AU2015243079B2

AU2015243079B2 - High-quality lipids and methods for producing by enzymatic liberation from biomass

Info

Publication number: AU2015243079B2
Application number: AU2015243079A
Authority: AU
Inventors: Joseph M. Kobzeff; Craig A. Weaver
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2002-05-03
Filing date: 2015-10-16
Publication date: 2017-07-13
Anticipated expiration: 2023-05-05
Also published as: AU2011202620B2; AU2015243079A1; AU2013273736B2; AU2011202620A1; AU2013273736A1

Abstract

A high-quality lipid composition is disclosed having low oxidative deterioration such as measured by low anisidine values. Also disclosed are methods of preparing the same from a lipid-containing material that include enzymatic degradation of protein and/or carbohydrate 5 components of the material. Lipid-containing materials include biomass, such as microorganisms. The invention further includes products containing the lipid compositions, such as dietary supplements, food products, pharmaceutical formulations, humanized animal milk, and infant formula.

Description

AUSTRALIA

Patents Act 1990

ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT

Invention title: HIGH-QUALITY LIPIDS AND METHODS FOR PRODUCING BY

ENZYMATIC LIBERATION FROM BIOMASS

This application is a divisional of Australian Patent Application No 2013273736 which is a divisional of Australian Patent Application No 2011202620 which is a divisional of Australian Patent Application No 2008252072 which is a divisional of Australian Patent Application No 2003237182 the Australian national phase entry of PCT/US2003/014177, which claims priority to US provisional patent application No 60/377,550 filed 3 May 2002. Each of these applications is herein incorporated by reference in their entireties.

The following statement is a full description of this invention, including the best method of performing it known to us:

MGH-QCJMJW IlPIBS AND MET MOHS: FOR FROPUCING BY ENZYMATIC

LIBERATION FROM BIOMASS

FIELD OF THE INVENTION

The owseni invention. is directed ίο high-quality lipids, and In particular, lipids with lew anisidine values. Methods are provided Par producing lugfoaonlity lipids that indude the step of liberating lipids from biomass, such as algal biomass, using enzymatic treatment.

'BACKGROUND OF THE INTENTION

Various methods have been employed Mr extracting lipids bom biomass. Techniques include direct extraction of the biomass with solvents, beating, pressure waves generated vis electric arcs, direct Saponitleatlon via K.CAI and ethanol, sonication, ffoemng and grinding and bead mills. For example, the biomass can be dried and tbs mad extracted with a solvent such as hexane. Alternatively, a microbial fermentation broth can be sub|ected to extreme conditions of pH and/or temperature er additional equipment such as a homogenizes· can be used to disrupt the ceils.

Problems with prior methods include poor product quality due to chemically aggressive conditions of high temperature and. high pH, high costs due to dm need to dry tbs biomass or far additional equipment such as homogen wars and pressure vessels.

The "fishy" and "painty" flavors associated with maty/ polyunsaturated fatty acids (PUi/As) found in lipids are primarily due to oxidation of the double bonds in the fatty acids. These flavor and odor notes are normally considered delects that can. preclude their use iss foods or other applications, Tire oxidative state and stability of a lipid or lipid-eontabiing material can be measured in a number of ways. Standard rneasurenient techniques include "attisidiae valued "peroxide value," "oxidative stability index," "Reoennai," ana gas chromatograph headspace analysis for oxidation products. Information on these different techniques is available Horn the AOCS (American Oil Chemists' Society) as well as train other sources.

The oxidative state of the bow or lipidwontaifong materia! is shongly impacted by toe processing conditions used to make the material. For food materials, foe conditions during processing as well as the actual ingredients and quality of the ingredients will affect the oxidation state. For fermentation-derived lipids (e.g., lipids obtained from 5 microbes grown in fermentors, ponds, etc.), the ingredients (fermentation and postfermentation) used as well as the conditions during the lipid extraction and fermentation will affect the quality. Other sources of PUFAs, such as agricultural crops and animal sources, will also be affected by the processing conditions used to obtain the lipids and lipid-containing materials.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides a lipid comprising polyunsaturated fatty acid, wherein said lipid has an anisidine value of 2 or less, and wherein the polyunsaturated fatty acid comprises at least 30 weight percent docosahexaenoic acid or at least 20 weight percent arachidonic acid.

In a second aspect the present invention provides a product selected from the group consisting of dietary supplement, food product, pharmaceutical formulation, humanized animal milk, and infant formula, wherein the product comprises the lipid of the first aspect.

In a third aspect the present invention provides a method of making a product selected from the group consisting of a dietary supplement, food product, pharmaceutical formulation, humanized animal milk, and infant formula, comprising adding the lipid of the first aspect to the product.

In a fourth aspect the present invention provides a method for obtaining a polyunsaturated fatty acid-containing lipid, comprising the steps: a. providing a biomass comprising a polyunsaturated-containing fatty acid; b. contacting said biomass with an enzyme; c. disrupting cells in the biomass by homogenization; and d. recovering said lipid.

In a fifth aspect the present invention provides a method for liberating a lipid from a biomass comprising liberating said lipid at a temperature of from about 10° C to about 80° C, a pH level of from about pH 5 to about pH 9, in the substantial absence of an extraction solvent.

The present invention also provides a refined, bleached, or deodorized microbial or plant lipid comprising polyunsaturated fatty acid, wherein the lipid has an anisidine value of 1.5 or less, wherein the lipid has been liberated from biomass enzymatically, and wherein the polyunsaturated fatty acid present in the lipid comprises at least 20 weight percent docosahexaenoic acid, at least 5 weight percent docosapentaenoic acid, or at least 20 weight percent arachidonic acid.

Preferably, the lipid anisidine value is 1 or less. More preferably, the lipid anisidine value is 0.5 or less.

Preferably, the polyunsaturated fatty acid is docosahexaenoic acid or docosapentaenoic acid or arachidonic acid.

Preferably, the polyunsaturated fatty acid comprises at least 30 weight percent docosahexaenoic acid or at least 35 weight percent docosahexaenoic acid.

Preferably, the polyunsaturated fatty acid comprises at least 10 weight percent docosapentaenoic acid or at least 15 weight percent docosapentaenoic acid or at least 20 weight percent docosapentaenoic acid.

Preferably, the polyunsaturated fatty acid comprises at least 30 weight percent arachidonic acid or at least 40 weight percent arachidonic acid or at least 50 weight percent arachidonic acid.

The present invention also provides a product selected from the group consisting of dietary supplement, food product, pharmaceutical formulation, humanized animal milk, and infant formula, wherein the product comprises the lipid of the invention.

The present invention also provides a method of making a product selected from the group consisting of a dietary supplement, food product, pharmaceutical formulation, humanized animal milk, and infant formula, comprising adding the lipid of the invention to the dietary supplement, food product, pharmaceutical formulation, humanized animal milk, or infant formula.

In accordance with another embodiment of the present invention, provides a lipid comprising polyunsaturated fatty acid wherein the lipid has an anisidine value of 2 or less, and in various embodiments the anisidine value can be as low as 0.3 or less. The polyunsaturated fatty acid in the lipid 15 can be a long chain polyunsaturated fatty acid, having a chain length of at least 20 or at least 22, and can have at least three or at least four double bonds. More particularly, the polyunsaturated fatty acid can be docosahexaenoic acid, docosapentaenoic acid, or arachidonic acid.

The lipid can be obtained from biomass, for example, from a plant or 20 microorganism. For example, the lipid can be obtained from algae, bacteria, fungi or protists.

In preferred embodiments, the lipid can be obtained from microorganisms of the genus Mortierella, genus Crypthecodinium, or order Thraustochytriales. Further, the lipid can comprise a monoacylglyceride, a diacylglyceride, or a triacylglyceride.

Further embodiments of the present invention include products selected from 25 dietary supplements, food products, pharmaceutical formulations, humanized animal milk or infant formula wherein the products include a lipid comprising polyunsaturated fatty acid and having an anisidine value of 2 or less. A further embodiment of the present invention is a method of obtaining a polyunsaturated fatty acid-containing lipid which includes providing a biomass 30 containing a polyunsaturated-containing fatty acid, contacting the biomass with an enzyme, and recovering the lipid. A further method of the present invention is a method for liberating a lipid from a biomass comprising liberating the lipid at a temperature of about 10C to about 80C at a pH level of from about pH 5 to about pH 9 This method is conducted in the substantial absence of an extraction solvent.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this specification.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one embodiment of the present invention, a high-quality lipid is provided. In particular, the lipid has a low anisidine value. Preferably, the anisidine value is 2 or less, more preferably 1.5 or less, more preferably 1 or less, more preferably 0.5 or less and more preferably 0.3 or less. Anisidine value can be thought of as a measure of the oxidative history of a lipid. Higher values indicate a lipid that has experienced more oxidative stress. As a lipid is oxidized, it is typically converted to a peroxide. This peroxide typically gets converted to an aldehyde or ketone. The anisidine value is a measure of these secondary oxidation products. Polyunsaturated fatty acid-containing lipids are very sensitive to oxidation and this oxidation can lead to off-flavors. Methods have been employed to remove these off-flavors, but these methods do not remove all of the oxidation products that can then act as off-flavor, and oxidation, precursors. As a result, these flavor improvement methods only lead to temporary improvement of the flavor. The anisidine value is a measurement of these oxidation and off-flavor precursors. The analytical method for measuring anisidine value is available from the AOCS (American Oil Chemists' Society).

In accordance with another embodiment of the present invention, a process for liberating lipids is provided. The process includes a step of liberating lipids using an enzymatic treatment for, e.g., degradation of cell walls of the lipid-containing material. Preferably, the lipids are liberated from a lipid-containing biomass using a protease enzyme whereby protein components of the lipid-containing material are proteolyzed, or other enzyme that is appropriate for breaking down or reacting with the lipid-containing material. A further embodiment of the present invention is a process that utilizes surfactants in addition to the enzymes to liberate the lipids from the lipid-containing material. The inventors have surprisingly found that the use of surfactants together with enzymatic treatment can allow for milder reaction conditions than with enzymes alone for liberation of the lipids. Surfactants, such as Polysorbate 80, mono- and diglycerides, or other surfactants, are preferably added at approximately the same time as the enzyme. Alternatively, surfactant can be added before or after the enzyme. In this embodiment, OH Ο ^ X Ο ' Ο, Γο' V *' Ν <Ν V vV --0 ν ' tetstperaiwe or pH and without using sJdtuurat equipment omn ou a homoeemni ot donig the biomass prior m lipid return ul. For example, the eit/Yumm. treatment two Ft conducted ut nmmetutuius beasw about hOC, more pretor&bb "νίοχχ ,*«vut ?0t, ,v\i ex to more pteietabiy, below «boat 65C, and. at pH conditions of approximately 5-9, 1¾ use of protease omsym.es, or protease enemies lo combination with scitkdtano, provide,' at economical and simple was of news me, the a pH isos tin t\Oi s» s ' 0 v ^ a n w'V *. ve m anting *.qm >.\ v *0 c-att then bo isolated from too rust of me fermentation broth by ctabilugaboo of the mix onto. In some cases* the lipid will ho incorporated into an emulsion-, For some eppBoatwnun the vmifision itself ought he the final product. For other applications, the emulsion would be treated to release die lipid for recovery separately, Techniques are taught m U,S, Patent Application Mo, 09/7h6fS0i> and include, hot are not limited to, dilution, addition of a solvent, temperature shifts., and freeriog.

The use of 0 protease enryme con help break down emnlsioinstahiltaiog proteins present, thereby aiding tn the breaking of an emulsion. In addition, the successful use of a protease far lipid liberation koto mierosigae is surprising because, tmcroalgao tend to have a low protein content (-.15-22% compared to -55% for if cob), nod have vet y robust cellular structure due to the presence of silica and polysaccharides such as cellulose.

This processing step also allows: the production of lipids comaimng long chain polyunsaturated fatty adds (FHFAs'l of exceedingly high quality as measured by anisidine value., The reason is that the process can easily be done under an inert atnmsphere, with low temperatures, and wan-reactive condition

In a particular embodiment* using proteolytic treatment of the llpidmontddog materia! without a surfactant, the proteolytic treatment can be conducted at higher tempemtums, sufficient to achieve desirable levels of lipid liberation. For example: m this embodiment, the enzymatic treatment is conducted at temperatures of at least about $0C, more preferably at least about 40C, and most preferably at least about 500, It. slouM be recognised however, that at higher temperatures, degradation of lipids can oec&o ThereKue, a temperature must be selected such that adequate lipid liberation is achieved without unacceptable levels of lipul degradation,

Preferred polyrpssfemfed fetif gfed be .&pyibbs?QSS: that are capable oil ihnrabott by ermyrnes as fe the present mvemioo. Preferred pofynrAaturated tatty areas scorees iareude biomass somces, such 3¾ animal, plant and/or tnictomsl sources, As used hereto, the term feipfrT secludes ptmsphohpfds: free lady acids, esters of lady adds; maeylt^yeemis; dlbpyi|lyrresd«sy soaps; pdosph&ddes; ^rdk.lpdys^Cfl esters; oarolidpidsi xadilM^bytls |s>f,, osyoardieooldsF byirdcartpsos; add etfere hplds Jmowe to ppe of ordinary skill Id. fee art Examples of animat sources iodutk agnatic animats (e.g., fish, marine mammals, crostaeeaos, roofers, ole,) add:lipids byes, esc,), Eysroplpsof pimresomyes ioehtde . . :*s0im# evoorng plmresk soy and borage. Examples of MlemarpolddS mplpde algae, prOilsts, Pc rods and feogl (iaclndfeg yeast). The nse of a microorganism source, such as algae., can provide organoleptic advantages, he., fatty acids from a microorganism source .may not have the fishy taste am! smell that fairy acids ffem a II ah source tend to have. More preferably the iong-chain fatty add source composes algae.

Preferably; when microorganisms arc: the source of loogOhaid idly acids, die microorganisms are cultured Is a fermentation medium in a ferroeniOA Alternatively, the microorganisms can be cultured photosymhedcally in a photobioreactor or pond. Preferably» the microorganisms are lipid-rich .microorganisms, more preferably, dm microorganisms are selected from the group consisting of algae, bacteria, lung; and protista, more preferably, the group consisting of golden algae, green algae, dlpoSaplIsfes, yeast, fehgl of feu pons Iferetetylfeand Sriameooplfes, feefembli;fur tnioroorgatnams. comprise mferOotptdsms: of the germs Cryptheoodbuum and order Thmustoahytnales and hfementoas fangs of the genus Mortlersikc and mote preferably, microorganisms am selected from the genus Thrm&oehyimm, 8b8ferie%feam or mixtures feerfetf, more preferably, the microorganisms are selected from the group consisting of odcroorporems oavmg the identifying characteristics of ATCC number 20888, ATCC oumber 2088th ATCC number Sdlfeh AfCfe number 20881 and ATCC oumber 2E892, strsiha of Motferfefb -^hmuckm·. &M·φ'«β, strains of C^ihecmMnkm mkmit midaot stralsrs derived from arty of the foregoing, and mixtures thereof. II should be noted that many experts agree that iMem& m not a separate genus from the genera Thrm$iochyttfitm/m4 Schk&ckyirium. Accordingly; as used herein, the poem and wifi tmlM® flMkin: regarding such algae can da found m U.S. Parent Nos. 5,4()7,957, 5,130,242 and 5,340,19¾ fehlcb am incomomted heroin by reflnence In their rotlrety.

Lipids recovered by lie present Invention Inclnde lipids comprising a :pl;|aMaabiraied Idly acid* imam fadlbblarlyjd Ibbffehfe even wore patticuferly, a polyousanoafed fatty add; present In said IpM Paving a carbon chain length of at least 20 or 22.. inch pnlyeeMipiated fatly acid^ pmsebt esp nave: at: feast 3 or at least 4 doable bonds. More particularly; the polyunsaturated fatty add cart Include doeosahexaeoolc add (at least 10, 20,30 or 35 weight peteem), docosspentaeaoic acid (at leasts* 10, IS, or 2d weightpercent), andfer araetddoale add fat least 2d, 30,40 or 50 weight percent), Ifelyonsataratsd fatty adds Include free fatty adds and cotnpoands eomptfemg PUPA residues. Including phospholipids; esters of fatty acids; triacylglycarols; dlacylglyoerides; monoacyiglycendcs; lysophosphohpids; phospnatldes; etc.

For different oil-containing materials, different enzymes and reaction conditions can he employed. For these dtflcrent materials, an hnposfeo! dbMadc selection criterion la to select an etetyrne tbaf dll!:: attack mu!degrade a portion of idte irpatdaal fauok as: the proteins, dell:MidrMdmbiPP%·layer, cellulose, chitio, hetnivcHubse, lignin, bgnlnenlated compounds, etc,) that <s otherwise knceding recovery of the off. Preferably, uoblpeethe protease enzymes snob as trypsin, chymoirypsm, or the like are used to degrade protein components of the oil'Containing osetetfels and. eatbobydmse enzymes such as amylase can be used to degrade carbohydrate components of the olhccntaobrig materials. The selection of .reaction conditions, Inc fading enzyme type, enzyme concentration, temperature, pM: water activity, other reapot concenfration, reaeboo dme, etc. will bepurm 1» part on the specific enzyme and rnatertaf that Ike lipid is being liberated from. These conditkms cars be readily defermfned Irhbs dsil&Miim <a^c|"^kaify amiable imp the supplier or in the literature), or determined by somebody skilled in the art. Typical temperatures may range between approximately 20-80*0, although some special enzymes may be anUclenffy active and stable fed fefe outride of fkia fenios Typical enzyme concentrations can be as low as 0,0!% to several percent, Tbs reaction rate is related to the enzyme concentration. with higher coooentrahons allowing lor shorter reaction times, In some sitontiobh. It may be: posslhlo to ose &n even lower concentration, such as when a papieolar eorprte :1s extmmcly active or smlde or when very loug: tebaclop tilths .may Be practical

Ibderably fee Ispkis are eileebveiy liberated born Sckizochyfnum sp organisms by treating the cells -With & protease enzyme. It is aotpislag:that libs pMeolar class of enaymes is effecbve lor: lips organism sice to the; mlabvefy small amount of promos mttmW found m the ceil wall of ilia orgamsm.: lipids ead be liberated Item biomass, and preferably mkroorganisme, by treating the sells with aozymes or other agents or by otbe? methods drat shack ether compcoeels of the bell wall, saetr as polysaeclwrffees, or the lipid beaver. Ibis treatment with -Mzjmm ihovtlea ode method nmler auM conditions onhgh gaahp. lends Otiau methods for liberation of

Spats that can be Pserl,. aloes or jo oombiMtlos wili ooayssaSc hpmrnph loolode trostmeet: with bemrgehts, osmetic shock, ffeeaiegltbasv eyebeg, aorelyek, bemoiebiatbpo, seokatloe, and mi Id heat Ooahaeni.)

One preferred embodiment of the process, of the present invention includes; » Obtaimng iipid-be&ring single cell organisms * Treating with protease cr a combination of surfactant aod protease x. Separating the lipid from the broth (may be an emulsion) o May mpolre addiiohal leeafmeat witb:ia polar organa; solvent, salt, o.\ ' x.vxv uv o s' cam (protease or other kind), hesnng, cooling * If the lipid fern the above step Is id fie term of an emulsion, this prodoel can be used Ms Ia*s or dried and used on treated to release the lipid Imre the emulsion o Treatment can Include treabdeot with a: polar organic solvent, salt, precipitating; agent, another enxyme (protease or other kind), beating, eoellug, etc. tie lipid cab: tfeah be sided, yefmed, bfoaebad, deodorised and/or reacted as needed.

The lipid can also fee treated with antioxidants and/or metal ion capturing agents (such as che.lat.lng agents, Ion exchange resin, precipitating agents) at any point before., during or alter die process. M noted shove., the present mvecdon eneofapaases tbe use of a pretense In the presence ef a surfactant to recover lipid from a bnnnass.ibltdble sorfaclante melode, but arc hot limited to; phospholipid, lysopbospholipid, rnonoglyccdde, diglyesrides, mixed glycerides·, partial glycerides·, soap, fatly acids, salts of idly piciy imipss .anltlbam, adds or salts of suitbmc acid, detergents, polvsorhaies (e.g,, polyethylene aorbibtn moeooleste), ylfifeie rmlis awl esters, polar organic molecules, alcohols, sulfates end sulfonates, ufUseptueouluiuiug compounds (e.g, wblnes, amides, polyamides;, ,'' '· ϊ On ,\ v- m- 0 v v , r > , t x O O'- \ \ v.0 vn ·, v teUu-ulley! ellaoep aftiewm polymer eoufsming slim bloteityl stiemte polpturas tbethyl silicones), sulfides and thio derivatives, halogenated compounds, triaeyi glycerols, long eltarn tally waaee (egg, V^^fl#fdl3i:-:§nd. odd sedated derivative of triueylglycemis and mineral ails, bentonite* -ami monosodlum phmphsle mixes! with bone acid and ethyl carbonate.

In a further embodiment, the process can be conducted with a combination of enzymes, More specifically, a protease ami a lipase nan be used- A lipase is an eeayme that hydrolyzes glycerides. Therefore, care needs to he taken to avoid unacceptable levels of degradation. of glycerides In the llpsd product, for euutople* a lipase Mill hydrolyre a triglyceride producing a fee fatty acid and a dtglyeeride. This mechanism Is believed, without Intending to he bound hy theory, to he beneficial tu an extent because poxlucts of the eneyrnadc degradation function as surfactants having the benefits described above In the embodiment of tlm Itwemten involving dlteot use of aurtaetahis, Howevet; them at the pplenief that the lipid product could: be tfoaeeapl#by degraded by the hnase. Therefore, addttiona! embodiments involve the use of small amounts of lipase or conditions under which the lipase is only active a small amount oft.be time. Such control of llfuse detrwly could: he controlled; ftir example, by the use of tosuperature setuidvo enzymes or the inboductioo of lipase inhibitors.

In another embodiment of the present iiweoion, the ptocesses of the present invention are combined with further oxldahon-redoomg techniques, including one. or mom of exofgslou of dlf (and. oxygen) and other oxidising agents, processing with mild conditions (moderate temperature, moderate pH, short processing times, etc ), exclusion of mess! teas such as copper and trots, exclusion of previously oxidized lipids {even if sutuegueutiy perilled), exclusion of oxidation pmettmms, pod life presence of ontloatdant compounds (such as tuccnborois, toootrlenels, BI-LA, carmsoi, carncslc acid, ascorbic pgaf: .foaaemiye aoM estpml (mcludiog: f^psoptfeyl galmdztes: Lmsefobyl steaMM, l, asoorhyi oleate), msemasy, etc, as well an esters or derivatives of these cempoonds), to obtain minimally oxidized lipids.

In μι» eases, alter the lipids' lie lltwtshgd Hem the biomass, the Bpids can Be separated directly tern: the uedcaircd materials (e,g.< cellular debris), such as By eenrriidgahon, or ether appropriate methods. 1« ether esaes* sit agent such an an alcohol nr other polar organic solvent can Be adder! to facilitate the separation of the liberated: lipid iom the otber materiaL Id still other eases, a solvent enu he added that will dissolve: the lipid andfaeittate the sapetpioi of the liBedied 1%¾ eig.,; By solvent extraction. Techniques fur separating the lipids Horn nndesired materials can be found la CBS. ΗΐΙΙο! Nwolier; IMdpih, IBS. tum&s and PCX Application Iddifhers TIS01/ITIM? nod: tiSdi/ldOdd, all; of which are incorporated herein By reference m their entirety. Another emhodmieot ot the invention involves the use of a combination ot the enzyme treatment, or the enzyme pins surfactant treatment, along wish homogenisation. This combination m some eases can achieve higher quality and/or higher yield than with ·ώ^ιβίϊι:\

If Is believed that horeopnlzaflon rad l^dlitatb·fhacion By allowing mow ii intimate contact between the enzyme and its substrate. It is also behoved that enzyme treatment cad: ilelitafc homogenization by weaken ing the cell walls and allowing the ose of less extreme (pressure or shear) homogenization conditions. The use of homogenization with the enzyme or enzyme'Surfitetant process can allow the use of conditions that are more chemically mild than would he possfhle without the homogenization. In other cases, this ooniblded process ean allow use of a lower pressure land rise Imre '.ovt howoeo'n umon

In accordance noth a farther embodiment of the present hivenhom the processes previously described ein Be linpliyed on hpid'-Bearmg material that has Been dried poor to Ipld removal. Wldle the: highest: -pally and lowest post process won id: normally: Be: :: expected from material that has nor been dried, there ere cases where It would he advantageous to dry the material either prior to or at some intermediate point during the process, poor to lipid separation. Use of the-previously -described processes-with drying ears provide a partial improvement in quality and/or cost over processes that mclode drying and do not iodide the invented processes. Some examples of when this drying step would he appropriate are when the facility .tor lipid separation Is located remote tmm the lUrmeatitldh: or other tspstrearn facility, or when there am scheduling drfBuulttos: between the lipid separation fncilty and the upstream facility,, or when the nyud-containing material must Be stored prior to separating die lipid.

Lunge aspect of toe preset InvehPOO, flic Ifpid is used in an eodprodact adeemed fmt&. ii$g group esmsutmg of a dhdMf supplement, a food product, a ifepppopuppa! formulation, u hontanlned animal ruill;., and; hn| infant Ibmmlm A pbannaceuucai fbfnfolntfdd can include, lit: Is oof IlipilMi to: no aotMubanunaiory fotntuhdnm, a chemotherapeutic agonh an active excipient, an osteoporosis drag, an armfoepressaab an iotooosaolstpp, an anciTfo/foofwcmr jufet drug, a drug for nnanneui of neinodegenendive disease, a drag tor treatment of degeoerahve liver disease, an antibiotic, and a cholesterol lowering formulation. In one aspect, the endproduct .is used to treat a condition peleeted Ifonr tbo pptip: coosisSog of; plnonfo rnflammation, acme iofiammation, gastrointestinal diadtiur,;. oaucop osebexia, cardiac resmoosis, neurodegensratlve disorder, degenerative disorder of the liver, blood lipid disorder, osteoporosis, osteoarthritis, autoimmune disease, preeclampsia,, preterm birth, sage related maobopathy, pulmonary disorder, schizophrenia, depression, weight maintenance and peroxisomal disorder. The following examples are provided for the purpose of iUostrrdioo and are not. intended to limit the scope of the present invention.

EXAMPLES

Emtmcdpofnm sp, termenMlon kolh was diluted and buffered as follows; 2$ mi of broth was combined with do nil PI watery then 10 to! of pfI 6.0 baiter (1,0 M 24.N-ruotphbllnol emaneanlfooic neid| was oddOd,

To diffbreut ahpsou of this broth mixture different combinations of eoiyme and serlecrant were added. Alter the eocyme and surfoenmt additions, the samples were Incubated at 45 € for LS hr, and then exarslued by microscope for degree of ffsifo Ice results are shown below;

*Ths enzymes are both from Novozyrnes North America, lee, ol Fmnkhotc»^ NC.

Tms example demonstrates both the successful lysis of the organism wills eoxymea aud She improvement of lysis with the feel when of a surfactant* Ifelysorbale 80.

SiMhtei:

etscl: bsslfbred as ip Example L A eetweseisl :pM®m (Aica!aeet& iA: L KE aeaiahte fkfer 'North.

Amesscw fee, of mokhnton* HC) and a eorometcfe! carbobydrase (Vtscoaymefe L:. avfelaMe fleet Novozyme» North Amenca, fee. of EssbEiotofe NO) were wMed to me ddbfed and buffered broth, Thfe ferefe mixture was divided mfe ilfhsresst:earlaetaoia wore added as follows: 1, dolyserbam$0 : 2 , Sodium laery! so date 2, Mono bob htefyefeidee (Dunodan CO»K born Damsco of Kuw Cfehiety Mf After fee surfactant addition, safes sample was beefed sit e bet water bath ('?$ C) tor approx. J rasa. Each sample seas thee held overnight at mom temgefemse with mixingm a Fisher Hematology/Chenhstsy Mixer. Thus samples warn exam feed under a microscope for degree of lysis. The resold are lhOAtihelrw;

This example demonstrates that different surfactants can be used. la thm ease both IbdysotL'alc $9 end mono wad dtglycendes (Outs aim) ware VAViWsicl boehmc iaaryl sulfate was sat as successful due to this particular chemical ahmAmg the anaymes.

Saha.x Gowns sp femwntahon booh was oewted a. ah earw&sme mm mb-1 palmitate and tocopherol s) and drunt dried. This dried biomass was teen treated as follows; * Added to distilled water (5.1 g of biomass to 300 g water) * The pH was adjusted to the range of 6.0-7.3 with i.N H2S04 * The mixture was heated to 60 C in a water bath * 1 „5 sal of Akalase 2.4L FG was added *. The broth mixture was thee purged with nitrogen to exclude oxygen and Incubated at60C for IS hoots * 12i> ml of isoprop&nol (99,9%) was added with gentle mixing .- The broth-alcohol mixture was then centrifuged at 4000 RPM .tor 5 minutes .- The lipid phase (supernatant) seas collected

The collected lipid was tested tor anistdine and peroxide value per AOCS (American Oil Chemists Society) methods Cd S-53 and €<t IS-90. A sample of the dried biomass was also hexane extracted by combining with hexane end ball milled In a Swedish tube extraction system. The Up id collected was tested for xmhwdma value and peroxide value as the other lipid sample.

The test results of the Upid collected by the two methods are shown below;

This example demonstrates the snceessthl lysis of the cells with enzymes, the isolation of the lipid that was present in the ceils and the very high quality of the lipid ifvesy lOWsphidlne value).

The ioregtoag discossfe of the mveotron has been preserved for purposes of Ifferahoo atsd desariptioo. Tie ioregomg is oof ih5leo|esl.io ffet the aovfeon to the fee or foies dfsclbfe heofe idthoagh the defeplprr of-fie feggirurrhas fersoed deecnptfe of see 05· mens embodiments ami csrtfe vanetfes aod ?ao<hfeuoos$ other vansdoos sod modifications are witbia the scope of the iovectioo.. eg., as rosy be w-thio the skit aod knowledge of those is the an; alter aoderstsodiog the present disclosure. It is itbeaded to obtfev rights which sod ado alternative eedodimeats to the esteat pepshtad; adtl/er epttrrolebf struetursa, feebons, rouges or stops to those ehhroed, whether or rmt seep alternate,, nncrehangcablc sodSor eqarvalcnt straetmes, Maotioos, roogsa or steps are dfseleseb herein, and without iroesdmg to pabliciy dedicate any patefehle Slbfe-itipfe*

Claims

The claims defining the invention are as follows:

1. A lipid comprising polyunsaturated fatty acid, wherein said lipid has an anisidine value of 2 or less, and wherein the polyunsaturated fatty acid comprises at least 30 weight percent docosahexaenoic acid or at least 20 weight percent arachidonic acid.
2. The lipid of claim 1, wherein said lipid has an anisidine value of 1.5 or less.
3. The lipid of claim 1 or 2, wherein said lipid has an anisidine value of 1 or less.
4. The lipid of any one of claims 1 to 3, wherein said lipid has an anisidine value of 0.5 or less.
5. The lipid of any one of claims 1 to 4, wherein said lipid has an anisidine value of 0.3 or less.
6. The lipid of any one of claims 1 to 5, wherein said polyunsaturated fatty acid comprises at least 35 weight percent docosahexaenoic acid.
7. The lipid of any one of claims 1 to 6, wherein said polyunsaturated fatty acid comprises at least 50 weight percent arachidonic acid.
8. The lipid of any one of claims 1 to 7, wherein said lipid is obtained from biomass.
9. The lipid of any one of claims 1 to 8, wherein said lipid is obtained from a plant or microorganism.
10. The lipid of any one of claims 1 to 9, wherein said lipid is obtained from at least one of the group consisting of algae, bacteria, fungi and protists.
11. The lipid of any one of claims 1 to 10, wherein said lipid is obtained from algae.
12. The lipid of any one of claims 1 to 10, wherein said lipid is obtained from microorganisms selected from the group consisting of golden algae, green algae, dinoflagellates, yeast, fungi of the genus Mortierella, and Stramenopiles.
13. The lipid of any one of claims 1 to 10, wherein said lipid is obtained from microorganisms selected from the group consisting of the genus Mortierella, genus Crypthecodinium, and order Thraustochytriales.
14. The lipid of any one of claims 1 to 10, wherein the lipid is obtained from microorganisms selected from the group consisting of the genus Thraustochytrium, genus Schizochytrium or mixtures thereof.
15. The lipid of any one of claims 1 to 14, wherein said lipid comprises a monoacylglyceride.
16. The lipid of any one of claims 1 to 15, wherein said lipid comprises a diacylglyceride.
17. The lipid of any one of claims 1 to 16, wherein said lipid comprises a triacylglyceride.
18. A product selected from the group consisting of dietary supplement, food product, pharmaceutical formulation, humanized animal milk, and infant formula, wherein the product comprises the lipid of any one of claims 1 to 17.
19. A method of making a product selected from the group consisting of a dietary supplement, food product, pharmaceutical formulation, humanized animal milk, and infant formula, comprising adding the lipid of any one of claims 1 to 17 to the product.
20. A method for obtaining a polyunsaturated fatty acid-containing lipid, comprising the steps: a. providing a biomass comprising a polyunsaturated-containing fatty acid; b. contacting said biomass with an enzyme; c. disrupting cells in the biomass by homogenization; and d. recovering said lipid.
21. A method for liberating a lipid from a biomass comprising liberating said lipid at a temperature of from about 10° C to about 80° C, a pH level of from about pH 5 to about pH 9, in the substantial absence of an extraction solvent.
22. The method of claim 21, wherein said temperature is from about 30° C to about 70° C.
23. The method of claim 21 or 22, wherein said temperature is from about 40° C to about 65° C.
24. The method of any one of claims 20 to 23, wherein said biomass comprises a plant or microorganism.
25. The method of any one of claims 20 to 24, wherein said biomass comprises at least one of the group consisting of algae, bacteria, fungi and protists.
26. The method of any one of claims 20 to 25, wherein said biomass comprises algae.
27. The method of any one of claims 20 to 25, wherein said biomass comprises microorganisms selected from the group consisting of golden algae, green algae, dinoflagellates, yeast, fungi of the genus Mortierella, and Stramenopiles.
28. The method of any one of claims 20 to 25, wherein said biomass comprises microorganisms selected from the group consisting of the genus Mortierella, genus Crypthecodinium, and order Thraustochytriales.
29. The method of any one of claims 20 to 25, wherein said biomass comprises microorganisms selected from the group consisting of the genus Thraustochytrium, genus Schizochytrium or mixtures thereof.
30. The method of any one of claims 20 to 29, wherein said step of contacting said biomass with an enzyme comprises treating said biomass with a protease or a combination of a surfactant and a protease.
31. The method of any one of claims 20 to 30, further comprising the step of separating the lipid from a fermentation broth.
32. The method of claim 31, wherein said separated lipid is in the form of an emulsion.
33. The method of any one of claims 20 to 32, further comprising at least one additional treatment selected from the group consisting of treatment with a polar organic solvent, salt, precipitating agent, another enzyme, heating or cooling.
34. The method of claim 32, wherein said emulsion is used as an emulsion and used as a dried emulsion or treated to release the lipid from the emulsion.
35. The method of any one of claims 20 to 34, wherein said lipid is further treated.
36. The method of any one of claims 20 to 35, wherein said lipid is further treated by at least one treatment selected from the group consisting of drying, refining, bleaching, deodorizing, and reacting.
37. The method of any one of claims 20 to 36, wherein said lipid is further treated by at least one treatment selected from the group consisting of treatment with antioxidants and/or metal ion capturing agents (such as chelating agents, ion exchange resin, precipitating agents).
38. The method of any one of claims 20 to 37, wherein said step of contacting said biomass with an enzyme comprises treating said biomass with a protease and a lipase.