NZ626764B2

NZ626764B2 - Method for processing crustaceans to produce low fluoride/low trimethyl amine products thereof

Info

Publication number: NZ626764B2
Application number: NZ626764A
Authority: NZ
Inventors: Inge Bruheim; John Cameron; Jon Reidar Ervik; Mikko Griinari; Even Remoy; Stig Rune Remoy
Original assignee: Rimfrost Technologies As
Priority date: 2012-01-03
Filing date: 2012-12-21
Publication date: 2016-08-02

Abstract

The present disclosure contemplates the creation of a low fluoride crustacean oil processed from a phospholipid-protein complex (PPC) formed immediately upon a crustacean (i.e., for example, krill) catch. Further, the crustacean oil may also have reduced trimethyl amine and/or trimethyl amino oxide content. The process comprises disintegrating the crustaceans into smaller particles, adding water, heating the result, adding enzyme(s) to hydrolyze the disintegrated material, deactivating the enzyme(s), removing solids from the enzymatically processed material to reduce fluoride content of the material, separating and drying the PPC material. Then, using extraction with supercritical C02 or supercritical dimethyl ether, and/or ethanol as solvents, krill oil, inter alia, is separated from the PPC. In the extraction the krill oil can be separated almost wholly from the feed material. Particularly disclosed is a crustacean oil composition, comprising phospholipids and where said phospholipids comprise phosphatidylethanolamine in the range of 1.4 to 4.9 w/w,resulting in a clear red colour due to a minimal oxidation, and/or degradation and formation of dark/brown colour, wherein said phospholipids are between 39-52 wt%, wherein said phospholipids comprise at least 2 wt % phospatidylethanolamine, 65 wt% phosphatidylcholine and maximum 2.4 wt% lysophasphatidylcholine. content. The process comprises disintegrating the crustaceans into smaller particles, adding water, heating the result, adding enzyme(s) to hydrolyze the disintegrated material, deactivating the enzyme(s), removing solids from the enzymatically processed material to reduce fluoride content of the material, separating and drying the PPC material. Then, using extraction with supercritical C02 or supercritical dimethyl ether, and/or ethanol as solvents, krill oil, inter alia, is separated from the PPC. In the extraction the krill oil can be separated almost wholly from the feed material. Particularly disclosed is a crustacean oil composition, comprising phospholipids and where said phospholipids comprise phosphatidylethanolamine in the range of 1.4 to 4.9 w/w,resulting in a clear red colour due to a minimal oxidation, and/or degradation and formation of dark/brown colour, wherein said phospholipids are between 39-52 wt%, wherein said phospholipids comprise at least 2 wt % phospatidylethanolamine, 65 wt% phosphatidylcholine and maximum 2.4 wt% lysophasphatidylcholine.

Description

PCT/m2012/003UU4 Method For Processing Crustaceans To Produce Low Fluoride/Low Trimethyl Amine Products Thereof FIELD OF THE INVENTION The invention relates to a method for processing crustaceans (i.e., for example, krill) rich in lipids to produce compositions low in ﬂuoride, trimethyl amine and trimethyl amine oxide comprising phospholipids, proteinaceous nutrients and oil (i.e., for example, neutral lipids and/or triglycerides).

, BACKGROUND OF THE INVENTION The crustaceans, especially hill, ent a vast ce as biological material. The amount of Antarctic krill (Euphausia superba), depending on the calculation method and investigation, is roughly 1 to 2x109 tons and the possible weight ofthe annual catch is estimated at 5 to 7x106 tons. These small ceans, which live in the cold waters around the Antarctic, are interesting as a source for proteins, lipids such as phospholipids, poly— unsaturated fatty acids etc, chitin/chitosan, astaxantbin and other carotenoids, enzymes and other materials.

Several methods for isolating above-mentioned als have been developed. One problem is that the products may contain unwanted trace al included in the exoskeleton (also called integurnent or cuticle) of the crustaceans. For e, krill accumulates ﬂuoride in their exoskeleton, thereby increasing the ﬂuoride amount of any produced material either through the inclusion of parts of the leton or through extraction ses not taldng into account the transfer of ﬂuoride to the ﬁnal material. In this case free ﬂuoride or loosely bound ﬂuoride may diffuse from the letal material and into the further processed material, making the end product high in ﬂuoride ions and/or fluorinatcd nds.

Fluoride is a nd that in high concentrations is detrimental for the health of land-dwelling s as well as all kind of ﬁsh and crustaceans and eSpecially ﬂesh-water ﬁsh species, since ﬂuoride atoms have the tendency of entering into the bone structure of such organisms and creating ﬂuorosis, or weakening of the bone structure similar in its effect to osteoporosis, but different since it is the bone structure itself, and not the porosity of the bone that is affected. Skeletal ﬂuorosis is a condition characterized by skeletal abnormalities nt pain. It is caused by pathological bone formation due to the mitogenic action of ﬂuoride on osteoblasts. In its more severe forms, skeletal ﬂuorosis causes kyphosis, crippling and invalidism. Secondary neurological complications in the form of myelopathy, with or without radiculopathy, may also occur. High fluoride intake has also been shown to be toxic to the male reproductive system in rat experiments, and in humans high fluoride intake and symptoms of skeletal fluorosis have been associated with decreased serum testosterone levels. Consequently, if krill material is used as a starting material for food or feed products, precautions have to be taken for removing fluoride through the processing steps. r, the diffusion of fluoride and the presence of miniscule particles of the exoskeleton represent a problem that is difficult to overcome when processing krill material in an industrial scale.

Polar lipids such as phospholipids are essential for cell membranes and are also called membrane lipids. For most known animal species the content of polar lipids is nearly constant. However, this does not hold for the Antarctic krill. The phospholipids content varies from 2% up to 10% depending on the season. The high content, e.g. more than 5%, of the phospholipids is in principle good, but means also a problem, e it may result in strong emulsions in rial processes. The ons complicate the tion of the lipid and naceous fractions in the processes, such as hydrolysis.

The krill oil is one the valuable products made from . It ns inter alia phospholipids, triglycerides and carotenoid astaxanthin while being ially free of protein, carbohydrates and minerals. Different portions of the krill material are separated from each other by, inter alia: i) crushing krill mechanically; ii) pressing them, iii) hydrolysis with heat and enzymes; iv) centrifugal force in ng devices; and v) solvent extraction.

What is needed in the art are significant improvements to these rather conventional approaches and are described within many embodiments of the present invention (infra). For example, a disintegrated raw crustacean material may be separated and/or extracted into various enriched low-fluoride, low trimethyl amine and/or low trimethyl amine oxide cean meal and/or oil compositions.

SUMMARY According to a first aspect the invention provides a crustacean oil composition, sing phospholipids and where said phospholipids comprise phosphatidylethanolamine in the range of at least 2 to 4.9 wt%, resulting in a clear red colour due to a l oxidation, and/or degradation and formation of dark/brown colour, n said phospholipids are n 39-52 wt%, wherein said phospholipids comprise 65wt% phosphatidylcholine and maximum 2.4 wt% lysophosphatidylcholine.

Unless the context clearly requires ise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

According to an embodiment of the invention provides a crustacean phospholipid-peptide complex (PPC) ition comprising protein, phospholipids and n approximately 200-500 ppm fluoride.

According to another embodiment of the invention provides a composition sing a mixture of a low fluoride crustacean PPC and a crustacean protein fraction, wherein said fluoride level ranges between approximately 200 - 500 ppm.

The invention s to a method for sing crustaceans (i.e., for example, krill) rich in lipids to produce compositions low in fluoride, trimethyl amine and trimethyl amine oxide comprising phospholipids, proteinaceous nutrients and oil (i.e., for example, neutral lipids and/or triglycerides).

In one embodiment, the present invention contemplates a crustacean oil composition comprising phospholipids and less than approximately 0.5 ppm fluoride. In one ment, the crustacean oil composition further comprises less than approximately 0.001% (w/w) trimethyl amine. In one embodiment, the crustacean oil composition further comprises less than imately 0.02% (w/w) trimethyl amine oxide. In one embodiment, the olipids are between approximately 39-52 wt%, wherein said phosPholipids comprise at least approximately 65% phOSphatidyicholine and at least approximately 2.4 wt% asphatidylcholine. In one embodiment, the crustacean oil further comprises triglycerides, neutral , approximately 20 - 26 Wt% Omega-3 (e.g., n-3) fatty acids, and at least approximately 0.8 wt% free fatty acids. In one embodiment, the crustacean oil composition is lcrill oil.

In one embodiment, the present invention contemplates a crustacean phospholipid- “IO peptide complex (PPC) composition sing a matrix of hydrolyzed protein, phospholipids and between approximately 200—500 ppm ﬂuoride. In one ment, the phOSpholipids are at least 40 wt%. In one ment, the crustacean PPC composition further comprises approximately 0.044% (w/w) nimethyl amine and approximately 0.354% (w/W) nimethyl amine oxide. In one embodiment, the crustacean PPC composition further comprises at least 40% (w/w) triglycerides.

In one embodiment, the present invention contemplates a crustacean de-oiled phOSpholipid—peptide complex (PFC) ition comprising a matrix of hydrolyzed protein, between approximately 0 ppm ﬂuoride, approximately 35% total fat, approximately 16.6% eicosapentaenoic acid, approximately 10.0% docosahexaenoic acid and at least 0.1 wt% free fatty acids. In one embodiment, wherein the total fat comprises less than 20% triglycerides, and approximately 69% other lipid components. In one ment, total fat ses approximately 35.2% fatty acids, wherein approximately 30 wt% of said fatty acids are n—3 fatty acids. In one embodiment, the total lipids further comprise at least 68% phospholipids. In one embodiment, the de-oilcd PPC further comprises approximately 2.2% lysophosphatidyl choline. In one embodiment, the de-oiled PPC further comprises approximately 115 nag/kg astaxanthin.

In one embodiment, the present invention contemplates a method for creating low ﬂuoride cean compositions, sing: a) disintegrating a crustacean catch into a material having a le size ranging between approximately 1 — 25 millimeters, and b) separating said egrated crustacean material into a phospholipid—peptide complex (PPC) composition subfraction, wherein said tion comprises a ﬂuoride content of less than 500 ppm. In one embodiment, the method further comprises extracting said PPC composition subfraction with a ﬂuid comprising a solvent wherein a low ﬂuoride oil is created, said oil having a ﬂuoride content of less than 0.5 ppm. In one embodiment, the 2012/003004 extracting further creates a low trimethyl amine/trimethyl amine oxide oil, wherein said trimethyl amine is less than approximately 0.001% (w/w) and said trimethyl amine oxide is less than approximately 0.02% (w/w). In one embodiment, the separating is performed without emulsiﬁcation. In one embodiment, the solvent comprises a non-polar solvent. In one embodiment, the t comprises at least one polar solvent. In one embodiment, the solvent comprises said non~polar solvent and said at least one polar solvent. In one embodiment, the non-polar solvent includes, but is not limited to, supercritical carbon dioxide and supercritical dimethyl ether. In one embodiment, the polar solvent includes, but is not limited to, ethanol and acetone. In one embodiment, the method further comprises hydrolyzing said cean material before said separating. In one embodiment, the extracting further creates a de-oiled PPC composition. In one embodiment, the polar solvent tes a phospholipid ition and a protein hydrolysate composition ﬁom said de- oiled PPC composition. In one embodiment, the extracting comprises less than ten hours. In one embodiment, the extracting ses less than ﬁve hours. In one embodiment, the extracting comprises less than two hours. In one embodiment, the crustacean material is krill material. In one embodiment, the separating comprises a centrifugal force ofbetween imately 1,000 - 1,800 g. In one embodiment, the separating comprises a centrifugal force of between approximately 5,000 — 10,000 g.

In one embodiment, the present invention contemplates a composition comprising a mixture of a low ﬂuoride crustacean PPC and a low e de-oiled PPC, wherein said ﬂuoride level ranges between approximately 200 —- 500 ppm. In one embodiment, the cean PPC is krill PPC. In one embodiment, the crustacean de—oiled PPC is krill de— oiled PPC. In one embodiment, the crustacean PPC and crustacean de—oiled PPC are in a 1:1 ratio. In one embodiment, the mixture comprises a milled ﬁne powder. In one embodiment, the powder comprises a particle size of approximately 250 pm. In one embodiment, the composition comprises a de level of less than 0.1 %;(mEq/kg). In one embodiment, the ition comprises ananiside level of less than 0.1 % (w/w). In one embodiment, the composition further comprises microencapsulated polyunsaturated Omega-3 fatty acids. In one embodiment, the composition further comprises zinc oxide. In one embodiment, the composition further comprises marine peptides. In one embodiment, the ition further comprises at least one supplemental amino acid.

In one embodiment, the present invention plates a method, comprising ating a composition comprising a low ﬂuoride crustacean PPC and a low ﬂuoride crustacean de-oiled PPC, wherein said ﬂuoride level ranges between approximately 200 —~ 500 ppm. In one embodiment, the method further ses milling said composition into a powder. In one embodiment, the method ﬁnther comprises tabletting said composition into a tablet. In one ment, the method ﬁirther comprises encapsulating said composition into a capsule. In one embodiment, the method further comprises mixing said powder with a food product. In one embodiment, the formulating further comprises microencapsulated polylmsaturated Omega—3 fatty acids. In one embodiment, the formulating further comprises zinc oxide, In one embodiment, the formulating further comprises marine es. In one ment, the formulating further comprises at least one supplemental amino acid.

In one embodiment, the t invention contemplates a composition sing a mixture of a low ﬂuoride crustacean PPC and a crustacean protein hydrolysate, n said ﬂuoride level ranges between approximately 200 — 500 ppm. In one embodiment, the cean PPC is krill PPC. In one embodiment, the crustacean protein hydrolysate is a krill n hydrolysate. In one embodiment, the crustacean PPC and crustacean protein hydrolysate are in a 1:1 ratio. In one embodiment, the mixture comprises a milled ﬁne powder. In one embodiment, the powder comprises a particle size of approximately 250 pm.

In one embodiment, the composition comprises a peroxide level of less than 0.1 %;(mEq/kg). In one embodiment, the composition comprises ananiside level of less than 0.1 % (w/w). In one embodiment, the composition further comprises microencapsulated polyunsaturated Omega-3 fatty acids. In one embodiment, the composition further comprises zinc oxide. In one embodiment, the composition further comprises marine peptides. In one embodiment, the composition further comprises at least one supplemental amino acid.

In one ment, the present invention contemplates a method, comprising ating a composition comprising a low ﬂuoride crustacean PPC and a crustacean n hydrolysate, wherein said ﬂuoride level ranges between imately 200 — 500 ppm. In one embodiment, the method r comprises milling said composition into a powder. In one embodiment, the method further comprises tabletting said composition into a tablet. In one embodiment, the method further comprises encapsulating said composition into a capsule. In one ment, the method further comprises mixing said powder with a food product. In one embodiment, the formulating further comprises microencapsulated polyunsaturated Omega-3 fatty acids. In one embodiment, the formulating further comprises zinc oxide. In one embodiment, the formulating ftnther comprises marine peptides. In one embodiment, the formulating further ses at least one supplemental amino acid.

W0 2013/102792 PCT/IBZOIZIDO3004 In one embodiment, the present invention contemplates a phospholipid—peptide complex (PPC) composition comprising a range between approximately 40 - 50% lipids and less than 0.5 mg/kg ﬂuoride. In one embodiment, the lipids comprise phospholipids. In one embodiment, the present invention contemplates an oil composition comprising approximately 400-500 grams/kg phospholipids, approximately 200-260 grams/kg Omega-3 fatty acids, less than 0.5 mg/kg fluoride, approximately 15 grams/kg lysophosphatidic acid, and less than approximately 8 kg free fatty acids. In one embodiment, the t invention contemplates a ed phospholipid-peptide x (PPC) composition comprising approximately 300~400 grams/kg lipids, wherein imately 0.1-1.0 % are ﬁ-ee fatty acids and a range between approximately 22—27 % (w/w) that are Omega-3 fatty acids. In one embodiment, the lipids comprise phosphohpids. In one embodiment, the present invention contemplates a crustacean lipid composition comprising at least 75% phospholipids. In one embodiment, the lipid composition comprises between approximately 75% - 90% phospholipids. In one embodiment, the lipid composition comprises between approximately 75% - 80% phospholipids. In one ment, the present invention contemplates a dried protein hydrolysate composition comprising approximately 70 — 80% protein, approximately 1.5 - 3.0% lipids, and imately 5 -7 % ash.

In one embodiment, the present ion contemplates a method, comprising: a) providing; i) a hydrolyzed and disintegrated crustacean material; ii) at least one horizontal centrifuge capable of separating said hydrolyzed crustacean material; and iii) a ﬂuid sing a solvent; and b) separating said hydrolyzed crustacean material into a high e solid fraction and a low ﬂuoride hydrolyzed material fraction with a ﬁrst horizontal centrifuge; c) separating said low ﬂuoride yzed material fraction into a phospholipid- peptide complex (PFC) ition subfraction and a trated hydrolysate subfraction with a second horizontal centrifuge; and d) contacting said PPC composition subtraction with said ﬂuid, wherein a low ﬂuoride oil is extracted. In one embodiment, the disintegrated crustacean material has particle sizes between approximately 1 - 25 millimeters. In one ment, the ﬁrst horizontal centriﬁige separates said hydrolyzed cean material Without emulisiﬁcation. In one embodiment, the solvent ses a non-polar solvent. In one embodiment, the non—polar solvent comprises supercritical C02. In one embodiment, the solvent comprises a polar solvent. In one embodiment, the polar solvent comprises ethanol.

In one embodiment, the second horizontal centrifuge comprises an extended separation pathway. In one embodiment, the contacting is med at a pressure of less than 300 bar.

In one embodiment, the non—polar solvent further extracts a de—oiled PPC composition from said PPC composition ction. In one embodiment, the ethanol separates a phospholipid composition and a protein hydrolysate composition from said de-oiled PPC ition. In one embodiment, the de-oiled PPC is separated from the PPC in less than ten hours. In one embodiment, the de-oiled PPC is separated from the PPC in less than ﬁve hours. In one embodiment, the rte-oiled PPC is separated from the PPC in less than two hours. In one embodiment, the hydrolyzed crustacean material comprises hydrolyzed krill material. In one embodiment, the separating said hydrolyzed crustacean al is med at a centrifugal force of between approximately 1,000 — 1,800 g. In one embodiment, the separating said low ﬂuoride hydrolyzed material traction is performed at a centrifugal force ofbetween 1O approximately 5,000 - 10,000 g. In one embodiment, the method es a phospholipid- peptide complcx (PPC) composition comprising a range between approximately 40%- 50% lipid and less than 05 mg/kg ﬂuoride. In one embodiment, the method produces an oil composition comprising imately 400—500 grams/kg phospholipids, approximately 200— 260 grams/kg Omega-3 fatty acids, less than 0.5 mg/kg ﬂuoride, approximately 15 kg lysophosphatidic acid, and less than approximately 8 grams/kg free fatty acids. In one embodiment, the method produces a ed phospholipid—peptide complex (PPC) composition comprising approximately 300-400 grams/kg lipids, wherein approximately 0.1— 1.0 % are free fatty acids and a range between approximately 20-28 % (w/w) are Omega~3 fatty acids. In one embodiment, the method produces a crustacean lipid composition comprising at least 75% phospholipids. In one embodiment, the lipid composition comprises a range n imately 75% - 90% phospholipids. In one embodiment, the lipid composition comprises a range n approximately 75% - 80% phospholipids. In one embodiment, the method produces a dried n hydrolysate composition comprising approximately 70 — 80% protein, approximately 1.5 - 3.0% lipids, and approximately 5 -7 % ash.

In one ment, the present invention contemplates a system comprising: a) a solvent unit comprising at least one non-polar solvent inlet; b) an extraction tank unit in ﬂuidic communication with the solvent unit, wherein the tank comprises an inlet conﬁgured to receive a phospholipid—protein complex composition; c) a tor unit comprising an Outlet conﬁgured to release a low ﬂuoride oil composition and residual co-solvent, wherein the separator is in ﬂuidic communication with the tank; d) an absorbent unit in ﬂuidic comintmication with the separator unit, wherein the absorbent unit is capable of recycling the non~polar solvent. In one embodiment, the non—polar solvent is a supercritical ﬂuid. In one embodiment, the supercritical ﬂuid comprises carbon e. In one embodiment, the PCT/lBZOlZ/003004 supercritical ﬂuid comprises dimethyl ether. In one embodiment, the solvent unit r comprises a eo—solvent inlet. In one embodiment, the vent is a polar solvent. In one embodiment, the polar solvent is ethanol or acetone. In one embodiment, the at least one non-polar solvent inlet comprises an unused non-polar solvent inlet. In one embodiment, the at least one non-polar solvent inlet comprises a ed non-polar solvent inlet. In one embodiment, the t unit further comprises a ﬂuid pump. In one embodiment, the tank unit is pressurized by the ﬂuid pump. In one embodiment, the solvent unit further comprises a heater. In one embodiment, the phOSpholipid-protein complex composition in the tank unit is heated by the heater. In one embodiment, the separator outlet is in ﬂuid communication with an evaporator. In one embodiment, the separator further comprises a horizontal centrifuge. In one embodiment, the horizontal ﬁlge is a decanter centriﬁige having an ed separation pathway. In one embodiment, the phospholipid—protein complex composition is a low e crustacean phospholipid-protein complex composition. In one embodiment, the low ﬂuoride crustacean phospholipid-protein complex composition is a low ﬂuoride Icn'll phospholipid-protein complex composition In one embodiment, the present invention contemplates a method for processing crustaceans, especially krills, in which method the crustaceans are disintegrated into smaller particles, fresh water is added to the disintegrated al, the water with the egrated material is heated and (s) are added for hydrolyzing the disintegrated material and said enzyme(s) is/‘are deactivated, the method further comprising steps: a) removing solids from the hydrolyzed material to reduce ﬂuoride content of the material; b) separating phosphoh'pid-peptide complex material and concentrated hydrolysate fraction from each other; e) drying said phospholipid—peptide complex material; and d) dividing the drying result, or PPC, to components by extraction(s) using at least a supercritical C02 as solvent, wherein the processing of crustaceans is d as soon as a crustacean catch has been decked on a ship or boat. In one embodiment, the ﬂuoride content solids are removed from the hydrolyzed material by a decanter. In one ment, the phospholipid—peptide complex material and concentrated hydrolysate fraction are ted from each other by a sedicanter with high centrifugal forces and long clariﬁcation/separation zones to avoid an emulsiﬁcation. In one embodiment, the method further comprises using in the extraction ethanol as a co-solvent in addition to the supercritical CO2 to separate: i) a krill oil consisting pholipids and triglycerides, or l oil, and ii) a protein hydrolysate from the PPC.

In one ment, the pressure of the solvent being at most 300 bar. In one embodiment, the extraction includes two steps: i) ﬁrst using only the ritical CD; as solvent to PCT/lBZOlZ/003004 te de—oiled PPC from the PPC; and ii) second using only ethanol as solvent to separate phOSpholipids and n hydrolysate from the de-oiled PPC. In one embodiment, the duration of the step When said de-oiled PPC is extracted from the PPC is at most three hours.

In one ment, the method produces a phospholipid-peptide complex (PPC) composition comprising approximately 40% - 50% lipid and approximately 0.5 nag/kg ﬂuoride. In one embodiment, the lipid comprises phospholipids. In one ment, the method produces an oil composition comprising approximately 400—500 grams/kg phospholipids, approximately 200-260 grams/kg Omega-3 fatty acids, approximately 0.5 mg/kg ﬂuoride, approximately 15 kg lySOphOSphatidic acid, and less than ’10 approximately 8 grams/kg ﬁee fatty acids. In one embodiment, the method produces a de- Oiled olipid—peptide complex (PPC) composition comprising approximately 300-400 grams/kg lipids, wherein approximately 0.1-1.0 % are free fatty acids and approximately 22- 27 % (W/W) are Omega-3 fatty acids. In one embodiment, the method es a crustacean phospholipid composition comprising approximately 75% polar lipids. In one ment, the method produces a dried n hydrolysate composition comprising approximately 70 ~ 80% protein, approximately 1.5 ~ 3.0% lipids, and approximately 5 -7' % ash.

DEFINITIONS The term “disintegrated material” as used herein refers to any biological material that has been subjected to a mechanical destruction and/or disruption that results in a composition having particle sizes ofbetween approximately 1 — 25 millimeters= preferably between approximately 3 — 15 millimeters, more preferably between approximately 5 - 10 millimeters and most preferably approximately 8 millimetels.

The term “hydrolyzed material” as used herein refers to any biological material that has been subjected to high heat and/or enzymatic ent. Such hydrolyzed materials would be expected to have phospholipid/peptide components that are physically separated from the components ofthe chitinous exoskeleton.

The term “crustacean” as used herein refers to any marine organism have a hard outside shell (e.g., a chitinous exoskeleton ed with a carbonate) encompassing a ﬂeshy interior that is a living organism. More cally, the crustaceans are usually considered a large class of mostly aquatic arthropods that have a chitinous or calcareous and ous exoskeleton, a pair of often much modiﬁed ages on each segment, and two pairs of ae. For example, a crustacean may include but not limited to, krill, lobsters, shrimps, crabs, wood lice, water ﬂeas, and/or barnacles.

The term “horizontal centrifuge” refers to any device that is capable of rotating a mixture in the Z-plane (as opposed to the e and/or Y—plane as with conventional centrifuges). This rotation is generated by a type conveyor element aligned horizontally within a tube shaped enclosure. The induced centrifugal forcethen layers the heavier particles to the outside edges of the enclosure, while the lighter particles form layers closer to the center of the enclosure. Some horizontal centrifuges are modiﬁed to comprise an extended separation pathway and induce high gravitational forces (cg, a sedicanter).

The term “polar solvent” as used herein refers to any compound, or compound mixture, that is miscible with water. Such polar solvent compounds include, but are not limited to, ethanol, propanol and/or ethyl acetate.

The term “non—polar t" as used herein refers to any compound, or compound mixture, that is not miscible with water. Such non-polar solvent compounds include, but are not limited to, hexane, pentane, dimethyl ether and/or C02. Either yl ether or C02 may be used in a supercritical phase. ’15 The term “supercritical” refers to any mixture comprising a chemical (e. g., for e, carbon dioxide (CO2) or dimethyl ether) in a ﬂuid state while held at, or above, its critical temperature and critical pressure Where its characteristics expand to ﬁll a ceniainer like a gas but with a density like that of a liquid. For example, carbon e becomes a supercritical ﬂuid above 31.1 °C and 72.9 aim/7.39 MPa. Carbon dioxide y behaves as a gas in air at standard temperature and pressure (STP), or as a solid called dry ice when frozen If the temperature and pressure are both increased ﬁom STP to be at or above the critical point for carbon dioxide, it can adopt properties midway between a gas and a liquid.

As contemplated herein, supercritical CO2 can be used as a commercial and industrial solvent during chemical extractions, in addition to its low toxicity and minimal enviromnental impact. The relatively low temperature of the process and the stability of CO2 also allows most nds (i.e., for example, ical compounds) to be ted with little damage or denaturing. In addition, because the solubility ofmany extracted compounds in C02 may vary with pressure, ritical CO2 is useful in performing selective extractions.

The term de” as used herein interchangeably and refer to any compound containing an organoﬂumide and/or an nic e.

The term “high ﬂuoride solid haction” as used herein refers to a composition containing the vast majority of a crustacean’s exoskeleton following a low g-force (e.g., between approximately 1,000 — 1,800 g) horizontal ccntrifugation separation of a hydrolyzed and egrated crustacean al. This fraction contains small particles of exoskeleton of the crustacean that retains the vast ty of e (i.e., for example, between 50 - 95%) in these organisms.

The term “low e” as used herein may refer to the product of any method and/or s that reduced the ﬂuoride from the original material by approximately d (i.e., for example, from 5 ppm to 0.5 ppm). For example, ‘a low ﬂuoride crustacean phospholipid- protein complex’ comprises ld less ﬂuoride than ‘a low e hydrolyzed and disintegrated crustacean material’.

The term “low ﬂuoride hydrolyzed material fraction” as used herein refers to a composition containing the vast majority of a crustacean’s ﬂeshy internal material following a low g~force (cg, between approximately 1,000 - 1,800 g) horizontal cenuifugation separation of a hydrolyzed and disintegrated crustacean material. This fraction contains small particles ofphospholipids, neutral lipids, proteins and/or peptides that is largely devoid of any ﬂuoride (i.e., for example, between 5% — 50% ofthe raw hydrolyzed and disintegrated material).

The term “a 10W ﬂuoride phospholipid—peptide complex composition subfraction” as used herein refers to a low e composition containing the vast majority of lipid material following a high g—force (cg, between approximately 5,000 - 10,000 g) horizontal fugation tion of a low ﬂuoride hydrolyzed material fraction.

The term “concentrated hydrolysate composition ction” as used herein refers to a low ﬂuoride composition containing the vast majority of water soluble lean material following a high gvforce (e.g., between approximately 5,000 - 10,000 g) horizontal centrifuge separation of a low e hydrolyzed material ﬂaction.

The term “low ﬂuoride oil” as used herein refers to a lipid-rich composition d by the extraction of a phospholipid—peptide complex composition subtraction using a Selective extraction process, such as with a supercritical carbon dioxide ﬂuid. Such a process s approximately ten-fold of the ﬂuoride from the raw yzed and disintegrated crustacean material.

The term “dc—oiled phospholipid-peptide complex” as used herein refers to a low ﬂuoride composition containing the vast: majority of dry matter composition d by the extraction of a phospholipid—peptide complex composition subfraction using selective extraction process, such as a supercritical carbon dioxide ﬂuid. A tie—oiled PPC generally comprises a reduced triglyceride content in comparison to PPC.

The term “phospholipid composition” as used herein refers to a low ﬂuoride composition comprising a high percentage of polar lipids (cg, approximately 75%) created ZOIZIDO‘3004 by the extraction of a de-oiled phospholipid—peptide complex using a co-solvent, such as ethanol.

The term “protein hydrolysate” as used herein refers to a low e composition comprising a high percentage of protein (e.g., approximately 70 - 80%) created by the extraction of a de-oiled phospholipid-peptide complex using a ecu—solvent, such as ethanol.

The term "immediately" as used herein refers to a minimum practical period between decking a crustacean catch in a trawl bag and/or net coupled with a direct transfer to a suitable disintegraor. For example, this minimum practical period should preferably not exceed 60 minutes, more preferred to not exceed 30 minutes, even more red to not ’10 exceed 15 minutes.

The term “hydrolysis” as used herein refers to any break and/or disruption made in a protein structure of a disintegrated crustacean material, wherein in the naturally ing protein sequences become shorter , for example, by breaking peptide bonds ofthe amino acid sequence primary ure) and/or denatured (i.e., for example, an unfolding of the ’15 amino acid sequence secondary, tertiary and/or quaternary structure). This process may be controlled by hydrolytic enzyme(s). For example, one or more exogenous proteolytic enzymes (cg. alkalase, neutrase, and s derived from microorganisms or plant s) may be used in the process. Co—factors such as speciﬁc ions can be added depending on the used enzymes. The selected enzyme(s) can also be chosen for reducing emulsions caused by high content of olipids in the raw material. Besides the temperature, the ysis takes place within optimal or ptimal pH and sufﬁcient time. For example, the exogenous enzyme alkalase the optimum pH is about 8, optimum temperature about 60°C and the hydrolysis time 40—120 s.

The term “solvent unit” refers to any enclosed volume conﬁgure to heat and pressurize a mixture of supercritical carbon dioxide ﬂuid and/or a (Jo-solvent (e.g., ethanol). Such an enclosed volume may be constructed out of any suitable material including but not limited to metals (e.g., steel, aluminum, iron etc), plastics (cg, rbonate, polyethylene etc), ﬁberglass (etc).

The term “extraction tank” refers to any enclosed volume conﬁgured to and heat and pressure sufﬁcient to perform lipid and protein extraction from a raw biomass using a supercritical carbon dioxide ﬂuid. As designed, the extraction tank contemplated herein is conﬁgured such that the solvents containing the extracted lipids and proteins rise to the tank top for er to a separator unit. Such an enclosed volume may be constructed out of any W0 2013/102792 suitable material including but not d to metals (e.g., steel, aluminum, iron etc), plastics (e.g., polycarbonate, polyethylene etc), ﬁberglass (etc).

The term ator unit” refers to any enclosed volume conﬁgured with a centrifuge capable of separating the components of the extracted. lipids and proteins received from an extraction tank. The tive tion components exit the separator unit via outlet ports such that the ing solvents (i.e., supercritical C02) are transferred to an absorbent unit for recycling. Such an enclosed volume may be constructed out of any le material including but not limited to metals (e.g., steel, um, iron etc), plastics (cg, polycarbonate, polyethylene etc), ﬁberglass (etc).

The term “absorbent unit” refers to any enclosed volume conﬁgured with materials that will remove contaminants from a supercritical C02 ﬂuid. Such materials may include, but are not limited to charchol, coal, ing gases, plastic polymer resins and/or tion dges comprising single or dual-ﬂat ed nets (Tenax UK LTD, Wrexham, North Wales LL13 9JT, UK). Such an enclosed volume may be constructed out of any suitable ’15 material including but not limited to metals (cg, steel, aluminum, iron etc.), plastics (cg, polycarbonate, polyethylene etc), ass (etc).

The term “in ﬂuidic communication” refers to any means by which a ﬂuid can be transported from one location to another location. Such means may include, but are not limited to pipes, buckets and/or troughs. Such means may be constructed out of any suitable material including but not limited to metals (e.g., steel, aluminum, iron etc), plastics (e.g., polycarbonate, polyethylene etc.), ﬁberglass (etc).

BRIEF DESCRIPTION OF THE FIGURES Figure 1 presents a ﬂow diagram of one embodiment of a method to produce a low ﬂuoride crustacean material.

Figure 2 presents a longitudinal centrifuge with an extended separation path. This specific example is a FLOTTWEG SEDICANTER horizontal decanter centrifuge.

Figure 3 depicts one example of an extraction plant suitable for use in the presently disclosed method. For example, the plant comprises a solvent unit (21), an extraction tank (22), separators (23) and ents (24).

Figure 4 present exemplary data showing the extraction efﬁciencies of two different runs in accordance with one embodiment of the present invention DETAILED DESCRIFIION OF THE INVENTION The invention relates to a method for processing crustaceans (i.e., for example, krill) rich in lipids to produce itions 10W in fluoride, trimethyl amine and trimethyl amine oxide comprising phospholipids, proteinaceous nutrients and oil 6.6., for example, neutral lipids and/or triglycerides).

Krill oil comprises lipids extracted with solvents from loill biomass. Krill biomass can be either fresh, whole krill (W02008/060163A1), frozen Whole krill (Neptune Technologies & Bioresources Inc, Canada), lyophilized whole krill (JP2215351) or krill meal (U820080274203). Solvents used in extracting lipids from krill biomass have been reported as acetone + ethanol (W02000/23 546; W02002/1023 94), ethanol + hexane (Enzymotcc Ltd), ethanol alone 5351; AkCI‘ BioMarine ASA, Norway) or supercritical C02 + ethanol co-solvent (U32008/0274203; W02008/060163). Solvent~free technology for obtaining krill oil has also been developed (U8201 10224450Al). Krill oil comprises a lipid fraction of raw krill biomass that is essentially free of protein, carbohydrates and/or ls.

Krill oil also comprises neutral lipids (e.g., mostly triglycerides), polar lipids (e.g., mostly phospholipids) and carotenoid astaxanthin. Although it is not necessary to tand the mechanism of an invention, it is believed that the lipid and/or fatty acid compositions of krill oil vary depending ofthe season.

In some embodiments, the present invention contemplates methods of processing crustacean biomass having cted ﬁndings ing, but not limited to: i) removal of most of the exoskeleton ﬁom the crustacean biomass that results in low level of ﬂuorides in a PPC composition and very low levels of ﬂuoride in krill oil extracted ﬁom the PPC composition by a non—polar solvent (e.g., n'tical C02) and, ally, a polar co- solvent (e.g., ethanol); ii) a level of ﬂuorides in the crustacean oil that is less than 0.5 ppm in contrast to tional krill oil with ﬂuoride content of approximately 5 — 100 ppm; iii) crustacean oil ted from PPC by ritical CO; and ethanol co-solvent has a minimal browrr color suggesting that minimal degradation of astaxanthin or formation of ry oxidation products has occurred; iv) a reduced dark/brown color as measured on a Hunter L* scale; v) a reduced e content as measured by absorption at 570 nm; v) minimal contents of free fatty acids (i.e., for example, 0.8 g/100 g of oil (~ 0.8% W/w)) and lysophosphatidylcholine (i.e., for example, 1.5 g/100 g of oil (~ 1.5% W/w)). These s suggest that the lipids of cean biomass have undergone minimal hydrolysis during the initial processing steps producing PPC PCT/IBZOIZ/003004 1. Historical Overview of cean Processing Methods Publication GB 2240786 discloses a method for processing krill including removing a part of the ﬂuoride content of krill. The ng is based on passing electric current through ized krill. However, ﬂuoride-containing solid les remain in the material.

Publication US 2011/0224450 (Sclabos Katevas et 31., herein incorporated by reference) discloses a method for obtaining krill oil ﬁom Whole raw krills using inter alia cooking, separating by decanter, and pressing. No solvents and extraction are used.

Publication (Pronova Biopharma AS) discloses a method for obtaining lcrill oil using supercritical C02 and either ethanol, methanol, propanol or isopropanol as co-solvent. Fresh or pre-heated (about 90 °C) Whole krills are used as the extraction feed material.

Publication WO 02/102394 (Neptune Technologies & Bioresources) discloses a method for obtaining krill oil using in diﬁerent phases acetone and ethanol or e. g. ethyl acetate as solvents. Frozen whole krill is used as feed material.

Publication JP 2215351 (Taiyo y) discloses a method for obtaining krill oil using ethanol as solvent. Lyophilized whole krills are used as feed material. ation US 2008/0274203 (Aker Biomarine ASA, Bruheim et al.)(herein orated by reference) discloses a method for obtaining krill oil from krill meal using supercritical ﬂuid extraction in a two—stage process. Stage 1 removes the l lipid by extracting with neat supercritical C02 or C02 plus approximately 5% of a co-solvent. Stage 2 extracts the actual krill oils using supercritical C02 in combination With approximately 20% ethanol.

There are a number ofproblems associated with these conventionally known technologies of extracting krill lipids, including but not limited to: i) whole crustacean biomass contains high ﬂuoride exoskeleton particles that results in the production of ﬂuoride- contaminated crustacean oil; ii) crustacean oil having a brownish hue color may arise from exposing astaxanthin to excessive heat during crustacean biomass processing. Speciﬁcally, the brown color can arise from degradation of nthin and]or from accumulation of the end products of non-enzymatic browning (e.g., Strecker degradation products or polymerized pyrroles). Although it is not necessary to understand the mechanism of an invention, it is believed that a brown color resulting from this non-enzymatic process results from oxidative degradation due to a on of secondary lipid oxidation products with amino groups from amino acids or ns creating so-called ry oxidation products; iii) freezing the cean biomass for transportation to an extraction plant can result in relative stability, but some changes in the product are known to occur over time, for example, one characteristic change in frozen krill is a partial hydrolysis of the lipids ing in the accumulation of free fatty acids (FFA) arising ﬁom degradation of triglycerides, phospholipids and/or lysophospholipids, speciﬁcally lysophOphatidylcholine (LPC), arising from ysis of phosphatidyleholine; and iv) the use of heat and frozen storage can induce oxidation of lipids and proteins in crustacaan biomass, where primary oxidation leads into formation of secondary ion products that are volatile and can be dctected in krill oil as off-ﬂavors or undesirable odor; and v) the separation of the krill oil from the feed material is quite ient, n only about a half of the oil can be extracted.

II. Production 0f Low Fluoride Crustacean Materials In one embodiment, the present invention contemplates a method comprising forming a phospholipid-peptide complex (PPC) composition from a crustacean (i.e., for example, krill) immediately after the catch has been brought upon on board (cg, decked) a boat and/or ‘15 ship (i.e., for example, a ﬁShing vessel). The process of ng the PPC ition ses disintegrating the crustaceans into a disintegrated material comprising smaller particles (i.e., for example, between approximately 1 - 25 millimeters), adding water, heating the disintegrated material, adding (s) to hydrolyze the disintegrated material, deactivating the enzyme(s), removing solids (i.e., for example, exoskeleton, shell, and/or carapace) from the enzymatieally processed material to reduce the ﬂuoride content of the material, separating and drying the PPC composition. Preferably, the PPC composition is erred to an On-shore facility (Le, a ﬁsh oil extraction plant) where a low-fluoride crustacean oil is separated from the PPC composition using solvents including, but not limited to, supercritical C02 and/or ethanol. Using alternative extractions, de-oiled PPC compositions, phospolipids and/or protein hydrolysate compositions are also separated from the PPC composition.

- An advantage of some embodiments of the invention is that these crustacean products, like krill oil, have a low e content. This is due to the fact that the solid crusteacean exoskeletal particles (i.e., for example, shell and/or ce) are effectively d from mass to be processed.

- Another advantage of the invention is that crustacean oil can be separated effectively, almost completely, from the egrated crustacean material (e.g., feed material) during the extraction. This is due to the fact that, in the extraction process with, for example, a supercritical C02 solvent, the feed material comprises a PPC composih'on. Although it is W0 2013/102792 2012/003004 not necessary to understand the mechanism of an invention, it is believed that the phospholipids of the feed material are embedded in a matrix ofhydrolyzed protein which means that the close association between the phospholipids and hydrophobic/phosphorylated proteins is broken thus facilitating the extraction of the lipids.

- An advantage of the invention is that relatively low re and temperature can be used in the tion, Which means lower production costs.

- A further advantage of the invention is that disposal of residual solvents, common when using other more conventional lipid solvents, is avoided when using ritical CO; as a solvent.

— A further advantage of the invention is that phOSphatidylserine (PS), free fatty acids (FFA) and osphocholine (LPC) contents are very low in the end products.

- A further advantage of the invention is that a low ﬂuoride cean oil product (i.e., for example, a 10W ﬂuoride krill oil) has very little brown color. It is believed in the art that apperance of a brown color in crustacean oil indicates that unfavorable processes are occuring during the the manufacture of the feed material (e.g., a disintegrated cean al).

A. Processing 0f Crustaceans The present invention provides an rial method for processing catches of ceans comprising a number of steps beginning with a very early and ntially complete removal of the crustacean’s exoskeleton (i.e., for e, the crust, carapace and/or shell). Although it is not necessary to understand the mechanism of an invention, it is believed that the crustacean exoskeleton comprises a vast majority of ﬂuoride in the organism. Consequently, this step thereby results in a substantial removal of ﬂuoride from C: the crustacean material. The method also uses udinal ﬁsgation techniques that prevents separation problems caused by emulsions When processing a raw material with high content ofphospholipids.

The method according to the present invention is initiated immediately after decking a catch of crustacean. It is of importance that the method according to the present invention is initiated as soon as possible after the crustacean catch has been decked since ﬂuoride starts to leak/diffuse immediately from the exoskeleton into the crustacean’s ﬂesh and juices.

When using the term ”immediately" in connection with starting the process according to the present invention this relates to the period from decking the crustacean catch and to the initial disintegration of the crustacean. This period of time should be kept to a minimum, and should preferably not exceed 60 minutes, more preferred not exceed 30 minutes, even more W0 2013/102792 ZOIZIOO3004 preferred not exceed 15 minutes, and should include a direct transfer of the crustacean catch from the trawl bag and/or net to a suitable disintegrator. A disintegrator of the crustacean material may be a conventional pulping, milling, grinding or shredding machine.

The crustacean catch is initially loaded into a disintegration appratus where the crustacean catch is subjected to pulping, milling, grinding and/or shredding to create a disintegrated crustacean material. The ature of the disintegration process is around the ambient temperature of the water ( i.e., for example, between approximately -2 and +1° C, but more ably n approximately +0° C to +6“ C) and may be performed by any ient disintegration method. This disintegration process is also conventionally done by the us known processing methods, and represents one of the les according to the prior art because it produces large amounts of exoskeletal particles from the crustacean mixing in the milled material and producing a disintegrated paste with a high ﬂuoride content. However, this high e content is one of the reasons Why the prior art processed crustacean material has limited ations and is less suitable for food, feed or corresponding food or feed additives compared to other marine raw materials e. g. c ﬁsh.

According to the present invention the crustacean material is separated into a particle size suitable for a further separation step that does not interfer with the subsequent extraction steps. The disintegrating process is performed continuously and produces particle sizes up to 25 mm, a red particle size range is between approximately 0.5 - 10 mm and a more preferred size range is between approximately 1.0 - 8 mm.

Although it is not necessary to understand the mechanism of an invention, it is believed that this small particle size bution represents one of advantages of the present invention because the ﬂuoride has a tendency to leak out of the milled material and mingle with the rest of the raw material. However, this leaking process takes time and is not rapid enough to negatively impact a subsequent enzymatic hydrolysis step, provided the ysis step is performed within Speciﬁc parameters with t to time and optimal, or near-Optimal ions, such as pH and temperature and optionally with the addition of co-l‘actors such as specific ions depending on the used enzymes.

The temperature ofthe disintegrated material may, according to the present invention, be elevated to a ature suitable for the subsequent enzymatic hydrolysis. Preferably, the temperature may be increased within seconds (e.g., 1—300 seconds, more preferred l—lOO s, even more preferred 1-60 seconds, most preferred 1-10 seconds) subsequent to the W0 2013/102792 disintegrating step for reducing the processing time and thereby preventing diffusion of ﬂuoride and for preparing the material for the enzymatic hydrolysis. ing to the present invention s may be added ly to the disintegrated material or through the added water or both, before, during or after the disintegration process.

According to the t invention, exogenous proteolytic enzymes (e.g., alkalase, neutrase, enzymes derived ﬁom microorganisms including, but not limited to, Bacillus is and/or Aspergillus niger, and/or or s derived ﬁom plant species) may be added before, during or after the disintegration, and before, during or after the heating of the disintegrated material. The added (s) may be in the form of one single enzyme or a mixture of enzymes. The conditions of the hydrolysis should match the optimal hydrolytic conditions of the added enzyme(s) and the selection of optimal conditions for the selected exogenous hydrolytic enzyme(s) is known to the person skilled in the art. As an example, the exogenous enzyme alkalase having a pH m of about 8, a temperature optimum of 60° C and a hydrolysis time of 40-120 minutes. The selected enzymes, or combination of enzymes, should also be chosen for ng emulsions caused by high content of phOSpholipids in the raw material.

An nt amount of proteolytic e(s) will be set after a process- and product zation process that depends upon the efﬁciency of a speciﬁc chosen cial enzyme or mix of enzymes. A typical amount by weight of commercial enzymes, as a ratio ofthe amount of the weight of the disintegrated raw material, are preferably between 0.5% and 0.05%, more preferably n 0.3% and 0.07% and most preferable between 02% and 0.09%. This hydrolysis step is aided by endogenous (natural) enzymes because rapid and uncontrolled autolysis is well known in fresh caught crustaceans.

In one embodiment, the ous enzymes breakdown the proteinaceous material in the disintegrated substance as well as speed up and/or accelerate the hydrolysis of the material to avoid and/or preclude the leaking of ﬂuoride from the shell, carapace and crust.

These ytic enzymes, or a combination of hydrolytic enzymes, should also be carefully chosen to reduce emulsion in the separation process. For example, such enzymes may be selected from 6210— and/or endopeptidases. If a mixture of enzymes is used, such a mixture may also include one or more chitinases for subsequently making the chitin—containing ﬁaction(s) more amenable to further downstream processing. If chitinases are used, care must be taken for not increasing the leakage of fluoride from the shell/crust/carapace of the crustacean into the other fractions. However, since such ﬂuoride leakage takes time, it is le to perform such an enzymatic treatment Within the preferred time parameters. A PCT/11320121003004 more convenient alternative to including chitinases in the enzyme mix of the initial hydrolysis step will be to process the separated chitin—containing fraction uently to the separation step.

In one embodiment, the leaking of ﬂuoride from the milled exoskeletal material into the milled ﬂeshy material is avoided by completing the disintegration/hydrolozing steps within a time interval of 100 s, preferably within 60 s, most preferred within 45 s calculated from the addition ofthe nous enzyme(s). The amount ofenzymets) added is related to the type of enzyme t used. As an e it may be mentioned that the enzyme alkalase may be added in an amount of 0.1-0.5% (w/W) of the raw material. This 1O should be taken into context with the added endogenous enzymes since the addition ofmore enzymes will reduce the time al of the hydrolytic step. gh it is not necessary to understand the ism of an invention, it is believed that a short hydroloysis duration reduces the diffusion time of ﬂuoride from particles of the exoskeleton into the proteinaceous material. ‘15 Subsequent to, or together with, the hydrolytic processing step the hydrolyzed and distintegrated crustacean material is passed through a particle removal device operating through a gravitational force such as a longitudinal centrifuge (i.e., for example, a decanter).

This ﬁrst separation step removes the ﬁne particles containing a considerable amount of the ﬂuoride from the hydrolysed or hydrolysing crustacean material to create a solids fraction.

The centrifuge is operated with a g force between 1,000 and 1,800 g, more ably between 1,200 and 1,600 g and most preferably between 1,300 and 1,500 g. Through this particle removal step a substantial amount of ﬂuoride is removed from the naceous crustacean fraction. The reduction of ﬂuoride on a dry weight basis as compared to conventional crustacean meal, with a typical ﬂuoride content of 1,500 ppm, may be up to 50%, even more preferred up to 85%, most preferred up to 95%.

The enzymatic hydrolysis may be terminated by heating ofthe hydrolysing material (incubate) to a temperature over 90° C, preferably between 92-98° C and most preferred between 9295" C, prior to, during or after the separation step, as long as the hydrolysis duration lies within the above given boundaries. The hydrolysis is terminated before, during, or after the ﬁne particle removal step, most preferred after the ﬁne particle removal step. The temperature of the ﬁrst centrifugation le l step in one embodiment, depend on the optimal activity temperature of the enzyme (in the case Where the enzymatic hydrolysis step is terminated by heating after the ﬁne particle separation step).

PCT/IBZOIZ/003004 The ﬂuoride content in the prior art processed krill protein material (eg, ~l,500 ppm) has limited applications and are less Suitable for food or feed or corresponding food or feed additives. In one embodiment, removal of the ﬂuoride content from the exoskeletal material may be followed by a r separation/puriﬁcation of materials such as chitin, chitosan and astaxanthin. Such isolation procedures are known within the art. Steps may also be taken to further reduce the e content from the isolated exoskeletal material using techniques including, but not limited to, dialysis, tmtion, electrophoresis or other appropriate logies. ytic enzyme(s) deactivation may be performed in different ways, such as 1O adding inhibitors, remOVing tors (cg, crucial ions through dialysis), through thermal inactivation and/or by any other deactivating means. Among these, thermal inactivation, is preferred by g the proteinaceous material to a temperature where the hydrolytic enzymes become denatured and deactivated. However, if a product where the relevant native proteins are not denatured is wanted, other means than heating for deactivating the hydrolytic ‘15 enzymes should be ed.

A ﬁrst centrifugation forms a tie—ﬂuoridated hydrolyzed and disintegrated crustacean material fraction and a solids ﬁaction (e.g., containing high ﬂuoride exoskeleton particles).

As described below, the low ﬂuoride hydrolyzed and disintegrated crustacean material fraction may be subsequently separated (cg, by a second centrifugation) to form a low ﬂuoride phospholipid-peptide complex (PFC) composition fraction and a lean low ﬂuoride concentrated ysate fraction (CI-IF) fraction that can be used as a food and/or feed additives, and a lipid on mainly ting of neutral lipids The PPC composition subﬁaction is rich in lipids, like a smooth cream with no particles, wherein the lipids are well suspended within the peptide components. This suspension results in small density differences between the ent PPC composition components thereby making it lt to further separate the PPC composition with common centrifugal tors and/or decanters.

This is especially accentuated with crustacean catches during the second half of the ﬁshing season.

Ordinary disc centrifugal separators (i.e., generating rotational force in the X and Y plane) do not work ly to separate a PPC composition subfraction into its respective components since emptying and necessary cleaning cycles with water will disturb separation zones. Conventional centrifugan'on separation processes result in the formation of unwanted emulsion products having a high olipid (PL) content and low dry matter concentrations. Standard decanters cannot separate the PPC composition subtraction into its PCTfIB2012/003004 respective components due to a low g force limitation, short separation zone and an intermixing of light and heavy phases at the discharge of heavy phase from the machine.

In one ment, the present invention contemplates a method sing separating a low ﬂuoride PPC material into subﬁactions using a horizontal decanter centrifuge with an extended separation path. See, Figure 2. Horizontal centrifuges (e.g., generating a rotational force in the Z plane) are useful for the present invention comprise modiﬁed convention decanter centrifuges. For example, a PPC composition subtraction would enter an ordinary er from a bowl through a central placed feed pipe in the middle of the separation zone. in contrast, when using horizontal centrifuges as plated herein, the PPC composition subfraction enters at the end and at the opposite side ofthe outlet (1). This modiﬁcation provides a signiﬁcant improvement in the separation process by providing a considerably longer clariﬁcation/separation zone than ordinary decanters and utilizes the total available separation length (2) of the machine. The drive is able to impart high g—forces: 10,000 g for small machines and 5,000 to 6,000 g for high capacity machines, facilitating the separation of very ﬁne, slow-settling PPC composition subfractions without the cations of emulsiﬁcation. The PPC ition subtraction will be ted to the highest g—force just before entering under the bafﬂe (3). The different liquid layers separated from PPC composition subfraction are concentrated gradually along the axis of the horizontal centrifuge thereby exiting the e under bafﬂe (3) by the g force pressure generated by the e (4). The separation of the PPC composition subfraction into a layer comprising about 27-30% dry matter makes the downstream processing nt in terms of operating/robustness and as well economically considering both yield and costs ofpreparing the dry matter into a meal composition. The PPC ition subfraction separation also s a layer comprising a lean hydrolysate that can be ated into a trated hydrolysate of greater than 60%.

B. Processing 0f Krill One embodiment according to the invention is depicted as a flow diagram for the processing of krill. See, Figure l. The function according to the method or the process according to the invention is initiated immediately as a krill catch has been raised to the ship.

Although it is not necessary to understand the mechanism of an invention, it is believed that ﬂuoride immediately starts to leak/diffuse from the chitinous exoskeleton into the ﬂesh and juices of the dead krills. “Immediately” means here a period at most 60 minutes, in practice, for example 15 minutes. During this period the krill catch is transferred from the trawl/net to a suitable disintegrator. In the disintegrator the krill material is crushed to relatively small W0 2013/102792 32012/003004 particles. The disintegrating can be performed by any convenient method: pulping, milling, grinding or shredding. The temperature in the disintegration process is around the ambient ature of the water, i.e. between ~2°C and +10°C, preferably between +0°C and +6°C.

The disintegration produces large amount of chitinous debris among the rest of the krill material, thereby contributing to a high ﬂuoride content.

The particle size ution of the disintegrated krill material is signiﬁcant because of the mentioned e leak from the chitinous debris and to the rest of the raw material. It is ed that the smaller particle sizes results in a more complete separation of the solids ﬁaction from the disintegrated krill material. For this reason the able range 1O ofthe particle size is 1.0 - 8 mm. However, the leaking process is relatively slow and has not time to be realized during the following s phases.

Next, fresh water is added to the disintegrated krill material (step 1 1). The volume/L of the water added is, for example, same as the /kg ofthe disintegrated krill material to be processed during the subsequent process phase of enzymatic hydrolysis. The temperature ofthe disintegrated krill material with the added water is increased such that it is suitable for the hydrolysis and enzyme(s) are added The heating is carried out fast, within at most ﬁve minutes, after the disintegrating step to reduce the processing time and y to prevent diffusion of ﬂuoride and to prepare the material for the enzymatic hydrolysis. The enzymc(s) can be added directly to the disintegrated krill material, or through the added water or both, before, during or after the heating step.

The term “hydrolysis” as used herein, means that breaks are made in the protein structure in the disintegrated substance, and the protein chains become shorter. This process is controlled by hydrolytic enzyme(s). For example, one or more exogenous proteolytic enzymes (eg. alkalase, neutrase, and enzymes derived from microorganisms or plant species) may be used in the process. Co—factors such as speciﬁc ions can be added ing on the used enzymes. The selected cnzymc(s) can also be chosen for reducing emulsions caused by high content of phospholipids in the raw al. Besides the temperature, the hydrolysis takes place Within optimal or near-optimal pH and ent time (e.g., for example, the ous enzyme alkalase the optimum pH is about 8, optimum ature about 60°C and the hydrolysis time 40-120 minutes).

The amount of proteolytic enzyme(s) can be set after a process/product optimization, and depends naturally on the efﬁciency of the chosen enzyme or mix of enzymes. A typical ratio of the weight of added commercial enzymes to the weight of the disintegrated krill material is between 0.05% and 0.5%, preferably between 0.1% and 0.2%. Fresh caught krill is known for rapid and uncontrolled autolysis, or the destruction of the cells by endogenous (natural) enzymes, for which reason the ent described here has to he ded t delays when the catch is not frozen.

The enzymatic hydrolysis also causes removing the bindings between the soft tissue of the krill and the exoskeleton. lfa mixture of enzymes is used, the mixture may also include one or more chitinases to facilitate the further processing of the chitin-containing fractions. ases are enzymes that break ycosidic bonds in chitin.

The enzymatic hydrolysis is d within 100 minutes ﬁom the addition ofthe endogenous enzyme(s). The red duration At of the hydrolysis is shorter, for example 45 minutes (step 12). Relatively short ysis duration is important, because in that case the diffusion ofthe ﬂuoride from the exoskeleton particles to the other material is reduced.

The hydrolysis is stopped by deactivating the hydrolytic enzyme(s) (step 13). There are many ways to deactivate the enzymes. Here it is used the thermal one: the temperature of the enzymatically processed material is increased over 90°C, preferably between 92—98°C, in '15 which case the hydrolytic enzymes become denatured. In practice the deactivating of the hydrolytic enzyme(s) can be performed also during or after the solid particle removal, The solid les (e.g., krill leton) are d from the enzymatically hydrolyzed and disintegrated krill material by passage through a device based on the centrifugal force such as a conventional horizontal centrifuge and/or decanter (step 14).

Although it is not necessary to understand the mechanism of an invention, it is believed that these solid particles, or solids, originate from the exoskeleton of krills and, as mentioned, contain a considerable amount of the ﬂuoride. The decanter is operated with a force between 1,000 and 1,800 g, preferably between 1,300 and 1,500 g. Through this particle removal step a substantial amount of ﬂuoride, more than 90 %, is removed from the krill material. The temperature in the decanter is for example 90°C, and if the deactivation of the enzyme(s) is done after the removal of solids, the temperatrn‘e in the decanter is then increased to e. g. 93°C.

Next, the hydrolyzed and disintegrated krill material with low ﬂuoride content is modiﬁed by passage through an ed separation path horizontal centrifuge (i.e., for example, a nter). See, Figure 1 step 15, and Figure 2. In the sedicanter, the yzed and disintegrated krill material, is ted into the valuable fatty portion, or PPC (phospholipid—peptide complex) material ﬁaction, and a CH? portion (concentrated hydrolysate fraction). 2012/003004 The separation of hydrolyzed and disintegrated loill material into the PPC material is difﬁcult because of the small density differences Within the krill material. The sedicanter is a modiﬁed horizontal fuge including a long horizontal clariﬁcation/ separation zone and generating high centrifugal forces (5,000 to 6,000 g). These features facilitate the separation of ﬁne; slow-settling PPC without emulsiﬁcation. The latter is a problem in the ordinary centrifuges with short separation zone and lower forces, and in which water is used in emptying and ng cycles. The dry matter concentration ofPPC material, pressured out from the sedicanter, is about 27-30%.

The PPC material may be then dried to a meal to avoid the lipid ion Figure 1, 1O step 16. The drying process is gentle with low temperature (O-l 5°C, preferably 2-8°C) and inert conditions, which give a reduced oxidative stress on the long—chain poly-unsaturated omega—3 fatty acids. A lyophilisation process would also be suitable since this avoids an ating of the t.

The PPC krill meal, or more brieﬂy PPC, is then packed in air tight bags under ’15 nitrogen atmosphere for later direct use and continuation process.

A typical mass balance ofthe processed raw lean Antarctic krill is shown below in Table I: Table 1: Typical Mass Balance Of Antarctic Krill Matter From SOOkg raw krill + water D_ry weight Wot PPC material 80 kg 28% PPC meal 25 kg 97% Hydrolysate 770 kg 6% CHF 78 kg 60% Fluoride-containing particles 45 kg 40% Neutral oils <5 kg The ﬂuoride content, prior to separation, in hydrolyzed and disintegrated krill material is 1.2 g/kg, whereas, after separation, the PPC is at most 0.5 g/kg and typically 0.3 g/kg. Thus, about two thirds of the ﬂuoride has been removed.

When the PPC is r processed, components may be ed by an extraction. In this phase, a t may be used. Figure 1, step 17. For example, to obtain krill oil from the PPC, ritical C02 and/or ethanol may be utiljlzed, either separately or in ation The extraction process yields, in addition to the krill oil, a protein hydrolysate (step 18).

Compressing and heating a material (e.g., for example, carbon dioxide or dimethyl ether) to above its critical temperature and pressure results in a supercritical ﬂuid. The W0 2013/102792 density is intermediate between a liquid and a gas and can be varied as a function of temperature and pressure. Hence, the lity of supercritical ﬂuids can be tuned so that selective extractions can be obtained. Due to the gas like properties, rapid extractions can be accomplished compared to liquid extractions as the diffusion rates are higher. C02 is a commorily utilized supercritical ﬂuid as its critical parameters can easily be reached. For example, one report has trated a low yield of krill phospholipids by using supercritical ﬂuid extraction at a pressure of 500 bar and a temperature of 100°C. Yamaguchi . A second report provides data on speciﬁc s conditions, which e pressure and temperature ranges (e.g., 300 to 500 bar and 60 to 75°C). These data are from a pilot scale process wherein an tion of 84 to 90% of krill total lipids was achieved. Bruheim et at, United States Patent Application Publication Number 200810274203 n incorporated by reference).

Supercritical C02 is also non-ﬂammable, cheap and inert, wherein such factors are relevant when considering industrial ability. ’Ihe inertness s in low grade of oxidation of labile compounds during extraction C02 also has a low surface tension which is an advantage so that the extraction medium can penetrate the material efﬁciently. In order to extract more polar substances, the CD; can be mixed with a polar solvent such as ethanol.

The level of modiﬁer can be varied to provide extra selectivity as well.

Consequently, currently available industrial scale supereritieal ﬂuid extraction processes using high temperatures and pressures has ed in a low extraction efﬁciency of conventional krill meal thereby ing an insufﬁcient oil yield to provide a commercially feasible solution for krill extraction Further, these currently available tion processes do not solve the problems discussed herein regarding providing improved low ﬂuoride meal and/or oil compositions.

Therefore, the improved solvent extraction methods described herein have been developed. In one embodiment, co-solvents are used with ritical C02 or supercritical dimethyl ether either alone or in various combinations of ethanol, hexane, acetone. For example, if ethanol is used alone as an extraction solvent, it has been observed that krill al is less selective than extraction with supercritieal C02. Pronova et a1., WO 2008/060163 A1. As a result, rable substances are extracted into the krill oil resulting in a need for additional post-extraction clean-up/processing. Further, ethanol-Only extracted krill oil tends to have higher viscosity and darker color which is independent of astaxanthin content ofthe oil.

In some embodiments, the present invention plates s that have cted s including but not limited to: i) PPC was extracted using low pressures (i.e., for example, between approximately 177 to 300 bar) and low temperatures (i.e., for example, between imately 33 and 60°C); and ii) high yield of lipid extract was produced (data available). It appears that krill meal sing hydrolyzed protein allows for easier extraction of the associated lipids in particular the phospholipid rich fraction of krill oil.

The data presented herein demonstrates that supercritical C02 was found to be a selective tion method as it ed high purity extracts containing triglycerides, ’10 phospholipids and astaxanﬂnin with minimal brown color and superior organoleptic quality as Ct ed to krill oils produced by ethanol-only extraction and/or acetone + ethanol extraction. Brown color of krill oil is considered to be undesirable. The exact origin of the brown color is unknown but it is believed to be associated with oxidation of krill lipids during the manufacture of krill meal phospholipids and/or degradation of the carotenoid astaxanthin.

The properties of such a supercritical ﬂuid can be altered by varying the pressure and temperature, allowing selective component extraction Extraction conditions for ritical C02 are above the critical temperature of 31°C and critical re of 74 bar. Addition of modiﬁers may slightly alter these values. For example, neutral lipids and cholesterol can be extracted from egg yolk with C02 pressures up to 370 bar and temperature up to 45°C, while using higher temperature, e. g. 55°C, would result in increased rate of phospholipid extraction.

CO; has a high industrial applicability because it is non-ﬂammable, cheap and inert. The inertness results in low oxidation of labile compounds during extraction.

As mentioned, either supercritical CO; or supercritical dimethyl ether is ﬂuid. lts density is intermediate between a liquid and a gas and can be varied as a ﬁinction of temperature and pressure. Hence, the solubility of supercritical fluids can be tuned so that selective extractions can be obtained Due to the gas-like properties, rapid extracn'ons can be lished ed to liquid-extractions. In the present method the extraction is effective; even 95% of the krill oil existing in the PPC is separated. AlthOugh it is not necessary to understand the mechanism of an invention, it is believed that the phospholipids of the feed material are embbded in a matrix of hydrolyzed protein which means that the close association between the phospholipids and hydrophobic/phosphorylated ns is broken thus facilitating the extraction of the . In addition, a minimal amount of ﬂuoride content is transferred to oil during the CO; extraction process. For example, the fluoride wo 2013/102792 PCT/IBZOIZ/003004 content ofPFC is about 0.3 g/kg, but after the C02 extraction the ﬂuoride content of the krill oil is less than 0.5 mg/kg. atively, when using only supercritical C02 as solvent, triglycerides and/or neutral oil may be separated from the PPC composition subfraction. Figure 1, step 19. In one embodiment, supercritical COz-only extraction also generates a low ﬂuoride 'de-oiled PPC‘ ition. Although it is not necessary to understand the mechanism of an ion, it is believed that de—oiled PPC is the most valuable portion of the PPC composition subfraction. When thereafter, the de-oiled PPC composition may be extracted using ethanol as a solvent, wherein a phospholipid subfraction and a protein hydrolysate fraction is also ted. See, Figure 1, step 1A.

In one embodiment, the present invention contemplates a system comprising an extraction plant, including but not limited to, a solvent unit 21, vertical tank 22, separators 23 and adsorbents 24. See, Figure 3. Normal 002 and possible co-solvent are fed to the solvent unit, which comprises a pump to generate a certain pressure (p) and a heater to generate a certain temperature (T). The supercritical C02 with possible co-solvent are then fed to the lower end of the tank 22. The feed material, in this case the PPC, is fed to the tank by means of a pump. Material affected by the solvent flows out of the upper end of the tank. The separators 22 separate the extract result, for example krill oil, to output of the system. If ethanol is used as co~sclvent, it follows the extract proper and has to be evaporated away. The CO; continues its circulation to adsorbents 23, where it is cleaned, and thereafter back to the solvent unit 2].

In one ment, the t invention contemplates low ﬂuoride PPC itions including, but not limited to, polar lipids (~ 43% w/w) and/or neutral lipids (~ 46% w/w). For example, the PPC neutral lipids may range between approxnnately 40 — 50% (w/w). In one embodiment, the polar lipids include, but are not limited to, phosphatidylethanoamine (~ 3% w/w), phosphatidylinositol (~ < 1% w/W), phosphatidylserine (~ 1% WW), phosphatidylcholine (~ 38% WW) and/or lysophosphatidylcholine (~ 2% WM). In one embodiment, the l lipids include, but are not limited to triacylglycerol (~ 40% w/w), diacylglycerol (~ 1.6% w/w), monoacylglycerol (~ < 1% w/w), terol (~ 2% w/w), cholesterol esters (~ 0.5% w/w), free fatty acids (~ 2% w/w) and fat (~ 48% w/w). In One embodiment, the l lipid fat ses approximately 75% fatty acids. In one ment, the neutral lipid fat fatty acids e, but are not limited to, saturated fatty acids (~ 28% w/W), monenoic fatty acids (~ 22% W/W), n—6 polyunsaturated fatty acids (~ 2% w/w) and/or n-3 saturated fatty acids (~ 26% W/W). See, Example XIII.

Phospholipid proﬁles have been created to evaluate low e krill oil extracted by the methods described herein For example, nuclear ic resonance technology has determined that phosphatidylcholine is the largest phospholipid component of lqill oil and its proportion is relatively . Several krill oil samples underwent independent analysis. See, Example XH. In one embodiment, the present invention contemplates a low ﬂuoride krill oil comprising approximately 39 — 52% (w/w) olipids, In one embodiment, the phospholipids comprise phosphatidylcholine g between approximately 65 ~ 80% (w/w).

In one embodiment, the phospholipids comprise alkyl acyl phosphatidylcholine ranging between approximately 6 — 10% (w/w). In one embodiment, the phospholipids comprise phosphatidyljnositol ranging between approximately 0.3 — 1.6% (w/w). In one embodiment, the phospholipids se phosphatidylserine ranging n approximately 0.0 — 0.7 % (w/w). In one embodiment, the phospholipids comprise lysophosphaﬁdylcholine ranging “I5 between imately 2.4 — 19% (w/w). In one embodiment, the phospholipids comprise lyso acyl alkyl phosphatidylcholine ranging between approximately 0.6 _ 1.3% (w/w). In one embodiment, the phospholipids comprise phosphatidylethanolamine ranging between approximately 1.4 — 4.9% (w/w). In one embodiment, the phospholipids comprise alkyl acyl phosphatidylethanolamine g n approximately 0.0 , 2.1 % (w/w). In one embodiment, the phospholipids comprise a combination of cardiolipin and N- acylphosphatidylethanolamine ranging n approximately 1 — 3% (w/W). In one embodiment, the phospholipids comprise lysophosphatidylethanolamine ranging between approximately 0.5 — 1.3% (w/w). In one embodiment, the phospholipids comprise lyso alkyl acyl phosphatidylethanolamine ranging n approximately 0.0 and 0.3% (w/w).

As described above, the non-polar solvent extraction of a low ﬂuoride cean oil results in the production of a low ﬂuoride de—oiled phospholipid-protein complex composition (dc-oiled PPC). Although it is not necessary to understand the mechanism of an invention1 it is believed that the low ﬂuoride de-oiled phospholipid-protein complex comprises a ﬂuoride content similar to the low ﬂuoride PPC complex (e.g., n approximately 200 — 500 ppm). A component analysis of de-oiled PPC includes, but is not limited to, polar lipids [~ 69% W/w) and/0r neutral lipids (~ 20% w/w). In one embodiment, the polar lipids include, but are not limited to, phosphatidylethanoamine (~ 4.2% w/w), phosphatidylinositol (~ < 1% w/w), phosphatidylserine (~ < 1% w/w), phosphatidylcholine (~ 62% W/w) and/or lysophosphatidylcholine (N 2% w/w). In one embodiment, the neutral W0 2013/]02792 lipids include, but are not limited to triacylglycerol (~ 17% w/w), diacylglycerol (~ 0.6% Why), monoacylglycerol (~ < 1% W/w), cholesterol (N 1% w/w), cholesterol esters (~ 0.5% W/w), free fatty acids (~ 1% WAN) and fat (~ 35% w/w). In one embodiment, the neutral lipid fat comprises approximately 69% fatty acids. In one embodiment, the neutral lipid fat fatty acids e, but are not limited to, saturated fatty acids (~ 21% w/W), monenoic fatty acids (~ 13% W/W), n—6 polyunsaturated fatty acids (~ 2% W/w) and/or n-3 polyunsaturated fatty acids (~ 31% W/w). See, Example DI.

III. Production Of Low Trimethyl Amine Crustacean Materials Trimethylamine (TMA) is an organic compound comprising a chemical formula of N(CH3)3. TMA is a colorless, hygroscopic, and ﬂammable tertiary amine that may have a strong "fishy" odor in low trations and an ammonia—like odor at higher concentrations.

TMA may be produced commercially and is also a natural by—product of plant and/0r animal decomposition. It is the substance mainly responsible for the odor often associated With rotting ﬁsh, some infections, and bad breath. It is also associated with taking large doses of choline and ne.

Chemically, TMA comprises a nitrogenous base and can be readily protonated to give hylammonium cation Trimethylammonium chloride is a hygroscopic colorless solid prepared from hloric acid. Trimethylamine is a good phile, and this reaction is 2O the basis ofmost of its applications.

Trimethylamine N—oxide (TMAO) is an organic compound comprising a formula (CI-I3)3NO. This colorless solid is usually tered as the dihydrate. TMAO is an oxidation t of TMA, a common metabolite in animals. TMAO is also an osmolyte found in saltwater ﬁsh, sharks and rays, molluscs, and ceans. Further, TMAO may function as a protein stabilizer that may serve to counteract urea, the major osmolyte of sharks, skates and rays. TMAO has high concentration in deep-sea ﬁshes and crustaceans, where it may counteract the protein—destabilizing effects of re. Yancey, P. "Organic osmolytes as compatible, metabolic, and counteracting cyt0protectants in high osmolarity and other stresses" J. Exp. Biol. 208(15):2819—2830 (2005). TIMAO decomposes to nimethylamine (TMA), which is the main t that is characteristic of degrading seafood.

Removal ofTMA/‘IMAO compounds from crustacean ts confers a useful advantage in that these compounds contribute to the strong, sant smell of crustacean oils. Consequently, low TMA/TMAO compounds have an improved industrial applicability as compared to traditionally prepared crustacean oils.

PCT/IBZO 12/003004 In one embodiment, the present invention contemplates a method comprising extracting a low ﬂuoride protein peptide complex (PFC) is a suitable raw material for krill oil production by extraction with any combination of solvents including, but not limited to, ethanol, acetone, ethyl e, carbon dioxide, or yl ether to produce a low ﬂuoride- 1ow trimethyl amine crustacean t. In one embodiment, the low fluoride—low trimethyl amine crustacean product comprises an oil. In one embodiman the low e-low trimethyl amine crustacean produce ses a de-oiled PPC.

Dimethyl ether (DME) has been previously ed as an extraction solvent for polyunsaturated fatty, but not for the preparation of low TMA products. Catchpole et al.

“Extraction OfHighly Unsaturated Lipids With Liquid yl Ether” W0 36281 _ When DME is in a supercritical form, the solvent has sufﬁcient solvent power to extract olipids resulting in rapid and gentle extractions. DME can be used on wet raw materials and can be ed at low res as compared to other supercn'tical ﬂuids such as C02. In one embodiment, the present invention contemplates a crustacean extraction product comprising hill oils with a low IMA/TMAO crustacean oil. In one embodiment, the low TMA/TMAO crustacean oil is a krill oil.

IV. Formulated Compositions In some embodiments, the present invention contemplates compositions comprising low ﬂuoride crustacean PPC or compositions sing low ﬂuoride crustacean de—oiled PPC itions and/or protein hydrolysatcs as described herein. In one embodiment, the itions comprises mixtures of the crustacean PPC x, crustacean de—oiled PPC and the protein hydrolysates in any combination. Although it is not necessary to understand the mechanism of an invention, it is believed that the mixed ratio can be any ratio but is preferably a ratio of approximately 1:1. In one embodiment, the mixture comprises a milled ﬁne powder. In one embodiment, the powder has a particle size of approximately 250 pm.

In one embodiment, the compositions have improved stability because of lower peroxide (e.g, < 0.1 %; mEq/kg) and/or aniside levels (< 0.1 %; w/w). In one embodiment, the compositions have improved stability because of lower microbiological contamination. In one embodiment, the composition further comprises microencapsulated polyunsaturated Omega-3 fatty acids. In one embodiment, the composition further comprises zinc oxide. In one embodiment, the composition further ses marine peptides. In one embodiment, the composition further comprises at least one supplemental amino acid. 2012/003004 In some embodiments, the present invention contemplates a method for formulating a composition comprising a low ﬂuoride crustacean PPC and/or a low ﬂuoride crustacean de— oiled PPC and/or a n hydrolysate as described herein. In one embodiment1 the composition is a powder. In one embodiment, the composition is a tablet. In one embodiment, the composition is a capsule. In one embodiment, the method further comptises mixing the powder with a food product. In one embodiment, the mixing r comprises a microencapsulated polyunsaturated Omega-3 fatty acids. In one embodiment, the mixing further comprises zinc oxide. In one embodiment, the mixing further comprises marine peptides. In one ment the mixing further comprises at least one supplemental amino acid.

EXPERINIENTAL Example I ”[5 Production 01’ Low Fluoride Krill Oil The feed material, ld lqill meal' granules (Olymeg® or low ﬂuoride PPC prepared as described herein), were supplied in a sealed plastic bag containing approximately 25kg. The feed al was kept frozen until used in cxtractions. The granules have a size distribution lly in the range 2 to 5mm, but a number of ﬁne fragments were also present. The granules are greasy to the touch but still break up under compression rather than Smear. kg batches of feed material in granular form, as processed using supercritical CO2 as solvent and azeotropic food grade ethanol as co-solvent, the weight ofthe ethanol being 23% C of the weight of C02. The plant was pre—pressurised to operating pressure with C02 only, and ethanol was added when CO2 circulation started. Solvent to feed material ratio was 25:1 or greater and co—solvent to feed material ratio was 5:1. Runs were carried out under two extraction ions; 300 bar at 60°C, and 177 bar at 40°C. See, Table II.

Table II —Krill Oil Extraction ions Run 1 Run 2 Feed Mass (g, as received) 5000.5 5000.9 Extraction pressure (bar) 300 177 Extraction temperature (°C) 60 33 First separator re (bar) 90 90 First separator temperature (°C) 41 41 W0 02792 Second separator pressure (bar) 48-50 48—50 Second separator temperature (°C) 39 39 C02 used with l cc-solvent (kg) 132.6 134.9 Additional CO; at end of run (kg) 33.1 44.5 Total ethanol used (kg) 31.65 32.19 The ted krill oil material was passed through two separation vessels in series, held at 90 bar and 45-50 bar respectively. The ﬁnal krill oil material collected from both separators was pooled together and the ethanol was evaporated. The residual feed material comprises a de- ‘10 oiled feed material (e.g., for example, de-oiled PPC) having a reduced lipid content in comparison to the starting feed material. See, Example ZX.

After ethanol evaporation, krill oil cumulative tion curves were generated for both Run 1 and Run 2 by independently analyzing each sample taken during the extraction runs. See, Table HI.

Table III — Progressive krill oil extraction sample points and yields.

Sample Number 1 2 3 4 5 6 Total Run 1 Cumulative C02 (kg/kg feed) 5.5 9.1 13.4 17.8 22.0 33.1 33.1 Extracted oil (g, dry) 1137 398 282 135 78 86 2115 Run 2 Cumulative C02 (kg/kg feed) 5.6 9.1 13.5 17.5 21.5 34.4 34.4 ted 011(g, dry) 715 496 368 220 149 129 2077 A total yield of 41-42 wt% of the feed material was achieved for all runs. The runs carried out at 300 bar and 60°C had a higher initial rate of extraction. The curves indicate that the extraction is virtually complete at Sample Number 5 after a tive C02 use 3O ranging between 21.5 — 22.0 kg per kg of feed material. Estimated maximum extraction is achieved at a point where the C022feed ratio is 26.5:1. See, Figure 3 (estimated maximum extraction is marked by an arrow); The ratio of azeotropic ethanol to C02 was 0.24:1 for the 300 bar runs, and slightly higher at 0.26:1 for the lower pressure run.

This method of krill oil production resulted in the near te extraction of total lipids from the krill meal (e.g., for e, approximately 95% of l lipids and 90% of phospholipids. The ﬁnal yield was similar for both the high and low pressure runs, but neutral lipids were more rapidly extracted at higher pressure. The phospholipid extraction rate was similar under both extraction conditions. As detailed below, in this extraction process, the pooled krill oil total lipid had an overall phospholipid level ofjust over 40 wt% and both phosphatidyl ol and phosphatidyl serine were poorly ted.

Phospholipid proﬁles of the various lqill material compositions were then determined using traditional column chromatography techniques. See, Table IV.

Table IV ~ Comparative Phospholipid Proﬁles Of Krill Compositions (run 1) Olymeg Residue e Sam» a 10mm: Extract 1 Emma! 2 Extracts Extract 4 Extract 5 Extract 6 - . Bouom was of total PL 77.1 753 75.9 73.5 72.7 40.2 325 9.0 9.8 9.1 10.6 9.0 7.5 7.8 0.7 0.6 0.6 6.2 10.! .5 8.1 . 5.6 5.7 6.0 88 7.5 15.4 8.9 LAAPC 13 12 12 m 13 12 1.4 3.2 26 PE 53 3.8 4.0 35 3.6 35 4.5 9.4 9.4 EMS 05 0.0 0.5 0.5 0.5 05 0.3 1.0 22 MPE 20 1.1 15 13 1.6 15 ac 4.4 43 LPS 0.7 1.9 CUNAPE 1.0 0.9 0.7 0.5 0.8 1,2 1.6 4.2 5.? LPE 0.8 0.3 04 0.4 0,4 0.4 0.4 32 4.5 Total FL (1.11% of ﬁld 40.88 81.46 00.96 183 2633 71:14 76.13 7w 4.0 4.8 The ﬁrst column shows the speciﬁc phospolipids that were analyzed. The second column show the ollpld proﬁle of the starting feed material (e.g., a low ﬂuoride PPC ‘10 prepared as described herein, or ‘Olymeg®'). Columns three — eight (Extracts 1 — 6) show the phOSpholipid proﬁle of each krill oil sample taken during the extraction process as described above. The last two columns show the olipid proﬁle of the residual extracted feed material sampled from either the top and/or the bottom of the phospholipid tion column.

The data show that the major phospholipid in the extracted krill oil samples is phosphatidyl choline (PC), ranging approximately from 72.7% to 80.4% of total phospholipids, including contributions ﬁom both alkyl acyl phosphatidyl e (AAPC) and lyso phosphatidyl cholines (e.g., for example, LPC and/or LAAPC). Smaller amounts of phosphatidyl ethanolamine (PE) are present in both the feed material (column 1, .~ 5.3%) and in the krill oil extract samples (columns 3 - 8), ~ 3.5 - 4.5%). Allcyl acyl and lyso forms of PE (AAPE, LPE) are also present in the feed material and krill oil extracts. Phosphatidyl inositol (PI) and phosphatidyl serine (PS) are present in the feed al, but because they are poorly soluble in ethanol, these phospholipids are poorly ted and are therefore concentrated in the extracted feed material residue (e.g., having a higher level in the residual PPC in comparison to the feed material, see columns 9 and 10).

W0 2013/102792 PCT/lBZOlZ/003004 Further analysis determined the overall relative lipid component proportions of the extracted krill oil. See, Figure V.

Table V e Main Lipid Components Of Extracted Krill Oil (%W/W) TAG Polar li-id FFA Astaxanthin Run 1 Run 2 The data show: i) a relative absence of free fatty acids (FFAs); ii) less than 2% of sterols; iii) 40 wt% oftriacylglycerides (TAGS); and iv) approxiately 50% phosphoh'pids (e.g., polar lipids). While FFA’s were not detected (ND) in this particular example, it is ed that extracted krill oils may se between approximately 0.01 - 0.1 % FFA of total lipids.

As described above, the extraction process results a yield ofbetween approximately 92.2 — 95.3% of the feed material total lipid.

The method and products according to the invention has been described above. The method can lly vary in its s from those presented. The inventive idea may be d in different ways Within the limits as described herein.

Example II Lipid tion Efﬁciency This example demonstrates an exemplary ical lipid extraction with the Soxhlet method comparing conventional krill meal with a low ﬂuoride krill meal (cg. low ﬂuoride PPC) as described herein Soxhlet method is a standard method in quantitative determination of fat content of foods and feeds and thus it can be used as a reference method to determine the extractabﬂity of various lcriH meals. For example, the Soxhlet method may be d out as below using petroleum ether (boiling point 30—60 °C). Conventional krill meal was prepared as described in US 2008/0274203 (Aker Biomarine ASA, Bruheim et a1.) and the low ﬂuoride PPC was prepared ing to the present invention.

The l lipids are often part of large aggregates in storage tissues, from which they are relatively easily extracted. The polar lipids, on the other hand1 are present as constituents ofmembranes, where they occur in a close association with proteins and polysaccharides, with which they interact, and therefore are not extracted so readily. Furthermore, the phospholipids are relatively tightly bound with hydrophobic ns and in particular With the phosphorylated proteins.

W0 2013/102792 PCTfIBZO 12/003004 The data show that l hydrolysis of the protein matrix in the preparation of a 10W ﬂuoride PPC compositiori as described herein improves the extraction ncy of total lipid by use ofnon-polar organic solvents (e.g., for example, supercritical C02, ethanol, and/or petroleum ether).

Brieﬂy, a 10 g sample of either conventional milled krill meal or low ﬂuoride PPC was weighed and placed in a Soxhlet apparatus and then continuously extracted for approximately eight (8) hours using 300 mL eum ether. After extraction, the solvent was evaporated at 60 °C under a nitrogen stream. Soxhlet R, “Die gewichtsanalytische bestimmung des ettes” Dingler ’s Polytech. J. 232:461—465 (1879). ’10 The s show that the proportion of residual (e.g., un-extracted) lipid was twice as large in the conventional krill meal compared to the low de krill meal. See, Table VI.

Table VI: Lipid Extraction Efﬁciency OfLow Fluoride Krill Meals Source material Extracted krill oil lipid Source Material Residual li id e. ., de-oiled meal) Conventional krill meal 79.6% Low ﬂuoride krill meal 88.9% ‘15 Consequently, the lipid extraction methods described herein have provided an unpredictable and surprising result that provides a superior product because of a y improved extraction efﬁciency.

Example HI Determination Of Fluoride Content This example presents one method of determining fluoride content of krill products as ﬂuoride by chemical analysis using an ion ive electrode.

A low ﬂuoride PPC krill meal was prepared as described herein and extracted in ance with Example I to create a low ﬂuoride krill oil were analyzed for ﬂuoride content and compared with conventional preparation processes. Biieﬂy, the method disclosed herein removes, in most part, the krill exoskeleton from the krill meal y reducing the ﬂuoride content. In contrast, the krill exoskeleton is included in the conventional krill meal thereby having vely high levels of ﬂuoride. Conventional processes are, for example, described in W0 2002/1023 94 (Neptune logies & Bioresources) and US 274203 (Aker Biomarine ASA).

WO 02792 The krill meals analyzed for ﬂuoride content were produced by: i) a low e method of present invention; and ii) a whole krill material produced by a conventional process. See, Table VII.

Table VII: Fluoride Content Comparison To Conventional Processes Anal zed Material Low Fluoride Preparation i Conventional Preparation 200 - 500 _-.-m The data demonstrate that by ng the exoskeleton in the process ofproducing krill meal (e.g., the low ﬂuoride preparation as disclosed herein), the ﬂuoride content of the krill meal and the krill oil produced from the meal have a markedly reduced ﬂuoride content (e.g., 3 — ‘10 10 fold reduction).

Example IV Krill Oil Color Comparison Krill oil has typically a streng red colour arising from the noid astaxanthin '15 present in the oil at levels varying from 50 ppm to 1500 ppm. Color of krill oil can be determined with a LabScan® XE spectrophotometer (Hunter Associates Laboratory, INC.

Resbon, VA, USA) and ed in CIELAB colour scales (L*, a* and b* values). Deviation from the red colour of astaxanthin can occur when the krill biomass is processed at high temperature and under conditions that induce oxidatiOn. Typical oxidation induced deviatiOn in krill oil color is an increase in the brownish hue. Brown color in krill oil arises from oxidatiou of lipids and formation of secondary and tertiary oxidation products with amino es. This process is also called non—enzymatic browning.

Strecker degradation products and pyrroles are products ofnon-enzymatic browning that have been characterized in samples of krill oil. For example, rization of pyrroles results in ion of brown, nin like macromolecules. Furthermore, pyrrole content of krill oil can be ined spectroscopically with absorbance at 570 nm.

Samples ofthree krill oils will be examined for color. One produced by the method of the present invention, one produced from frozen krill by a method described inWO 2002/102394 (Neptune logies & Bioresources) and one extracted from dried krill meal with l alone as described in US 2008/0274203 (Aker Biomarine ASA). It is to be found that krill oil produced by the method of the present invention has the lowest level of W0 2013/102792 2012/003004 brown color determined spectrophotomehically by using CLELAB colour scales (L*, a* and b* values) and/or the lowest level ofpyrroles ined spectroscopically.

Example V Organoleptic Krill Oil Qualiﬂ Determination Organoleptic quality of krill oil is conventionally determined by al analysis of volatile nitrogenous compounds arising from the decomposition of krill proteins and trimethyl amine oxide (TMAO). enous compounds analyzed are total volatile nitrogen (TVN) and trimethylamine (TMA). In simpliﬁed terms the level of nitrogenous compounds correlate with the level of spoilage in the raw al i.e. krill biomass used for extraction of the oil.

It has become evident that, in addition to the volatile nitrogenous compounds, a large number of volatile components with distinct odour bute to the sensory properties of krill oil. Many of the volatile components arise from the oxidation of lipid and proteinaceous compounds of krill biomass. Thus, a method that limits the level of oxidative degradation in the krill biomass, Will reduce the amount ofvolatile components in krill oil.

Assessment of the leptlc quality of different types of krill oil is to be performed by a panel of trained duals. The sensory properties to be determined include several pro—deﬁned parameters of smell and taste. It is to be found that the novel krill oil has an ed sensory proﬁle compared to the other oils tested. The other oils to be tested include one extracted from frozen lm'll by a method descnbed in W0 2002/1023 94 (Neptune logies & Bioresourc es) and one ted ﬁ'om dried krill meal with ethanol alone as described in US 2008/0274203 (Aker Biomarine ASA).

Example VI Production Of Low Trimethyl Amine Crustacean Products ' This example describes one method to produce low TMA crustacean products using a krill meal material composition. One having ordinary skill in the art, upon reading this 3O speciﬁcation would understand that this krill meal material composition may have variable ﬂuoride content, including ﬂuoride contents below 05 ppm, in addition to the basic components described below. See, Table VIII.

PCT/IBZOIZIOO3004 Table VIII: Unextracted Krill Meal Composition Eicosapentaenoic Acid (EPA) _(11 % w/w) Docosahexaenoic acid DHA) 7 % WNW 22.7 g/lOOg (22.7% MW) Phosholi lids ’LS) 45 g/100g (45% WW) Trimcthylamine TMA) | 44 mg N/lOOg (0.044% WW) Tnimeth lamine oxide TMAO I 354 mg NflOOg % w/W) A krill oil may then be prepared from the krill meal using ethanol extraction as described above that has the basic components described below. See, Table IX.

Table IX. Krill Oil Com onents After Conventional l Extraction Of Krill Meal Omega-3 Fatty Aoides 22,1 g/lOOg (22.1% w/w) Phos - holipids I 44 g/lOOg (44 % W/w) Trimethylamine 50 m N/lOO (0.05 % W/w) TrimethylamineOXide 216 mg N/lOOg (0.216% W/W) 1O Alternatively, krill oil was prepared by krill meal extraction at 40 bars and 40°C using ritical dimethyl ether (SC DME). The DME extract composition was dried on a Rotavapor® and then ﬂushed with nitrogen. The ents of the resultant dried composin'on is listed below. See, Table X Table X: Krill Oil Components After SC DME Extraction Of Krill Meal I Parameter Value EPA I 10,4 g/IOOg (10.4% w/w) DHA 6,8 100 (6.8% W/W) | 011135.43 Fatty Acids 21,7 g/lOOg (21.7% w/W) | Phospholipids | 45,7 g/ioowjryo w/W) Trimethyl amine <1 111g N/100 g (< 0.001% w/W) mg N/100 g 0.02% w/w These data clearly show that supercritical DME extraction of krill meal itions result in a preferential 10 — 100 fold reduction ofTMA and TMAO levels.

WO 02792 PCT/1132012/003004 Example VII Nuclear Magnetic Resorranee Phospholipid Proﬁles Of Low Fluoride Krill Oil This example presents representative data of the phospholipid composition of 10W ﬂuoride krill oils prepared by the methods described . See, Table XI.

Table XI: Phospholipids in Low de kn'll oil analyzed using 31? NMR.

Sample #1 (color; orange) PmmﬂrﬁﬂllM..--wwww-wmw-mﬂtzéﬁmﬁﬂlﬁﬂg§3ﬂ£ Eh-gizhﬂéxmﬁcémww.WWW,.,....W-EELMWddummzlﬁinmmr”0.33m ﬁkyleathmbaimmgﬁnsMMMMWMM‘L-“wwﬁwwﬂ i’méewﬂﬂwwmmwww...-.n.w..m.,£_l_m........nnnnnn”unmemmizé.........“9.3.

Emybgﬁsixlsgéne_________________ PS Mnnw-___.-9_-Z ............pg W‘Wmmwmgﬁg.m___,_w-.,_.~%wl“W- .m Lw.§m_sﬂiphs§mgﬁiﬂcwi&mm ., - ....LA.&P£.,.,_~.___--E~§.~_~__'__;'____’;§Z’ZI.

PmémgﬁgxigmaaemlaamwWWW-“ ELM.”-WWaj_.,.__1;§.- @ﬁﬁhagn,mh§mmwm_ﬂ§nh._,_:,..,_v,.,,,,m_.,‘,9:§,.w..w”Mia, ﬁawN-amﬁmwmmmnmmwmmmmmm9-5. gmmdmmmewew.........EPE.......,.......Wmﬂé........... 4. wwwegxlpms nﬁlemrlqlsmng...... .-Ww_§_..w.-,_-will______________93..

‘ Tom phospholipid comm 2.9.0 mung sample 385 gﬂﬂﬂg solids n.d. = not detected ' 3am 9H1» identified phoephcllpid emu: Sample #2 (color; orange) Phosrbeﬂpwm..............................Wunﬁagttgelﬂwanmms wnnaﬁsymimwwmWW,.W.,ﬁzmmmm,-H.d.§_§;?__m.u..,.mm Nmmyﬂéﬁrwglnim__________Magma-m_~£._9..-,m_mw%§, a!“..........WWWmew-.MWMWWMWWWoMW.” WEQ‘E‘ﬁ‘EEM __.-,,,-A_-..m,-WM-k.r.ELMW,WWWWW .7 -_ Lemﬂaﬁwﬁnsw.................. - - Evymwjnhglﬁgwnnwwwg____________Wm.........n.“_ LPCMM ahgghrzﬂdrlemmenngw._._._. EL“--- ___..___ _ ﬁalmmmeﬁﬁlwmﬁammﬁ NJMPEH + N- “whosgyaﬁd Ieﬁwanéiémem CUNAPE ﬁmmgaﬂeeémametfgwm” . 2mm; t s hadﬂeﬂlﬂl‘i‘ﬂlﬂwlﬁﬁpﬁ......... ,,,,,,.w Tom! phosphollpld canton? 49.5 #1009 sample ~ 42.: 9mm; was: mi. = not detected ' Sum o! the Bdenxiﬁeé phoephnlipld classes PCT/[82012/003004 Sample #3 (COIOI; orange) Phosphonpia 19L! WW______________.mwwmwmmwmutgmgoﬂamm Phosmaﬁézisho "9 ...\,_.M..,_.....,...._EE=M_ “WWW 1’23 31-1 «my...

Alk [a has hatld choﬁne ___mmw"5313““ mw_w_~_v§w1_‘::l_wm§:éh m‘ﬂﬁmﬁﬂ“muw Pl; a V 4, ‘ 913i: ,. ”0'1 P5 . .¢.A._M..m.w..._:w Lysommmm ” LPG ‘18-? ﬁg -_ .,.,._,.,.Y..,~._J,.._.N...~ [Jig9W1:1! Phosgalgnqﬁcﬁoméw we o.é‘""“""""”‘”ﬁ?§‘ Enpsghatidylethamkamine“MW ....—.~nw.~ _-.....~..___-M._.. ﬁlly! acylpbggghaﬁdyieihanéiamlne’f ,u..--w_€.._--w~_._m-_m__.._4_~w_.w..__~_ my.“ AAA-kn H‘~._._M.«_...._ , C..ﬁ_.r.d._nailpir11,__eyN-amm “W .ﬁi‘éﬁiﬁiﬂdjtﬂmﬁfﬁ-\MZ:.‘__§:;:§§§::ZL“ffaﬁ' memnﬁammeuJE§__~_M~_MHN.. 553"?"@1315,wﬁhngaﬁdyieﬁIanoiamh-a WPE n.d.

Total phasphollpld contant" 43.0 911009 sample 45.1 gﬂﬂﬂg solids “1d. 1' not dﬂmd ‘ Sum affha ﬁed phosphaﬂpfd dam Sample #4 (color; orange) lmmmﬂwww.mmm...-..,n,._nm.w..ﬂAméﬁmztﬂﬂhﬁfﬂﬁﬂaﬁlama. qugphﬁﬁﬂchollne 77.4 39.5 ‘v-v‘mwwrwlv . _. PG“. _...._-‘.-_ _ my"rsmmﬁauawm .iLMaaagmmW,;§g:t;::::::::;:§§; Phosphatwincsm PI 0.9 0.5 "Wm .—m—..m.m.-\ mum-ﬁﬁﬂwm ‘ _ , u Phosphatidylsezine WWW» WWWWWM.-.,..._"...mm-.._9£ QwWXIMW.MZLF:§=____W_-‘W_§J§Wmmwgﬁ LIE?EM;cyiatrgmxgmuaaw,.,..v HJ-AAPEWM‘WMWWOAB.........,. 03: fhpsphanggethanolamgnq PE 2.6 ”‘1' 3. ,. 0* 4.“... ”.~ m. “H.“‘ A.U..h-n»mm—M~c~l~.wv¢uw\~—u“Wuw‘m‘cum. mu.““ “._ “Mm.“ Aikyi acyighgggﬂgﬁﬁgtgnanolamhe” ﬂaps 1. 0,7 Eaﬂﬁﬂjﬂwjeﬁeépﬂﬁeﬁiﬂiﬁ.QQBE_E§.;W]?“95 Ly§ggggl§pﬂg§ylemanoiamine LPE 0.5 0 3 LEGaw!awewhosphaﬁdﬂeméﬁigma-.,-..~$MPE__..,,.~§;'.III”'"Iii?”"w“"7391n«w»..mwmw.-mm- .........‘.....

Total phasphoﬂpid contensi" 51.1 911009 sampie 52.3 woos; solids ad. a not detached E' Sum oftht Idgnf‘rﬁad phuiphoipid dam 7 '“ May contain some glycmphmphocbnam (GPO) Sample #5 (color; orange) PhEEP‘lE’Eﬂlg.lBlal_mmtmwwmmmwwmsm.mﬁlMﬂElﬁhmﬂﬂﬁggm Ellospﬁaﬂdglchollne ' ' PC 65.5 7258 ’“ ” " ggwmdginosizol l5! 1:6 0.6 ”at ”“mmxmA—HWH_-~q.w.r-Pwmmn=nnmmRim-sew».Ms.Hmwmunmvlwtnuu ﬁgcépggggpserlne PS 0.? 0.3 twjbgssbslgncnﬁm'.nmw.m_‘ New” e um ,W. ,. t _-n—. ~«~wW‘u>ra—ankl n._-u,—.mmz.~.m.nu.__. LPG 4,2 _ . -W “10-1 .EZEﬂMEEX‘EBﬁWE‘JDEWMWWMMEMW... ”"73 . . ....,0-5 Phosnhslunstbenslsmlnem...._t.w,...-...~...-..n-l3§s ,, “WAS. -W '20 menenmnnmmanmmmtenses”-.- 22.1 31:5? Gennlinnjhsnlah..°.sﬁefine.n§mhnins.W..§.!:£Nn£§mm;m_m;_2;é,mm; W13: tnnneamennymlamnszmwsea”some..-i.iit.~32 enemiwnsnnnenmenn W-._-._Lﬂ-€LP§.H Wm wW_ﬁ________.s9»l Total phospholipld content“ 41.0 911009 sample 43.0 911909 solids ml. 5 not detected ' Sum (lithe Wed phesphoﬁpld classes These data are consistent with those obtained using traditional column chromatography techniques shown in Example I.

Example VIII Lipid Compositional Analysis OfLow Fluoride PPC al The example presents data showing the lipid compositional analysis of a low ﬂuoride phospholipid-protein complex composition created by the methods bed herein.

Consequently, it would be expected that the ﬂuoride content of the compositions described below are less than 500 ppm.

The PPC comprises approximately 46.7 g/ 100 g (e.g., ~ 47%) total fat, 11.8 g/100 g (e.g., ~ 12%) pentaenoic Acid (EPA) and 6.7 g/ 100 g (e.g., ~7%) docosahexaenoic acid (DHA). The total lipid t of the PPC total fat was approximately 87.7 % (w/w) and comprises between approximately 115 - 260 mg/kg astaxanthin and between approximately .2% —‘ 46.7% unextracted oil.

PCTfIB2012/003004 Table XII: Low Fluoride Krill PPC Fat: l Lipid Content (45.2% MW of total fat): Sam .10 Number lMG Components lglycerol Diacylglycerol Monoacylglycerol Free fatty acids Cholesterol Cholesterol Esters | _ _ __ Table X111: Low Fluoride Krill PPC Fat: Neutral Lipid Content (46.6% w/w of total fat): Sample Number ZMG Components Triacylglycerol Diacylglycerol Monoacylglycerol Free fatty acids Cholesterol Cholesterol Esters m Table IXV: Low de Krill PPC Neutral Lipids: Fatty Acid Content (49.7% W/w of neutral lltldS : Samde Number lMG Components % (w/w) neutral lipid Saturated 27.4 C» Monoenoic 21 .9 N-6 Polyunsaturated 1.8 N—3 Polylmsaturated 22.7 74.4 Table XV: Low Fluoride Krill PPC Neutral : Fatty Acid Content (46.7% w/W of neutral li nid): Sample Number ZMG Components % (w/w) neutral lipid Monoenoic N-6 Polyunsaturated N—3 Polyunsaturated Total i 76.9 WO 02792 Table XVI: Low Fluoride Krill PPC Polar Lipid Content (42.6% W/W of total lipids): Sample Number 1MG Components Phosphatidylethanolamine Phosphatidylinositol Phosphatidylserjne Lyso PhOSphatidylcholine Table XVII: Low Fluoride Krill PPC Polar Lipid Content (42.8% W/W of total lipids): Sample Number ZMG ents % (xv/w) olar liid Phosphatidylethanolamine | Phosphatidylinositol Phosphatidylsen'ne Phosphatidylcholine Lyso Phosphatidylcholine Example IX Lipid Compositional is 01° Low Fluoride De—Oiled PPC Material The example presents data showing the lipid compositional analysis of a low ﬂuoride de-oiled phospholipid-protein complex composition created by the methods described herein.

Consequently, it would be expected that the ﬂuoride content ofthe compositions described below are less than 500 ppm. The ed PPC comprises approximately 35 g/ 100 g (e.g., ~ 35%) total fat, 16.6 g/100 g (e.g., ~ 17%) eicosapentaenoic Acid (EPA) and 10.0 gf 100 g (e.g., ~10%) docosahexaenoic acid (DHA). The total lipid centent of the tie—oiled PPC total fat was imately 87.7 % (W/W) and comprises approximately 115 rug/kg astaxanthin and approximately 35.2% nnextraoted oil.

Table XVIII: Low Fluoride Krill De-Oiled PPC Fat: Neutral Lipid Content (20.1% w/w of total fat : Sam 16 Number SMG Components Triacylglycerol Diacylglycerol ylglycerol Free fatty acids Cholesterol Cholesterol Esters Table IXX: Low Fluoride Krill De-Oiled PPC l Lipids: Fatty Acid Content (35.2% W/W of neutral lipids): Sample Number SMG Components % (W/w) lipid | Saturated | 21.3 | Monoenoic l 3.9 N~6 Polyunsaturated N—S Polyunsaturated Table XX: Low Fluoride Krill PPC De-Oiled Polar Lipid Content (68.9% w/w of total fat): Samde Number 3MG Components % (W/W) polar lipid Phosphatidylethanolamine Phosphaticlylinositol Phosphatidylserine Phosphatidylcholine Lyso Phosphatidylcholjne Compositional Analysis Of PFC/Protein Hydrolysate Mixtures The example presents data showing the lipid itional analysis of a 10W ﬂuoride phospholipid—protein complex mixed with a protein hydrolysate composition d by the methods described herein in an approximate 60/40 ratio. It would be expected that the ﬂuoride content of the compositions described below are less than 500 ppm. The mixture comprises between approximately 28-30 g/100 g (e.g., ~ 30%) total fat, approximately 98 PCT/I32012/003004 mg/kg astaxantine esters, imately less than 1 mg/kg astaxanthine, a peroxide level of less than 0.1 %;(mEq/kg) and/or an anam'side level of less than 0.1 % (xv/W).

Table XXI: Low Fluoride PFC/Protein Mixture Fat: Neutral Lipid Content (28% w/w of total fat) ents Triacylglycerol Cholesterol Cholesterol Esters Table XXII: Low Fluoride PFC/Protein Mixture Neutral Lipids: Fatty Acid Content Components % (w/w) Monoenoic 19.2 {id—6 Polyunsaturated I 2 O N-3 Polyunsaturated 24.9 Table XXJII: Low Fluoride PFC/Protein Mixture Polar Li id Content Components % (W/w) := olar 1i id Phosphatidylethanolamine atidylinositol Phosphatidylsen'ne Phosphatidylcholine Lyso Phosphatidylcholine

Claims

1. A crustacean oil composition, sing phospholipids and where said phospholipids comprise phosphatidylethanolamine in the range of at least 2 to 4.9 wt%, resulting in a clear red colour due to a l oxidation, and/or degradation and 5 formation of dark/brown colour, wherein said phospholipids are between 39-52 wt%, n said phospholipids se 65wt% phosphatidylcholine and maximum 2.4 wt% lysophosphatidylcholine.

2. The crustacean oil ition according to claim 1, further comprising triglycerides, neutral lipids, 20 - 30 wt% polyunsaturated Omega-3 fatty acids of the 10 lipid fraction, and at least 0.8 wt% free fatty acids.

3. The crutacean oil composition according to any one of the preceding claims, wherein the level of astaxanthin is at least 115 mg/kg.

4. The crustacean oil composition according to any one of the preceding claims, wherein said oil is krill oil. 15

5. A crustacean oil composition according to claim 1 substantially as herein described with reference to any one of the embodiments of the invention illustrated in the accompanying examples.