or
paper) the residue is optionally washed with pure acetone, preferably two volumes
10
(original volume of material) to recover yet more lipids. The combined filtrates are
evaporated
under reduced pressure. Optionally, flash evaporation or spray drying
may be used. The water residue obtained after evaporation is allowed to separate
from the oil phase (fraction I) at low temperature.
15
The solid residue collected on the filter is suspended and extracted with alcohol,
such as ethanol, isopropanol, f-butanol or altemativelv with ethvl acetate, nreferablv
two volumes (orininal volume of material). The filtrate is evaporated leaving a second
fraction of lipids (identified as fraction II).
Although the extraction period is not
critical, it was found that an extraction time of about 30 m inutes is sufficient
20
temperatures
below about 5’C.
Temperature
of the organic solvents, except f-butanol, and temperature
at
of the
sample are not critical parameters, but it is preferable to be as cold as possible.
However, in the case of t-butanol which is solid at room temperature, it is important
25
to warm it before using it and to perform the extraction at 25 “C immediately.
Comoarative
examples
To compare the efficiency of the extraction process, a classical technique (Folch et
al. 1957) using chloroform and methanol was applied to krill. This method is the
30
reference
for measuring
efficiency
been made with a technique
of the extraction process. Another
using hexane
as the extraction
solvent.
comparison
has
Lipid recovery
PCTlCA99l0098l
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7
was estimated by suspending lipid fractions in small volumes of their original solvents
and measuring by gravimetty small aliquots after evaporation.
For all examples provided herein, the method of the present invention involving
.
5
acetone extraction followed by extraction with a second solvent (ethyl acetate, for
example) gave a translucent oil having appearance and properties more attractive
than any oil obtained by the classical technique of Folch et al. (1957).
To analyze lipid composition, 780 ug of each extract was loaded on silica-gel plates
10
and fractionated by thin layer chromatography, TLC (Bowyer et al. 1962) with the
following solvents. Neutral lipids: hexane, ethyl ether, acetic acid (90:10:1, v/v) and
phospholipids: chloroform, methanol, water (80:25:2, v/v). Fatty acid composition of
E. pacifica was analyzed by gas liquid chromatography, GLC (Bowyer et al. 1962,
see bibliography) including some modifications to the original technique: 2h at 65OC
15
instead of 1 h at 80°C, three washes with hexane instead of two and no wash with
water.
To get rid of traces of organic solvents, lipid fractions I and II are warmed to about
125°C for about 15 minutes under inert atmosphere.
20
Fat was analyzed according to the American Oil Chemist’s Society (AOCS). The
following criteria have been used to analyze the lipids extracted: saponification and
Wijs iodine indexes and moisture-volatile matter levels. Cholesterol content has also
been determined by the method of Plummer 1987 (see bibliography). The same
25
analyzes and others have been made by an independent laboratory under Professor
Robert Ackman’s supervision (Canadian Institute of Fisheries Technology, DalTech,
Dalhousie University, Halifax, Nova Scotia, Canada). This includes Wijs iodine index,
peroxide and anisidine values, lipid class composition, fatty acid composition, free
fatty
30
acid
FAME,
cholesterol,
tocopherol,
asthaxanthin and canthaxantin contents.
all-trans
retinol,
cholecalciferol,
PCl’ICA99t00987
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Table 1 shows that higher levels of lipids are extracted from dry krill by acetone
followed by ethanol as compared to the classical procedure of Folch et al. (1957).
Table 2 shows the results of lipid extraction
5
from frozen Euphausia pacifica,
a
species of kriil from Pacific Ocean. Assuming an eighty percent content of water, the
lipid content is comparable to dry krill as shown in Table 1. Isopropanol, f-butanol
and ethyl acetate, as solvent for the second extraction, give a yield less important
than ethanol, but are not necessarily less effective in lipid recovery since ethanol
carries more impurities than isopropanol, t-butanol or ethyl acetate. Then, they can
10
be used as second solvent after acetone as well. Variations between results from
acetone extractions are mainly due to the water-oil separations. These separations
are influenced
by the quantity of residual acetone in the water-oil solution after
acetone evaporation. This quantity of acetone varies from an experiment to another;
because the evaporation system used at a small scale is less reproducible (at the
15
industrial scale, the evaporation step will be optimized).
Single solvents have also
been tested to extract the totality of lipids from krill. This shows that ethyl acetate
(1,37% extraction rate), as hexane (0,23% extraction rate) are not good solvents,
compared to acetone alone (1,86% extraction rate, and even greater extraction rates
with an efficient acetone evaporation system).
20
One of the main advantages of the procedure is the removal of bacteria from extracts
(lipid fraction
and solid protein-rich
material).
Indeed, samples of E. pacifica
incubated in different ratios of acetone at 4°C for 112 days have been inoculated on
NA medium containing Bacto” beef extract 0,3%, BactoTM peptone 05% and BactoTM
25
agar 1,5% (Difco Laboratories, Detroit, USA) then incubated at room temperature or
4°C for 18 days. No significant bacterial growth was observed at a ratio of 1 volume
of acetone per gram of krill. At higher proportions
of acetone (2 volumes and 5
volumes), there was no bacterial growth at all, which means that acetone presences
krill samples.
30
Acetone is known as an efficient
(Goodman et al. 1980).
bactericidal
and viricidal agent
,
PCTKA99l00987
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Table 3 shows the yield of lipids from M . norvegica. The percentage of lipids (3,67%)
is comparable
. Variations
,
to the one obtained with E. pacifica (3,11%) shown in Table 2.
can be attributable to diet and time (season) of collection, which are
different for those two species.
\
5
Table 4 shows the influence of grinding on the efficiency
of extraction
of M .
norvegica lipids. These extractions were carried out under optimal conditions and
show the definite advantage of the procedure over the classical method (4,46 %
versus 3,30 %). It also shows that grinding may be an important factor when the
10
species is large (4,46% versus 3,53 %).
Table 5 reports on lipid extraction from Calanus. Considerable quantities of lipids
were obtained. Some variations in Calanus species composition may explain the
variations between experiments 1 and 2 (8,22 % and lo,90 % of fresh weight).
15
Tables 6-8 report the total amount of lipids extracted from fish tissue. The method
of the present invention was demonstrated
method was demonstrated
Advantageously,
20
on mackerel, trout and herring. The
on peripheral tissues (mainly muscles) and viscera.
the present method would permit the recovery of valuable lipid
fractions from parts of fish that are usually wasted after the withdrawal of fillets of the
fish. Those fish tissues not used after the transformation
consumption
could be stored in acetone,
of the fish for human
and lipids extracted
therefrom
in
accordance with the present invention even if the method Folch [I9571 recovers
more lipid than our method. Indeed small amounts of lipids from mackerel (0.52%
25
from viscera and 1,45% from tissues) have been extracted by the method of Folch
after a first extraction with acetone and ethanol as described in the present invention.
Comparative extractions with the method described in the present invention carried
out in parallel with the method of Folch on trout and herring show superior recovery
:
with the latter. However, it is noteworthy that the Folch method can not be applied
..
30
for the recovery of lipids for commercial uses (because of toxicity).
PCTICA99/00987
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IO
In Tables 9 to 11, are shown results of lipids extraction
from shark liver tissues.
There is no marked difference in results between techniques within a species.
Table 12 shows the fatty acid composition of krill oil (e. pacifica) following extraction
in various solvents.
5
Tables 13 shows some characteristics
features of fraction I (acetone) and fraction
First, the saponification index of
II (alcohol or ethyl acetate) for krill oil (e. pacifica).
fraction I (130,6) indicates that this fraction contains fatty acids with longer chains, L/
compared to fraction II (185,7). The Wijs iodine index of fraction I shows that this
10
fraction contains high levels of polyunsaturated fatty acids. As compared to olive oil
which has an index of 81 .I. It explains why fraction I is liquid at room temperature.
It is well known that unsaturated fatty acids have a fusion point inferior to the one
of their satyrated homologues. The same observations are made for fraction II which
has a iodine index of 127,2.
15
corroborates
The fatty acid composition
shown in Table
12
these iodine indexes: fraction I has a high percentage (30,24%) of
polyunsaturated
fatty acids (pentaenes+hexaenes)
and so fraction II (22,98%).
Finally, Table 13 shows also that fraction I is comprised of 1 O,O% of volatile matter
and humidity after evaporation of the solvent. For the same test, the fraction II gives
a value of 6,8%. To get rid of traces of solvents, it is important to briefly heat (to ,-,,-’
20
about 125’C, for about 15 min) the oil under nitrogen.
Results on krill oils obtained in accordance with the method of the present invention ‘1
(fraction I extracted with acetone and fraction II extracted with ethyl acetate) are’ /
provided in Tables 13, 14, 15, 16, 17 and 18.
25
It is noteworthy to mention that in
Table 18, the carotenoids content was signiftcantly high as measured in terms of two
carotenoids
namely asthaxanthin and canthaxanthin.
Indeed, duplicates analyzes
revealed values of 92 to 124 pgig of lipid fraction for asthaxanthin and 262 to 734
pg/g for canthaxanthin. Thus, for the purpose of the present invention it may be said
that the krill extract comprises asthaxanthin at least 75 and preferably at least 90
30
uglg of lipid fraction.
In the case of canthaxanthin,
least 270 pg/g of lipid fraction.
advantageous
Low values
at least 250 and preferably at
for peroxide
and anisidine
are
and are due to the presence of high levels of natural antioxidants
PCTlCA!l9/00987
WO OOR3546
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11
(astaxanthin
and canthaxanthin).
pharmaceutical
. of carotenoids
or cosmetological
These compounds are indicative of favourable
properties of the krill extract whereby high levels
indicate excellent transdermal
extract is a good candidative for transdermal
migration characteristics.
Thus, krill
delivery of medicines.
5
Table 19 shows the best mode of the method in accordance with the present
invention for lipid extraction of aquatic animal tissues.
Table 20 shows that the enzvme activity of the solid fraction is maintained following
10
the method of the present invention.
Indeed, the demonstration was completed for
solid krill residue obtained after successive
Proteolytic
activities
spectrophotometric
concentrations
15
were
assay
measure
using
acetone and ethvl acetate extraction.
by the liberation
o-pthaldialdehyde
were measured by the Bradford method.
of amino
as
groups
reagent.
by
Protein
Solubte proteins were
extracted with water and added to a 10% lactoserum protein concentrate obtained
by ultrafiltration.
At the end of incubation at 37’C in 50mM potassium phosphate
buffer, trichloroacetic
acid was added and the amount of NH3 group was measured
in the supernatant according to the method of Church et al. 11983, J Dairy Sci 66:
1219-12271.
20
Figures 1 to 6 show chromatograms
of fatty acid composition
of E. pacifica lipids.
On each of them, high proportions of 205 and 22:6 fatty acids (characteristic
of
marine and aquatic oils) are noticeable and represented by two distinct peaks. Data
are shown in Table 12.
25
Variations
in lipid patterns of neutral lipids (from Figure 7 to Figure 11) from one
species to another are attributable
to the differences in food sources. Within a
species (E. pacifica, for example) there is no marked variation between lipid patterns
obtained from different techniques
30
of lipid extraction. Concerning
phospholipids
(Figure 12 to Figure 16), the opposite is observed: variations are explained by the
different extraction processes of lipids since the same species do not lead to the
same lipid pattern. Lipids from shark species (extracted by the mentioned methods)
Wo
YCTiCA99l0098l
OOf23546
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12
and commercial
Quebec,
cod-liver oil (sample available from Uniprix drugstores, Province of
Canada)
are
mainly
composed
of neutral
lipids
as opposed
to
phospholipids.
5
The influence of the volume of solvent and incubation time on the efficiency of the
acetone
to extract lipids from E. pacifica
is illustrated
in Figures 17 and 18,
respectively. A ratio of 1:6 (w/v) produced optimal yield with near complete extraction
after 2h. The second extraction
step has been experimented
with ethanol. The
volume of this solvent does not appear to be critical since the same yield was
10
obtained with different volumes
of ethanol (Figure 19), but incubations time in
ethanol should be at least 30 minutes as indicated by the results on Figure 20.
One of the inventors, Dr. Adrien Beaudoin, has ingested the different lipid fractions
of krill. No side effect profile was observed.
Although the invention has been described above with respect with one specific form,
it will be evident to a person skilled in the art that it may be modified and refined in
various ways. It is therefore wished to have it understood that the present invention
should not be limited in scope, except by the terms of the following claims.
20
Demonstration
that krill residue, obtained after acetone and ethvl acetate extraction,
contains enzvme proteolvtic activities. Proteolvtic activities were measured bv the
liberation of amino oroups bv spectrophotometric
assay using o-phthaldialdehyde
as reagent. Protein concentrations were measured by the Bradford method.
25
The enzvme source was the residue obtained after acetone and ethyl acetate
extractions of lipids. Soluble proteins were extracted with water and added to a 10%
lactoserum protein concentrate obtained bv ultrafiltration.
30
At the end of incubation
trichloroacetic
at 37°C in 50 mM potassium
phosphate
buffer,
acid was added and the amount of NH3 groups were measured in the
supernatant according to Church and al. 1983.
W O OOf23546
PCTlCA99f~0987
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13
BIBLIOGRAPHY
Bowyer,
D.E.,
determination
5
Leat, W.M.F.,
Howard,
A.N.
and Gresham,
G.A.
1962. The
of the fatty acid composition of serum lipids separated by thin-layer
chromatography; and a comparison with column chromatography. BBA. 70: 423431.
Chandrasekar, B., Troyer, D.A., Venkatraman, J.T. and Femandes, G. 1996. Tissue
specific regulation of transforming growth factor beta by omega-3 lipid-rich krill oil in
autoimmune murine lupus. Nutr Res. 16(3): 489-503.
10
Christensen, M .S., Hoy, C-E. and Redgrave, T.G. 1994. Lymphatic absorption of n-3
polyunsaturated
fatty acids from marine oils with different intramolecular fatty acid
distributions- BBA. 1215: 198-204.
15
Church,
F.C.,
Swaisgood,
H.E.,
Porter,
D.H.
and
Catignani,
G.L.
1983.
Spectrophotometric assay using o- Phthaldialdehyde for determination of proteolysis
in m ilk and isolated m ilk proteins. J Dairy Sci. 66: 1219-1227.
Difco laboratories. 1984. Difco Manual Dehydrated Culture Media and Reagents for
20
M icrobiology. 1 Orhed. Detroit.
Folch, J., Lees, M . and Sloane-Stanley, G-H. 1957. A simple method for the isolation
and purification of total lipids from animal tissues. J. biol. Chem. 226: 497-509.
25
Goodman Gilman, A., Goodman, L.L. and Gilman, A. 1980. The Pharmacological
Basis of Therapeutics. 6rh ed. Collier Macmillan Canada Itd, Toronto.
:
Harwood,
H.J. and Geyer,- R.P. 1964. Biology Data Book. The Federation
of
American Societies for Experimental Biology, Washington.
30
Hellgren, L., Karlstam, B., Mohr, V. and Vincent, J. 1991. Krill enzymes. A new
concept for efficient debridement of necrotic ulcers. lnt J Dermatol. 30(2): 102-l 03
PCTICA99(00981
W O OOl23546
- .-
14
Plummer, D.T. 1987. An introduction to practical biochemistry. 3rh ed. McGraw-Hill
Book Company, London.
Rawn, J.D. 1990. Trait6 de biochimie. De Boeck-vesmael,
Bruxelles.
5
Runge, J.A. and Joly, P. 1994. Rapport sur l’etat des invertebres en 1994: 7:0
Zooplancton (Euphausiaces et Waanus) de I’Estuaire et du Golfe du Saint-Laurent.
Sargent, J.R. 1997. Fish oils and human diet. Br J Nutr.78 Suppl 1: S5-S13.
10
PCTICA99l00987
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15
TABLE 1. EXTRACTION
5
OF DRY KRILL LIPIDS (E. pacifica)
EXP. No.
Techniaue
Yield (%)
Total (%)
1:
acetone a’
ethanol b,
8,00
7.60
15,60
19,70
6,90
26,60
8,15
11,20
19,35
6,80
13,60
20,40
2-
I,
Mean (O/o)+ s.d.
10
,,
3-
4-
0
15
20,49+3,95
20
1 !i,50
chlor : MeOH ‘)
5
6-
II
,
14,90
15,20&0,30
25
Determinations in triplicates (variation c 5 %).
a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), no incubation.
b, *Extraction
made with a sample-solvent ratio of 1:4 (w/v), incubated 1 night at 4°C following
.
a first extraction with acetone.
‘) :Folch et al. 1957.
30
TABLE
OF FROZEN
KRILL
LIPIDS (E. pacifica)
EXP. No.
Technique
Yield (%)
Total (%)
l-
acetone a’
ethanol b’
1,17
1,23
2,40
3,05
1,09
4,14
1,53
1,26
2,79
35
2-
40
2. EXTRACTION
3-
,,
,t
Mean (%) +_s.d.
3,11-10,91
4-
acetone a’
isopropanol b,
2,45
0,70
3,15
1.80
0,80
2,60
1.60
0,80
2,40
45
5-
650
I,
#I
2,72+0,39
WOOOIZ3546
PCTiCA99l00987
- _-
16
TABLE 2 (continued).
5
Ek.
7-
10
No.
EXTRACTION
OF FROZEN KRILL LIPIDS (E. pacifica)
Technique
Yield (%I
Total (%I
acetone a)
t-butanol ‘)
2,15
0,47
2,62
2,ll
0,40
2,51
2,37
0,45
2,82
8-
9-
n
15
Mean (%J + s.d.
2,65f0,16
10-
acetone a)
ethyl acetate b,
ll20
12-
”
2.28
0.21
2,49
I,09
0,16
1,25
2,54
0,09
2,63
2,12f0,76
25
13-
14-
combined
acetone-ethanol
”
d,
3,28
3,02
30
15
”
3,25
3,181tO,l4
1635
ethyl acetate ‘)
17-
,,
18-
,I
1,32
1,49
1,31
1,37+0,10
40
19-
hexane e,
20-
,t
21-
,#
0,31
0,18
0,20
45
0,23+0,07
22-
chlor:MeOH
r,
2,37
PCUCA99l00987
WO 00123546
17
TABLE 2 (continued).
Exp. No.
5
EXTRACTION
OF FROZEN KRILL LIPIDS (E. pacifica)
Technique
Yield P??)
Total C%)
23-
*
2,07
24-
”
2,62
Mean (%I + s.d.
2,35*0,28
10
15
Determinations in triplicates (variation c 5 %).
‘) :Extraction made with a sample-solvent ratio of
b, *Extraction made with a sample-solvent ratio of
‘following a first extraction with acetone.
‘) :Extraction made with a sample-solvent ratio of
following a first extraction with acetone.
d, :Extraction made with a sample-acetone-ethanol
e, :Extraction made with a sample-solvent ratio of
0 : Folch et al. 1957.
I:6 (w/v), incubated 2 h at 4°C.
I:2 (w/v), incubated 30 min at 4°C.
1:2 (w/v), incubated 30 min at 2SC,
ratio of 155 (w/v/v), incubated 2 h at 4%.
I:9 (w/v), incubated 2 h at 4°C.
20
TABLE 3. EXTRACTION
25
OF FROZEN KRILL LIPIDS(M.
norvegica)
EXP. No.
Techniaue
Yield (%I
Total C%\
l-
acetone a)
ethanol b,
I,82
I,82
3,64
230
3-
,,
R
Mean (%I + s.d.
I,15
2,35
3,50
I,68
2,19
3,87
3,67*0,15
35
Determinations in triplicates (variation < 5 %).
a) :Extraction made with a sample-solvent ratio of I:9 (w/v), incubated 1 night at 4°C.
b, :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 h at 4°C following a first
extraction with acetone.
PCTICA99190987
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-2
18
TABLE 4. INFLUENCE OF GRINDING ON EXTRACTION OF FROZEN KRILL LIPIDS
(M. norvegica)
EXP.
No.
Yield (O/o)
Krill around before lfiextraction
Technique
5
acetone ‘)
.:- , t i ethanol b,
l-
”
2-
yes
3.10 -1.07
no
2,14
1,39
3,53
3,32
1,14
4,46
10
3-
15
.
I,
4-
I
( ‘r"
I I,,'?, <iA
- i-
'>*'It
12
yes
chlor : MeOH ‘)
5
20
Total C%) ,
v’,
.>:&Y;‘ , 1‘. *, 7
4,17
yes
3,30
yes
3,26
Determinations in triplicates (variation < 5 %).
af :Extraction made with a sample-solvent ratio of 1:6, incubated 2 h at 4°C.
b, :Extraction made with a sample-solvent ratio of 1:2, incubated 30 min at 4°C following a first
extraction with acetone.
‘) :Folch et al. 1957.
25
TABLE
30
40
OF FROZEN Calanus
EXP. No.
Technique
l-
acetone a)
ethanol b,
235
5. EXTRACTION
,,
.
LIPIDS (Calanus
sp.)
Yield (%I
Total (%I
6,18
2,04
8,22
8,64
2,26
IO,90
Mean (%) + sd.
9,56+_1,34
Determinations in triplicates (variation c 5 %).
“:Extraction
made with a sample-solvent ratio of 1:9 (w/v), incubated 1 night at 4°C.
b, :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 h at 4°C following a first
extraction with acetone.
PCTKA99l00987
WOOOf
- .:-
19
TABLE 6. EXTRACTION OF FRESH FISH LIPIDS (Mackerel)
5
Exe. No.
Technique
1 - viscera
fish 1
acetone a’
ethanol b’
2- tissues
fish 1
c
3- viscera
fish 2
”
4- issues
fish 2
I
10
15
Yield (O/k)
30
”
7- viscera
fish 4
,,
8- tissues
fish 4
n
9- viscera
fish 1
lo- tissues
fish 1
35
40
6,70
3,78
0,91
4,69
lo,46
057
11,03
8,06
8,39
6- tissues
fish 3
25
6,ll
0,59
6,65
1,41
5- viscera
fish 3
20
Total (Ok)
chlor:MeOH
II
‘)
0,66
9,05
527
0,97
6,24
8,47
0,69
$16
8,40
1,02
9,42
0,52
I ,45
a’:Extraction made with a sample-solvent ratio of 1:9 (w/v), incubation time:
.
fish 1 viscera: 4h, fish 1 tissues: 23h
.
fish 2 viscera: 23h45, fish 2 tissues: 45h30
.
fish 3 viscera: 8 days 2h20, fish 3 tissues: 8 days 22h30
fish 4 viscera: 17 days 23h, fish 4 tissues: 18 days 2h25.
b, :Extra&ion made with a sample-solvent ratio of 1:4 (w/v), incubated 1 h at 4”C, following a first
extraction with acetone.
‘) :Folch et al. 1957, followina extractions with acetone. then ethanol..
PCT/CA99/00987
WOOOi23546
- .20
TABLE
7. EXTRACTION OF FRESH FISH LIPIDS (Trout)
Techniaue
acetone a1
ethanol ‘)
I- viscera
5
2- tissues
,r
34,70
2,18
36,88
5.53
1,17
6,70
10
3- viscera
chlor:MeOH ‘)
,,
4- tissues
15
39,81
14,93
Determinations in triplicates (variation < 5 %).
a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 1 night at 4°C.
b, :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 h at 4°C following a first
extraction with acetone.
‘) :Folch et al. 1957.
20
TABLE
8. EXTRACTION
OF FRESH FISH LIPIDS (Herring)
Exp. No.
Technique
Yield (%)
Total (%)
acetone a)
ethanol b,
2,09
0,68
a77
25
l-tissues
and viscera
chlonMeOH ‘)
2-tissues and viscera
5,95
30
35
Determination in triplicates (variation < 5% ).
a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 1 night at 4”.
b, :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 h at 4°C following a first
extraction with acetone.
‘) :Folch et al. 1957.
TABLE
40
45
50
9. EXTRACTION
OF FRESH SHARK LIVER LIPIDS (M. schmitti)
EXP. No.
Technique
Yield (%)
l-
acetone a)
ethyl acetate b,
36,39
4,48
Total (%)
40,87
2-
ethyl acetate ‘)
36,68
3-
chlor : MeOH d’
41,86
Determinations in triplicates (variations <5 %).
a) :Extraction made with a sample-solvent ratio of I:9 (w/v), incubated 2h at 4°C.
b, :Extraction made with a sample-solvent ratio of I:2 (w/v). incubated 30 min at 4”C, following
a first extraction with acetone.
” :Extraction made with a sample-solvent ratio of 1 :9 (w/v), incubated 2h at 4°C.
d, :Folch et al. 1957.
wo
PCTICA99l00981
OOl23546
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21
T’ -LE
10. EXTRACTION
OF FRESH SHARK LIVER LIPIDS (G. galeus).
Exe. No.
Technioue
Yield (%)
Total (%)
l-
acetone a)
ethyl acetate ‘)
21,39
5,27
26,66
2-
ethyl acetate ‘)
25,89
3-
chlor : MeOH d,
29,99
Determinations in triplicates (variations c5 %).
‘) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 2h at 4°C.
b, :Extraction made with a sample-solvent ratio of 1:2 (w/v), incubated 30 min at 4°C following
a first extraction with acetone.
‘) :Extraction made with a sample-solvent ratio of 1 :9 (w/v), incubated 2h at 4°C.
d, :Folch et al. 1957.
TABLE
11. EXTRACTION
OF FRESH SHARK LIVER LIPIDS (Angel Shark)
Exp. No.
Techniaue
Yield (%)
l-
acetone a)
ethyl acetate b,
19,23
Total (%)
8,98
z&21
e
ethyl acetate ‘)
39,22
3-
chlor : MeOH d,
39,23
Determinations in triplicates (variations c5 %).
“:Extraction
made with a sample-solvent ratio of 1:9 (w/v), incubated 2h at 4°C.
b, :Extraction made with a sample-solvent ratio of 1:2 (w/v), incubated 30 min at 4°C following
a first extraction with acetone.
‘) :Extraction made with a sample-solvent ratio of 1 :9 (w/v), incubated 2h at 4°C.
d, :Folch et al. 1957.
TABLE
8
12. FATTY ACID COMPOSITION
Solvent
Saturated
chlo-meth
26.18
acetone
21.4
acetone
Unsaturated
(E. pacifica)
Mono
Qi
polv
H-Poly
22.54
1.91
3.23
26.34
19.8
-22.18
1.75
3.7
24.52
26.46
19.09
22.11
2.03
3.48
30.24
23.03
ethanol
28.07
22.92
2.14
3.07
27.78
16.03
t-butanol
32.63
24.96
1.86
2.86
17.86
19.83
I acetate
,-La expressed
22.68
in percentage
25.77
2.17
of total fatty acids (%).
2.88
22.98
23.51
Unidentified
1
Pcr/cA99~00987
WOOO/23546
a :-
22
TABLE
5
13. CHARACTERISTICS
Saoonification
OF KRILL OIL (E. pacifica)
independent
laboratory a)
handbookb
130,6
105,7
192,o e,
--
189,7
185,2
127.2
85,3@
172,5
139,2
-
81,l
Zl
3,7
0,2 @
1.9
380
-
-
QO
w
-
index
Fraction I ‘)
Fraction II ‘)
Olive oil
10
Wiis iodine index
15
Fraction I ‘)
Fraction II d,
Olive oil
Cholesterol
20
content
(%I
Fraction I ‘)
Fraction II d,
Olive oil
Volatile matter and moisture levels (%\
25
Fraction I ‘)
Fraction II d)
Peroxide value (mea peroxide/ko
30
Fraction I ‘)
Fraction II d)
p-Anisidine
35
40
45
10,o
638
oil\
-
value (a*’ absorotionl
Fraction I ‘)
Fraction II d,
-
a): Professor Robert Ackman’s laboratory, Canadian Institute of Fisheries Technology,
Halifax, Nova Scotia.
b, : Harwood and Geyer 1964.
‘) : Extraction made with a sample-acetone ratio of 1:6 (w/v), incubated 2h at 4°C.
d, : Extraction made with a sampleethyl acetate ratio of I:2 (w/v), incubated 30 min at 4°C
following a first extraction with acetone.
e, : Extra virgin olive oil cold compressed from Bertolli Th’.
PCTICA99100987
WO 00123546
- I-
23
TABLE
14. LIPID CLASS COMPOSlTlON
OF KRILL OIL (AREA ‘1’0)(E. pacifica)
Trialvcerides
5
19,0&0,7
66,5& 2,3
Fraction I a)
Fraction II b,
Hvdrocarbons
10
trace
1,3+ 0,l
Fraction I a)
Fraction II br
Free fattv acids
15
23,7& I,1
20,3+ 0,3
Fraction I ‘)
Fraction II b,
Monoolvcerides
20
Phosoholipids
25
1,4c 0,3
0,s 0,l
Fraction I a)
Fraction II b,
or other polar material
54,lk 6,l
8,5 +_1,6
Fraction I a)
Fraction II b,
30
Data from Professor Robert Ackman’s laboratory, Canadian Institute of Fisheries Technology,
Halifax, Nova Scotia.
a) : Extraction made with a sample-acetone ratio of 1:6 (w/v), incubated 2h at 4°C.
b, : Extraction made with a sample-ethyl acetate ratio of 1:2 (w/v), incubated 30 min at 4°C
following a first extraction with acetone.
35
TABLE
40
45
50
55
15. FATTY ACID COMPOSITION
OF KRILL OIL (VVT/WT%) (E. pacifica)
Fattv acids
Fraction I a)
12 :o
13 :o
IS0 14 :o
14:o
IS0 150
ANT 15:O
15:o
IS0 16:0
ANT 16:O
16:0
7MH
ANT 1710
17:o
18:0
20:o
Saturates
Q,O
02
64
42
03
02
096
02
02
14,l
‘X6
O,l
u3
l,O
0.1
25‘2
Fraction II ‘)
61
O,l
0,6
7,6
0,7
0.2
14
63
02
21,6
0.9
0,3
3,7
136
O-3
39,2
YCTICA99/0098~
WOO0123546
- :-
24
TABLE 15 (continued).
(E. pacifica)
FAllY
ACID COMPOSITION
OF KRILL OIL (WTiWT%)
Fattv acids
Fraction I a)
Fraction II b,
14:l
151
16:l n-7
16:l n-5
17:l
18:l n-9
18:l n-7
18:l r-i-5
2O:l n-9
2O:l n-7
2O:l n-5
22:l n-11 +13
Monoenes
Q4
0,l
686
W
0,6
88
4,2
Q1
083
0,3
0,3
O,l
21.6
56
O,l
0,4
0,4
0,4
02
26,3
W
1.3
O,l
20
0,l
02
12
I,3
02
18
O,l
02
5
10
15
20
25
30
35
40
16:2
16:2
18:2
18:2
18:2
20:2
n-6
n-4
n-7
n-6
n-4
NMID
03
02
78
02
0,7
93
.
2012 n-6
Dienes
O,l
434
081
43
16:3 n-4
18:3 n-6
18:3 n-4
18:3 n-3
18:3 n-l
20:3 n-3
Trienes
I,4
0,4
02
3,2
0.1
O,l
584
92
0,3
02
300
0.1
O,l
43
16:4 n-3
16:4 n-l
18:4 n-3
18:4 n-l
20:4 n-6
20:4 n-3
Tetraenes
03
18
92
Q1
0,7
0,7
12,6
0,7
0.8
704
0.0
0.5
0.3
9.7
20:5 n-3
21:5 n-3
22:5 n-6
22:5 n-3
Pentaenes
17,4
0.7
02
0.5
18,8
88
Q5
45
50
0,1
0,3
985
:
PCTICA99l00987
W O O O f23546
I
-
:-
25
T A B L E 1 5 (continued).
:
F A T i Y A C ID C O M P O S ITIO N O F KRlLi
Fattv acids
Fraction I ‘)
22:6 n-3
13,2
O IL ( W T I W T % )
Fraction II b,
5
683
Hexaenes
10
15
Iodine value calculated
214,8
145,l
Data from Professor Robert A & m a n ’s laboratory, C a n a d i a n Institute of Fisheries Technology,
Halifax, N o v a Scotia.
a) : Extraction m a d e with a sample-acetone ratio of 1:6 (w/v), incubated 2 h at 4°C.
b, : Extraction m a d e with a sample-ethyl acetate ratio of 1:2 (w/v), incubated 3 0 m i n at 4°C
following a first extraction with acetone.
T A B L E 16. K R ILL LIPID F R E E F A l T Y A C ID F A M E ( W W V T % ) (E. pacifica)
20
Fattv acids
25
30
35
40
45
12:o
13:o
lSOl4:O
14:o
IS 0 15:o
A N T 15:0
15:o
IS 0 16:0
A N T 16:0
16:0
7MH
A N T 17:0
Phytanic
17:o
18:0
20:o
22:o
S a turates
14:l
15:l
16:l
16:l
55
03
02
02
I,3
0.3
a1
n-9
n-7
Fraction II b,
a1
a0
02
Z6
02
a3
Ql
0.5
Q1
02
02
3,3
05
02
02
10,6
098
02
05
02
0,3
08
684
02
02
Q1
08
03
0,6
02
O ,l
17,4
02
0,5
Q1
a0
5.2
68
16:l n - 5 + 1 1 7 : 0
0 ,1
O ,l
17:l
W
Ia :1 n - 9
50
Fraction I a)
18:l
18:l
i-7
n-5
7,o
43
O ,l
2 O :l n - 1 1
02
2 O :l n - 9
221 n-11+13
24:l n - 9
Monoenes
OS1
O ,l
080
19.2
Q7
11.4
93
033
Q3
0.3
02
O ,l
29.8
YCTiCA99~00987
WO OOl23546
- .:-
26
TABLE
16 (continued).
Fattv acids
5
7 6:2
16:2
18:2
18:2
18:2
KRILL LIPID FREE FAlN
ACID FAME (WT/WT%)
Fraction I ‘)
(E. pacifica)
Fraction II b,
n-6
n-4
n-7
n-6
n-4
Of4
12
0,l
24
081
o,g
20:2 n-6
Dienes
081
4,3
O,l
4,9
16:3
16:3
18:3
18:3
18:3
18:3
20:3
20:3
1,4
02
Ov4
O,l
3,3
O,l
081
a1
03
0,5
083
O,l
3,4
O,l
OS1
02
Trienes
5,7
56
16:4 n-3
1614 n-l
18:4 n-3
18:4 n-l
20:4n-6
20:4 n-3
22:4 n-3
Tetraenes
W
1 ,o
38
Q1
1,7
033
0,3
14,l
O,3
W
62
O,l
I,4
18:5 n-3
20:5 n-3
21:5 n-3
22:5 n-6
22:5 n-3
Pentaenes
02
26,4
03
080
0,7
28,2
17.4
05
Q1
03
18,7
2216 n-3
Hexaenes
20.5
20,5
14,4
14,4
291,6
220,3
14
.
02
2,6
O,l
10
15
20
25
30
35
n-4+117:1
n-3+118:0
n-6
n-4
n-3
n-l
n-6
n-3
03
0,3
34
Q1
40
Iodine value calculated
45
50
Data from Professor Robert Ackman’s laboratory, Canadian institute of Fisheries Technology,
Halifax, Nova Scotia.
.
‘) : Extraction made with a sample-acetone ratio of I:6 (w/v), incubated 2h at 4°C.
b, : Extraction made with a sample-ethyl acetate ratio of 1:2 (w/v), incubated 30 min at 4°C
following a first extraction with acetone.
PCT’/CA99/~0981
W O OOL23546
- :-
27
TABLE 17. TOCOPHEROL,
IN KRILL OlL (E. pacifica)
5
10
aloha-tocopherol
Fraction I a’
Fraction II b,
RETINOL AND CHOLECALCIFEROL
CONTENT
bv HPLC (IU)
0,91
063
aamma-tocooherol
Fraction I a)
Fraction II b,
delta-tocooherol
Fraction .I a)
Fraction 11b,
ALL-tram
bv HPLC us/q
Tf
Tr
bv HPLC ualg
N.D.
N.D.
15
20
25
30
all-trans retinol bv HPLC (IU)
Fraction I a’
Fraction II bt
395,57
440,47
cholecalciferol
Fraction I a)
Fraction II b,
N.D.
N.D.
bv HPLC (IU)
Data from Professor Robert Ackman’s laboratory, Canadian Institute of Fisheries Technology,
Halifax, Nova Scotia.
Data expressed per gram of kriil oil.
a) : Extraction made with a sample-acetone ratio of 1:6 (w/v), incubated 2h at 4°C.
‘) : Extraction made with a sample-ethyl acetate ratio of 1:2 (w/v), incubated 30 min at 4°C
following a first extraction with acetone.
TR
= trace
N.D. = not detected
Conversion : Vitamin
alpha-tocdpherol
mg/g oil x 1,36 = International Unit
All-trans retinol
pg/g i 0,3 = International Unit
35
TABLE 18. ASTAXANTHIN
(E. pacifica)
Asthaxantin
AND CANTHAXANTHIN
CONTENT OF KRILL OIL
(us/a oil)
40
Fraction I ar
Fraction II b,
Canthaxanthin
93,l
121,7
(us/q oil)
45
Fraction I a)
Fraction II b,
50
55
270,4
733,0
Data from Professor Robert Ackman’s laboratory, Canadian Institute of Fisheries Technology,
Halifax, Nova Scotia.
‘) : Extraction made with a sample-acetone ratio of I:6 (w/v), incubated 2h at 4°C.
b, : Extraction made with a sample-ethyl acetate ratio of 1:2 (w/v), incubated 30 min at 4’C,
following a first extraction with acetone.
PCTICA99~00987
W O OOl23546
- _-
28
TABLE 19. OPTIMAL
TISSUES (suggested
CONDlTlONS
procedure)
FOR LIPID EXTRACTlON
OF AQUATIC
ANIMAL
STEP
CONDITIONS
Grinding (if particles > 5mm)
4°C
Lipid extraction
sample-acetone ratio of 1:6 (w/v)
2h (including swirling 20 min)
4°C
Filtration
organic solvent resistant filter
under reduced pressure
Washing
sample-acetone ratio of 1:2 (w/v)
pure and cold acetone
Filtration
organic solvent resistant filter
under reduced pressure
Evaporation
under reduced pressure
Oil-water separation
4°C
Lipid extraction
~
5
10
15
20
25
ethvl acetate ratio of 1:2 (w/v)“)
!EF
4°C b’
30
35
40
Filtration
organic solvent resistant filter
under reduced pressure
Evaporation
under reduced pressure
a): Ethanol can be replaced by isopropanol, t-butanol or ethyl acetate.
b! 25 “C when using f-butanol.
TABLE 20: PROTEOLMIC
ACTIVITY OF KRILL RESIDU USING LACTOSERUM
SUBSTRATE, AT 37 “C, PH 7.0 FOR A RATIO ENZYME:SUBSTRATE
OF 1:43
Time
(min)
Amino acids released
(umoles)
15
30
170
255
28.76
43.74
98.51
177.26
Enzymatic rate
(umoleslmin)
Specific
enzymatic
activity
(umoles/min/mq*)
45
.
50
* total quantity of enzymes in hydrolysis media
1.917
0.999
0.322
0.308
AS THE
0.164
0.125
0.050
0.060
PCI-lCA99/00981
WO OOf23546
- .-
29
We claim:
* 1.
:
A method
for extracting
lipid fractions
from marine
and aquatic animal
material, said method comprising the steps of:
..
(a)
5
placing marine and aquatic
animal
material
preferably acetone to achieve extraction
in a ketone solvent,
of the soluble lipid fraction
from said marine and aquatic animal material;
(b)
((2
10
(d)
separating the liquid and solid contents;
recovering
a first lipid rich fraction
from
the liquid contents
evaporation
of the solvent present in the liquid contents;
by
placing said solid contents in an organic solvent selected from the
group of solvents consisting of alcohol, preferably ethanol, isopropanol
or t-butanol
and esters of acetic acid, preferably
achieve extraction of the remaining
ethyl acetate to
soluble lipid fraction from said
marine and aquatic animal material;
15
(6
(9
separating the liquid and solid contents;
recovering
a second lipid rich fraction by evaporation
of the solvent
from the liquid contents;
(9)
recovering the solid contents.
20
2.
A method as in claim 1 wherein during step (a), the solvent and animal
material are homogenized.
3.
A method as in claim 1 wherein during step (d), the solvent and solid contents
are homogenized.
25
4.
A method as in any of claims 1 to 3 wherein steps (b) and (d) are conducted
under inert gas atmosphere.
30
5.
A method as in any of claims 1 to 4 wherein steps (b) and (e) are effected by
techniques selected from filtration, centrifugation
and sedimentation.
wo
PCTICA99f00987
00/23546
- .-
30
6.
A method as in any of claims 1 to 5 wherein steps (c) and (f) are effected by
techniques selected from vacuum evaporation, flash evaporation and spray
drying.
5
A method as in any of claims 1 to 6 wherein after step (b) and before step (c),
7.
the method additionally comprises the intervening step of washing the solid
contents with the solvent and adding the resulting washing solution to the
liquid contents of step (b).
10
8.
A method as in any of claims 1 to 7 wherein after step (e) and before step (f),
the method additionally comprises the intervening step of washing the solid
contents with the organic solvent selected in step (d).
9.
A method as in any of claims 1 to 8 wherein prior to step (a) the marine and
aquatic animal material is finely divided, preferably to an average particle size
15
of 5 m m or less.
10.
A method as in claims ? to 9 wherein steps (a) and (b) are conducted at
solvent temperatures of about 5’C or less.
20
11.
A method as in claims I to 10 wherein said marine and aquatic animal is
zooplancton.
12.
A method as in claim 11 wherein said zooplancton is krill.
13.
A method as in claim 12 wherein said zooplancton is Calanus.
14.
A method as in claims.1 to 10 wherein said marine and aquatic animal is fish
25
filleting by-products.
30
PCTICA99l00987
W O 00123546
31
15.
A method for extracting lipid fractions from marine and aquatic animal material
selected from zooplancton and fish filleting by-products, preferably viscera,
said method comprising the steps of:
(a)
placing said animal material in a ketone solvent, preferably acetone &I
achieve extraction of the soluble lipid fraction from said marine and
5
aquatic animal material;
(b)
separating the liquid and solid contents;
w
recovering a lipid rich fraction from the liquid contents by evaporation
of the solvent present in the liquid contents;
IO
(d)
recovering the solid contents.
16.
A method as in claim 15 wherein the animal material is krill.
17.
A m&hod as in claim 15 wherein the animal material is Calanus.
18.
A method as in claims15 to 17 wherein during step (a), the solvent and animal
15
material are homogenized.
19.
A method
as in any of claims 15 to 18 wherein steps (b) and (d) are
conducted under inert gas atmosphere.
20
20.
A method as in any of claims 15 to 19 wherein step (b) is effected
by
techniques selected from filtration, centrifugation and sedimentation.
25
21.
A method as in any of claims 15 to 20 wherein step (c) is effected
by
techniques selected from vacuum evaporation, flash evaporation and spray
drying.
22.
30
A method as in any of claims 15 to 21 wheiein after step (b) and before step
(c), the method additionally comprises the intervening step of washing the
solid contents with the solvent and adding the resulting washing solution to
the liquid contents of step (b).
PCUCA99~00987
WOOO/23546
- :-
32
23.
A method as in any of claims 15 to 22 wherein prior to step (a) the marine and
aquatic animal material is finely divided, preferably to an average particle size
of 5mm or less.
5
24.
A method as in claims 15 to 23 wherein steps (a) and (b) are conducted at
solvent temperatures of about 5’C or less.
25.
A krill lipid extract characterized in that the carotenoid content in asthaxanthin
is at least about 75 and preferably at least about 9O:g/g of krill extract.
10
26.
A krill lipid extract
characterized
in that
the
carotenoid
content
in
canthaxanthin is as least about 250 vglg and preferably at least about 270
pg/g of krill extract.
15
27.
A method of lipid extraction as in claims I or 15 wherein the solid contents
recovered in the last step consists of a dehydrated residue containing active
enzymes.
PCT/CA99/00987
W O OOi23546
7/20
,‘--.I-
10
1.263
1.455 - 12:o
1.625
1.812
1.876
2.056
2.173
2.331 - 14:o
2.505
2.591 - 14:l
2.682
2.802
2.855
3.078
- std 150
3.309
3.586
3.810
4.176
- 16:0
20
--s----a
30
4.521 4.684
4.891 -.
5.121
5.426
5.570
6.037
6.662
6.871
7.235
7.925 8.439 8.640 9.544
9.801 10.491
10.825
11.042
SUBSTITUTE
40
16:l
16:ltr
18:0
18:l
18:ltr
18:2
SHEET (RULE
50
11.637
12.145 13.458
15.626 16.045
16.482 17.017 19.344 22.606 24.103
26.247
28.287
31.295
40.655
49.721
56.3’73
62.225
26)
60
18:3
2O:O
2O:l
2O:l(cisll)
20:2
20:4(6.10,14,18)
PCTKA99~00987
- :-
W O 00123546
PA
60
55
50
45
40
35
30
25
20
10
20
30
40
50
trr
1.217
1.264
1.454
1.624
1.812
1.876
2.055
2.171
2.330
2.505
2.591
2.680
2.800
2.854
3.077 3.306
3.585
12:o
14:o
14:l
std 15:0
3.806
4.157
4.515
4.680
4.891
5.028
5.109
5.421
5.562
6.031
6.642
6.870
7.230
7.910
8.419
8.622
9.529
- 16:0
7 16:l
- 16:ltr
- 18:0
- 18:l
- 18:ltr
SUBSTITUTE
70 m m
60
i
-2
9.786 - 18:2
10.484
10.813
11.590
12.136 - 18:3
13.447
15.623 - 20:o
16.025
16.466 - 2O:l
17.021 - 2O:l (cisll)
22.585 - 20:4 (6,10,14,18)
24.100
26.217
28.241
40.622
56.417
62.086
SHEET (RULE 26)
wo
I
PCTICA99/00987
OOl23546
_-
3/20
:
PA
60
55
50
45
40
35
30
25
20
1.216
1.262
1.454
1.624
1.811
1.875
2.016
2.054
2.174
2.330
2.505
2.589
2.679
2.799
2.854
2.981
3.074
3.304
3.580
3.804
4.169
4.296
in
- 12:o
- 14:0
- 14:l
- std 15:0
- 16:0
13.457
13.943
15.053
15.572
16.016
16.486
16.999
18.762
19.303
20.474
21.027
22.575
24.071
26.215
28.333
31.180
40.560
46.595
49.513
56.292
62.250
4.520 - 16:l
4.683
4.884 7 16:ltr
5.030
5.111
5.420
5.561
6.031
6.642
6.868
7.226
7.908 - 18:O
8.444 - 18:l
8.639 - 18:ltr
9.005
9.5‘36
9.788 - 18:2
IO.267
10.481
10.807
11.626
12.140 - 18:3
SUBSTITUTE
SHEET
(RULE
26)
- 2O:O
- 2O:l
- 2O:l (cisll)
- 20:2
- 20:3
- 20:4 (6,10,14,18)
PC'T/CA99/00981
w000/23546
4/20
PA
60
55
50
45
40
35
30
25
20
50
1.552
1.749
1.968
2.095
2.262
2.485
2.582
2.784
2.886
3.004
3.145
3.478
3.720
4.088
4.325
4.793
5.196
5.406
12:o
- 14:0
- 14:l
- std 15:0
- 16:0
- 16:l
5.675
5.964
6.284’
6.533
6.655
7.009
7.159
7.440
7.874
8.019
8.462
9.411 10.000
10.249
10.716
11.357
11.647
16:l tr
12.888
13.386
14.017
14.524
16.107
19.275
20.112
20.781
27.553
29.529
32.161
34.614
39.240
50.374
61.892
70.568
77.894
18:O
- 18:l
- 18:ltr
- 18:2
12.519
SUBSTITUTE
SHEET
(RULE
60
26)
70
min
- 18:3
- 20:o
- 2O:l (cisll)
- 2O:l
PCTICA~9l00981
- -
WO 00/2X46
5/20
PA
60
55
50
45
40
35
30
25
20
10
I .‘. I..
20
30
. s..
40
-. t..
50
‘. 3 ‘. -. 1.
60 .
‘70
tzt
1.556
1.753
1.972
2.104
2.304
2.590
2.892
3.012
3.153
3.485
3.710
4.096
4.203
4.333
4.800
5.206
5.417
5.711
- 12:0
- 14:o
- 14:l
- std 15:0
- 16:O
- 16:l
- 16:lt.r
5.977
6.294
6.546
7.020
7.166
7.889
8.030
8.473
9.425 10.010 10.260 10.735
11.394
11.661 . 12.540
12.909
13.402
14.010
SUBSTITUTE
14.540
16.133
16.805
18.131
19.154
19.875
20.099
20.820
23.903
27.583
29.570
32.195
34.597
39.334
50.452
70.660
77.895
18:0
18:l
18:ltr
18:2
SHEET
(RULE
26)
f
min
-5
y 18:3
- 20:o
- 2O:l(cisll)
- 2O:l
- 24:0
woOon
PCTICA99/00987
_ _-
6/20
PA
60
55
\
A.,
, . . . .
20
.,
40
.ru
. . . .
60
sb
100
li0
Go
trr
1.564 1.762
1.977
2.080
2.273
2.591 2.887 3.008
3.470 4.108
4.341
4.803 5.210 5.683
6.292
6.514
7.030
‘7.810
12:0
14:o
14:l
std 15:0
16:0
16:ltr
ii;0 min
I -6
8.025
34.677
8.485
38.373 - 22:0
9.360. - 18:0
40.163
9.407
50.157
10.147 - 18:ltr
57.532
11.618 - 18:2
61.436
12.858
70.271
14.515 - 18:3
77.784 - 24:0
161162
110.694
18,077
127.696
19.355 - 2O:l
20.182 - 20:4 (cisll)
20.311
23.205 - 20:2
24.678
27.411 - 20:4 (6.10,14,18)
29.307
31.990
SUBSTITUTE
SHEET
(RULE
26)
WOOOl23546
SUBSTITUTE
7/20
SHEET
I
..:..
a”
.*:
cd
?‘
I
-2
26)
W
(RULE
PCWCA99/00981
_
wo
W/23546
SUBSTITUTE
8/20
SHEET
(RULE
26)
?CTICA99/0#98~
._
wo OOf23546
SUfETl-WTE
9/20
:. ...
.:
... . ..
;
1.
SHEET (RULE 26)
PCT/CA99/00987
_
A
B
c
D
E
F
G
H
WO OOlZ3.546
II/20
SUBSTlTUTE
SHEET
(RULE
26)
PCTICA99/00981
__
wo
00/23546
ul
c
/z/20
.. .. .. . .
cfzF=E=
SUBSJTI-UTE SHEET (RULE 26)
PCTICA99100982
._
WOOOi23546
. ::..
_-
SUBSTITUTE
(RULE
/3/20
SHEET
26)
PCTICA99I00981 _
n
“1
I’
II
1‘4
”
I
b
I
15
I
I
I
I
I
20
25
30
35
40
I-
45
-- I
50
--
1
55
Volumeof acetone(mL).
Incubationtime of 2 h.
Determinations in triplicates (variation 1ess than 5 S,).
trr
I -17
1
P.A
tiu
Il-
010
2I
4I
6I
I
8
I
10
I
12
I
14
I
16
I
18
I
20
I
22
I
24
Incubationtime in acetone(h).
Sample-acetone
ratio of 1:9 (w/v).
Determ inations
in triplicates(variationlessthan 5 X).
I
26
:
10 ]
?-
65432101
0
I
25
I
50
I
75
I
100
Volumeof ethanol (ml.&
Incubationtime of 30 min.
Determinationsin triplicates (variation less than 5 X).
tri
I -19
1 5
0
I
15
I
30
I
45
I
60
I
75
--
I
90
I
--
103
.A^
I
1ZU
1;
Incubationtime in ethanol (min).
Sample-ethanolratio of 1:4 (w/v).
Determinationsin triplicates (variationless than 5 %).
trr
I G?u
INTJZRNATIONAL
SEARCH REPORT
baa xrlb$4p6emIb
PCT/CA 99/009B7
PATENTABSTRACTSOF JAPAN
vol. 1996, no. 12,
26 December 1996 (1996-12-26)
-b JP 08 198754 A (YAKULT HONSHACO LTD),
6 August 1996 (1996-08-06)
abstract
b2.5,
14.15,
18,20
27
WO 84 01715 A (HELLGRENLARS G I ;#OHR
VIGGO (NO); VINCENT JAN GUSTAV (SE))
10 Ray 1984 (1984-05-10)
page 7, line 29 - line 31
examples 1,2
27
-I--
31 January 2000
Nmwmdmdhgaddrsadbel34
Etrqwt~
pslai
OfIke, P.B. 6318 Parendaan
NL-22&JWRl~Jc
Td. (M-70)
34b204aTx.31
fsl cporl.
Fax (4.31-70) aa-me
28/02/2000
AJamlxedomar
2
Dekeirel,
M
page 1
of
3
- .-
INTERNATIONAL
SEARCH
REPORT
.
hLlr mdb#iatano
PCT/CA 99/00987
_ --
DATABASEUP1
Sectton Ch, Ueek 197845
Derwent Publications
Ltd., London, GB;
Cl ass C03. AN 1978-80681A
XPOO2129262
-b JP 53 112195 A (NIPPON PAINT CO LTD),
30 September 1978 (1978-09-30)
abstract
PATENTABSTRACTSOF JAPAN
vol. 009, no. 160 (C-2891,
4 July 1985 ( 1985-07-04 1
-& JP 60 035057 A (SANE1 KAGAKU KOGYO KK),
22 February 1985 (1985-02-22)
abstract
15916.25
PATENT ABSTRACTSOF JAPAN
vol. 016, no. 249 (C-09481,
8 June 1992 (1992-06-08)
-& JP 04 057853 A (CHLORINE EN6 CORP
LTD;OTHERS: 011,
25 February 1992 (1992-02-25)
abstract
25
26
EP 0 732 378 A (NIPPONOIL
26
CO LTD)
18 September 1996 (1996-09-18)
column 1, line 37 - line 47
column 4, line 49 - line 53
DATABASEUP1
Section Ch, Week 197633
.
Derwent Publications
Ltd., London, 68;
Class D13, AN 1976-62648X
XPO02129263
4, JP 51 076467 A (KYOWA HAKKO KOGYO),
2 July 1976 (1976-07-02)
abstract
25,26
CHEMICAL ABSTRACTS, vol. 98, no. 21,
23 May 1983 (1983-05-23)
Columbus, Ohio, US;
abstract no. 177859,
RAA, JAN ET AL.:
“Isolation
of
astaxanthin
from crayfish
or shrimp waste
25
for use as a coloring
page 531; column 2;
agent
in fish
feed"
.
XPO02129261
abstract
-& NO 147 365 B
20 December 1982 (1982-12-20)
page 2 of 3
INTERNATIONAL
SEARCH REPORT
-
nJLppGatbn&
PCT/CA 99/00987
PAIENT ABSTRACTSOF JAPAN
vol. 009, no. 059 (C-2701,
15 March 1985 (1985-03-15)
-& JP 59 196032 A (FUN10 NISHIKAUA;OTHERS:
021, 7 November 1984 (1984-11-07)
abstract
27
page 3 of 3
- -
*,
i
‘-
.,
tAA
15:5&
UI(~‘LlIUU
&lY
$11
U”“.
t)LLL-CUt?~
0215
-
:
..-!‘:
,’
PATEljIT COOPERATION
TREATY
Framm:
WTEPNATIONAL
PRELlMINARY E%&UNtffi
AVT~IOR’TY
PCT
TO;
DUBUC, Jeen H.
GOUOREAU GAGE DUBUC
The Stodc Exchange Tower
800 Pke Victoria, Suite 34ao
P.O. Bax 242
Montr~l, Quibec H4Z 1 Es
WRIITEN
(PCT
OPINION
Rule66)
CANADA
Oab of milling
Apprits w t+nt~ no rat~cm
WI
bfm’+=WvYW
11.0&2000
REPLY
wilhin 3 month(s)
ContmmrabomrMedfdlhg
DUE
0857274
Inbndand
appUcedonNo.
PCTIcA99100987
InInmationU Patent clrrsi%ab~
brbmebonu 8Ungdab f&ytwx@*)
Piiority dar &sq/lnwlu@q
21/70/1999
umi99a
(IPC] or born Wonal
ciaoi~n
WI IPC
Cl lBlll0
Applit
UNIVEASITE DE SHERBROOKE et al.
.;
. Thk w&ton opinion is the fii
!. This oplnbn
1
II
111
IV
V
W
VII
VIII
contains
drawn up by this lntornational Pralmhy
Ir~Ucatiofu
rolathg
Examining Authorlr/.
to the foilowing items:
S
0
0
Cl
b
Battsof the opinion
Priority
Non-rstabllshment of cpinion with regard tc novelty, invan~jvr step and indutial applic.abiKy
Lack of unity of inve~tiin
Reasoned statement undrr Rule 662(a)(ll) with regard to noveXy, Invornive step or indWriJ a?plicabirity
citations and explanations rupporthg such statoment
0
B
m
Cortaindocurn6ntcitad
Cenaln defect3 19
inrernrUonal appltion
Cetialn observations onlhe imernatiinal rppllcation
me
3. The applicant Is hereby invited to reply to thk opinion,
whell7
4.
6~ the Ume h;t indbbd abc4a ma appkmt may, bdwr bw exp!faflailon
of Kaf Urn0firnIt
hqusrt 81h Ab’Jwty to grantm exienslon, sea hb KU(d).
lha !hal dak by Mich he inlnnutiond
wanif atiaq repm must b3 osWshod
~timincvy
ucordrg to iMa IiS2 ir: 2 1!02&!001.
Nun0 r?d mailing acb30as of Uw intamllicwl
prrlirhury l xamlting aulhuity
-Elnc+un Patwe CMca
O-10291 &k&h
0
TeL4SRS23s-0
lx:5236%opmue
- .- 2
Fax+49832399-485
I
Form PCTslPEAJ&%J (ccvoc hat)
(Januq
19%)
1 6 AOUT
2000
.
-
_. .
“““I
.-
W R ITT’EN OPINION
lntrmatbnal
appriticn
NO. PCT/CAWOO987
I. Basis of the opinion
1. This op.&on has been drawn on lhe bask of (substr%to sheers wtrid,
in mspomr lo an invitation under A-tide 74 are
Destrlption,
fd2fr2d
to h tfik
ha112
opinion
been fomirhed to the recei/ivg O lfice
a.9 ‘of@ta~~y
f&S.):
pages:
1-28
as originally filed
Claims, No.z
l-27
as orighaliy filed
Drawlngr, aheats:
l/20-20120
as originally filed
2 The amendments have resulted in the cancellation of:
Cl
the description,
pages:
0
thecfaims,
NOS.:
Cl the drawings,
sheek
3. This opinion has been established as II (some of) the amendments had not been made, s ince they have been
considered to go beyond the dbctosure as fiid (Rule 70.2(c)):
4. Additional observations. if necessary;
V. Reasoned ststcmant under Rule 66.2(a)(ii) whh regard to novehy, inventive step or industrial
applicnbilhy; c iMions and wcptahations supporting such statement
1. Statement
(N)
Claims
15.25
Inventive step (IS)
Claims
l-27
Novelfy
Industrial aQpliibility
(IA)
Claims
2. Citations and explanations
see reprats
Form PCTAFEA/la)
sheet
(Boxes I-W , Shad 1) (Jrn~
1994)
. ;
.
WRITTEN OPINION
International appkatlon No.
VII. Certain detects in the intmational
PCT/CA99/00987
application
The folowing dsf@cls in the form or contents of the international application have been noted:
SW separate sheel
Wll. CMoin obuvationr
on the Intemtionsl
appliution
730 folbwing obse~atbns on the ckuity of the claims, desctiption, and drawings or on the question whslher the
.
. .._.
. . _.
claims aru fulfy supported wthe description, are made: . . . .
sue wperrts
Fom PCTIIPEN~~
sheet
(3oxms I-VIII. Shec(2) (Jjnuwy lS94)
BLEL-LcieS
WRIT-I-EN OPINION
SEPARATE SHEET
International
appliiatianNo. PCT/CA99/00987
Re Item V
Reasoned statement under Rule 66.2(a)@) with regard to novelty, inventive step
or industrial applicability; citations and explanations supporting such statement
Reference is made to the following documents:
01: JP+A-53112195
02: Chem. Abs. 177659
.
.
.
Noveltv (Art. 33(21 PCTI
Claims 15 and 25 are not novel.
Document 01 descrfbes a method to extract fat from, e.g. Horse Mackerel by placing
said animal preferably in acetone followed by drying at 40-60 “C. The step of
separating the liquid and solid content before drying is obvious.
The subject matter of claim 15 is therefor not new.
Document 02 discloses a shrimp extract (knll is a shrimp) containing 445 mg!L of
carotenoid In astaxanthin. The krill extract is less than 200 kg, taking into account that
the density of soybean oil is less than the density of water. Hence, the krill lipid extract
carotenoid in astaxanthin is at least 22OugIg of krill extract.
The subject matter of claim 25 is therefor not new.
Inventiveness (Art 3331 PCT)
The present set of claims is not based on an inventive step. The proposed method of
extraction does not seem to improve the amount of extracted products. The results are
often worst than when using the method of Folch et al. (see Table 7 to 11).
P.9 I. 29-30, the applicant writes that the lipids extracted with the Folch method are
toxic. No documents are cited to support this affirmation. Also, the applicant has not
shown a particular selectivity of his method over the method of Folch (see Table 12).
Because of the absence of any quantitative or qualitative technical effect, neither the
objective problem nor the solution to this problem can be defined. The present application cannot be considered as involving an inventive step (Article 33(3) PCT).
Form PCT/Sapanb
shnth.3
(sheet
I) (tPo.~p:111937)
OQ/ll/OO
.;
15:58
si UUJ
BLEL-CldeS
FAX 819 821 8215
.. .:
WRITfEN OPINION
SEPARATE SHEET
International
appticati NO. PCT/CA99/00987
Re Item VII
Certain defects in the international application
The symbol “:’ in claim 25 is obviously an error and has been interpreted as “JL-.
.-
.
.
.-
pe Item VIII
Certain observtiions
.
_
.
.
-.
on the international application
Claims 25 and 26 are not supported by the description as required by Article 6 PCT, as
their scope is broader than justified by the description. The reason therefor is the
following: the amount of carotenold in asthaxanthin or in canthaxanthin per weight of
krill extract has not been mentioned in the description or in the examples.
Form PCT/Sqmrato
S’weV4ob (Shaet 2) (EPO-*gid 1997)
T&i. : (450) 562-8525
Fax : (450) 562-1433
E-mai\ : lye-sari 0 lye-san.ca
Internet : www.tyo-san.ca
Lye-San Inc.
500. bout. de I’Adroparc, C.D. 598
Lachute (Qu&ec) Canada I
JEW 4G4
TEE PREE%E-DRYING TECANOLOGY'
AND THE LYO-TECH FREEZE DRIERS
General
L
Information
MANUFACTURIER DE PAOOUITS LYOPHILtStS &CHtS
FREEZE-DRIED PRODUCTS MANUFACTURER
A FAOIO)
LYIJ-bW4
lllYKlLUN!zGN
rc-tbt
It
FAX
LYO-SAN &c.
500 Akroparc, C.P. $8
Lnchute, Qukbec, Ca@da
,
J8H 4G4
-rCl.: (450) 562-8525 /
Fax.: (450) 562-14333
------
--_--M-m
A:
A/S:
e-mail: celine@lyo-sanxa
Internet: www.Iyo-sanca
Neptune tecl$nologle UL bioresoutce,
M . Ro@rC
_---we
----------------
De:
C&line St-Pierre
Dirtctrice gb&alc
(Poste 28)
Fax:
Date: 12 avril2002 j
----
-------
Nombrc total de pagesincluant celle-ci: 12
_--_c-SW
Sujet : Document sti lyophilisation et lyopbilisateur de Lye-San.
Bonjour M . Corriveap,
Tel que convenu lors$ie notre conversationttlkphonique de ce matin, vous trouverez ci-joint ks
documentssuivants : :
- the fkeze-dtying technology and the Lyo-Tech fiecze driers
- Specifkatiobs , Lye-Tech freeze drier Y5 model.
Vow pouvezk&xn&
aiter sur le site de I’ACIA (Agence canadiennede I’inspection des
aliments), les noms &s entreprisesapprouvkessont ins&s.
Voici le site internet k yww.cfia-(icia.ag
Veuillez rioter qu’il $ a bexwcoupd’information sur cc site et ce n’est pas toujours &idenI de
trouver rapidement c+ que I’on veut. Je vais vow envoyerun messagepar courriel qui vous
donne le lien direct @ourarriver sur la section qui vous permettra de voir lc nom de Lye-San dans
lcs compagniesappr&es pour les l&s-Unk et 1’Union Eufop&enne.
J’esp&reque ccs inflations
vont vous aider pour votre dossier. N’b&tez pas B m e contacter si
des renseignementsoompkmentaires sont nkcessaires.
Recevez,M . Corriveau, mes salutationsdistingukes.
PA&
Ctline St-Pierre
01
LYU-b&&N
What is
freeze-drvinq
HUKlLUNXV’iIt
exactly?
to a stage
Freeze-drying
is a process by which we bring a product
low content
of residual
moisture.
where it will
contain
a very
W a ter
extraction
from the product
will
assure
a much better
stability
and conservation
on a long term basis
will
then be
possible.
physical
transformation.
This
Freeze-drying
Ls using a simple
transformation
is called
sublimation
and occurs when ice changes
to water vapor directly
by-passing
the liquid
phase so there is no
presence of water at any tim e in the process.
To achieve
fre&ze-drying,
- the
product
- the
environment
four
conditions
need to be met:
has to be frozen
has
to be under
a negative
pressure
(vacuum)
- an energy
-
a
water
source
vapor
trap
for
sublimation
must be available
must be available
Lyo-Tech freezd-driers
are equipped to realize
all those conditions
efficiently.
Following
is a short description
of the different
equipment
and kxplanation
of their
individual
functions.
-l-
Description
and
function
of
freeze-drier
comoneqts
I
4
Sublimation
;
chamber:
which
takes
Cylindrical
tank
in
place the sublimation.
When the
door is closed the tank is airtight
and can be put under vacuum.
Trays:
Recipient
be dried
Shelves:
Surface
in the
shelves
product
Condenser:
1
i
on which
is placed.
the
product
to
on which we put the trays
sublimation
chamber.
The
may be cooled
to freeze
the
and heated to dry it.
Heat exchanger , placed
inside
the
sublimation
chamber,
that can be
cooled to recuperate
the water
vapor after
sublimation.
,
Compressor:
t
Mechanical
cooling
condensers.
-20
unit
of
that
the
achieves
shelves
the
and
Mechanical
Vacuum pump:
unit
vacuum inside
the
that
achieves
sublimation
the
chamber.
Circulating
Heating
p 4 mp:
elemebts:
Mechanical
unit that takes the
heating
fluid
and sends it to the
By heating
that fluid
we
shelves.
can increase
the temperature
of the
shelve8 and thU8 supply the
necessary energy for sublimation.
Immersed
heat the
electrical
heating
elements
fluid.
that
I
Control
paneli
I
Panel on which
instruments
-3-
are all
of the
control
freeze-drier.
and
113;
LYU-3-W
it3
W h v is
Freeze-drying
freeze-drvina
tiUKlLUNbW
rcIut
It
the best?
&as numerous advantages if
.of drying and conservation
compared
process.
to
other
types
l-
when
Freeze-dried
products
can be stored at room temperature
Transportation
and storage
packed inlairtight
contsinera.
cost are thus lower.
2-
Freeze-dr$ing
produce6
an important weight loss that
facilitates
the handling.
A6 an example, some food6 can
lose as m$ch as 90 % of their initial
weight.
3-
The freete-drying
process will
achieve
temperatufe.
The product is thus protected
overheating
that
will
alter
its
quality.
drying
against
at
4-
Once recohstitued
the freeze-dried
product will recover
its initibl
color,
flavor and texture in a few m inutes.
5-
One of th& most in\pOrtant
low
advantage6 of freeze-drying
is
that this:process
can dry food6 while keeping their
vitamins,
m inerals and protein almost intact.
Vitdns
and
protein are very sensitive
to heat- Low temperature
involved 'in
this
process will
deterioration
of
prevent
those impbrtant elements. Also since the drying is done under
vacuum this will
prevent oxydation of the vitamins.
-4-
UC
LYO-SAN
The
freeze-drvinq
Foods
filled
preparation:
l-
Food
2-
Freezing::
Vacuum:
4-
Sublimatibn:
S-
Opening:
6-
Defrosting:
The normal
cycle
are processed
and
with the product.
trays
are
Trays are placed in the rsublimation
started,
compressor
is
chamber,
ahelvee begin to cool down and the
product
ie freezing
slowly.
I
3-
tiORIZONSANTE
Vacuum pump is started
and pressure
in the 6Ubli8natiOn
chamber drops.
'
Heat
is
starts.
Freeze-drier
is removed
'
switched
ie
from
on
and
opened and product
the traye.
While the empty trays
are sent
freeze-drier
preparation,
defrosted
and cleaned.
cycle
time
for
a freeze-drier
-5-
drying
is
24 houre.
to
is
04/12/2002
10:20
4505621433
LVO-SAN
PAGE
HORIZONSf\NTE
TN : (450) 562-8525
Fax : (450) 562-1433
E-mail : lye-sanOtyo-san.ce
Internet : www.lyo-san.ca
Lye-San hc.
500. boul. de I’A6roparc. C.P. 598
Lachute (Qu&ec) Canada
JBH 4G4
SPECIFICATfONS
LYO-TECB FREEZE
DRIER
Y5 MODBL
*NUFACT~RIER
DE PRODUITS LYOPHILIS~S tsbts
FREEZE-DRIED PRODUCTS MANUFACTURER
I
08
A FROID)
84/12/2682
LM-FAN
4505621433
18: 20
Eauioement
. One vacuum
. Two heating
and accessories
list
pslmp
klementa
. Two circula&g
. Three
HORIZONSANTE
pumps
condenmerm
. One automatit
defrosting
. One control
@cl
. One Mcleod
g+uge
. One digital
with
for
kemperature
eyatera
electromechanical
lecture
of vacuum
control
.
-l-
ewitch
PAGE
89
04/12/2002
10:20
BC hnical
Freeze-drier
information
for bulk products
interior
condensers
wall mounted
trays
tkpe:
from 66 to 88 kg of wet
product
per cycle depending on product
to dry.
Capacity:
Total
sublimation
Refrigeration
Condensing
P&GE 18
LYO-SAN HOf?IZONSnNTE
4505621433
!
12.25m
surface:
5.61tw compressor,
using freon
R502, water cooled.
l
minimum water consumption:
1100 1 / hr
* maximum water consumption:
1900 1 / hr
* water at 20C
fystem:
3 condensers
total
capacity
system:
minirmam
O-75?
gyy
Vacuum eystem:;
of 85 kg of water
teqerature:
vacuum pump
s',""iW
of
eupplying
a vacuum of
microns
in 60 minutes
approximately.
-2-
-6X
100
Heating
2 electrical
system4
elements
of
6KW
each
Quantity
of
trayat
44 (45 cm X 60 cm)
eteel
eteinleee
Quantity
of
shglves:
11
* Space
2.h
deep
21&n wide
2.51~ high
requirbd:
Maximum power
tequiredt
22.exw
Total
electricity
consumption
peti cycle:
(approx. 1
3OOXWh
Voltage:
240 Volts,
600 Volts,
l
Could
vary
d*ending
on layout.
-39
one
three
phase,
phase,
6082
6OH2
04/12/2002
10: 20
4505621433
r
I.
<
u
~‘fo-S&N
t,
V
t
r
HXIZCNSANTE
r
al
,
J
P&E
12