CN101389749A - Eukaryotic microorganisms for producing lipids and antioxidants - Google Patents

Abstract

Disclosed are compositions and methods related to eukaryotic microorganisms that can produce unsaturated fatty acids which can be purified and used.

Description

Produce the eukaryotic microorganisms of lipid and antioxidant

The application requires the U.S. Provisional Application No.60/688 with submission on June 7th, 2005, the U.S. Provisional Application No.60/751 that on December 16th, 207 and 2005 submitted to, 401 as basis for priority, and the full text of these two parts of U.S. Provisional Applications is included this paper in by the mode of quoting.

Background technology

A large amount of scientific evidence show, (n-3) highly unsaturated fatty acids for example docosahexenoic acid (DHA) cardiovascular disorder, chronic inflammatory diseases and cerebral disorders are had active effect.On the other hand, (n-6) lipid acid is considered to for example mesostate of prostaglandin(PG), leukotriene etc. of 20 acids sterol.

At present, the main source of these highly unsaturated fatty acidss is fishes, has noticed that the amount of multiple mazarine fish (for example sardines and tuna) intravital DHA and timnodonic acid (EPA) is respectively about 20% and 10%.Yet, if will use fish oil to remain in some shortcomings, for example taint of odour, can not stablize the problems such as pollutent that utilization, natural fish oil content there are differences and may accumulate hostile environment as unique source of these lipids.In addition, if want to obtain from these sources highly purified (n-3) or (n-6) oil, preferential separation and purifying be difficulty very.

Summary of the invention

Disclosed composition and the method that relates to eukaryote thraustochytriale (thraustochytriales) and thraustochytriale section (Thraustochytriaceae), described eukaryote can produce a large amount of unsaturated fatty acidss for example ω 3 (n-3) and/or ω 6 (n-6) oil when cultivating, as DHA, EPA and DPA, because all these compositions can be used, and because its mode of production, they all can be purified and use.

Description of drawings

The accompanying drawing that is included into and constitutes the part of this specification sheets has been set forth several embodiments, and with specification sheets disclosed composition and method is set forth.

Fig. 1 illustrates and provides the figure that the lipid that derives from ONC-T18 is carried out the result of fatty acid methyl acquisition.

Fig. 2 has described at the original strain isolated of the ONC-T18 that Advocate Harbor collects with graph mode and has been preserved in the comparable situation of fatty acid methyl ester between the strain isolated of ONC-T18 genus thraustochytrium kind (Thraustochytrium sp.) of ATCC with numbering PTA-6245.All peaks are all identified by gas-chromatography and mass spectrum.

Fig. 3 illustrates and carries out the scatter diagram that ONC-T18 biomass optimization is tested the result who obtains.The technology that is called as the Taguchi method is used in these experiments, and purpose is to determine the optimal growth condition of ONC-T18 under different culture medium condition.

Fig. 4 is illustrated in the histogram of the lipid acid condition of production of the ONC-T18 of the growth Ninth Heaven under the top condition (embodiment 4).

Fig. 5 illustrate as this paper other local as described in the figure of the oleaginous microorganism that obtains of separation.

Fig. 6 illustrates the branching system tree of the relation between ONC-T18 and other thraustochytriale purpose 18S rRNA gene.

Fig. 7 is illustrated in the lipid and the DHA condition of production of ONC-T18 under the different condition.

Fig. 8 illustrates by the information that discloses Eukaryotic growth conditions about this paper and carries out improved figure (to Ratledge, C. (2004), the method for Lipid Technol.16:34-39 is improved).

Fig. 9 illustrates the pathways metabolism of inferring that disclosed eukaryote produces PUFA.

Figure 10 is illustrated in the comparable situation of lipid acid maximum yield and component under the situation of various alternative low-cost carbon sources.

Figure 11 illustrates the grouping based on its C20 and the C22 PUFA condition of production of the strain isolated collected.The result compares with two reference strain ATCC 20891 and MYA-1381.

Figure 12 illustrates the 18S rRNA of bacterial strain ONC-T18 in abutting connection with tree (Neighbour-joiningtree).Horizontal line is represented genetic distance, and square brackets are given in the sequence of using in this genealogical tree that derives from GenBank.

Figure 13 is given under the following 3 class different fermentations environment in containing 2g L ^-1Yeast extract, 8g L ^-1L-glutaminate, 6g L ^-1Sea salt and 60g L ^-1The lipid acid condition of production of the ONC-T18 that grows in the substratum of glucose: agar plate (1.5% agar, 25 ℃, 27 days), flask (in the 250ml flask 50ml being housed, 120RPM, 25 ℃, 3 days) and 5L bio-reactor (4 lpm air, pO ₂90%, 25 ℃, 3 days).

Figure 14 gives and comes from the HPLC color atlas that genus thraustochytrium kind ONC-T18 separates the carotinoid compounds that obtains.For example in genus thraustochytrium kind ONC-T18, isolate astaxanthin, zeaxanthin, canthaxanthin, myoxanthin and β-Hu Luobusu.

Figure 15 is illustrated in canonical biometric amount, total fatty acids (TFA), the DHA condition of production and the glucose utilization situation of the genus thraustochytrium kind ONC-T18 that keeps 168h in the 5L bio-reactor that substratum is housed, and described substratum contains 60g L ^-1Glucose, 2g L ^-1Yeast extract, 8g L ^-1L-glutamic acid and 6g L ^-1Salt (4lpm air, pO ₂90%, 25 ℃, pH7-9).

Figure 16 provides that astaxanthin forms the related approach of inferring among the genus thraustochytrium kind ONC-T18.

Embodiment

Before compound of the present invention, composition, article, device and/or method are carried out disclosure and description, be understood that, except as otherwise noted, they are not limited to specific synthetic method or specific reorganization biotechnological means, perhaps except as otherwise noted, they are not limited to concrete reagent, therefore naturally can change to some extent.It will also be appreciated that its purpose of term that this paper uses only is specific embodiments is described, and have no intention to limit.

One of ordinary skill in the art will recognize that and maybe can determine not only can use normal experiment, also can use the multiple equivalence of the particular of method and composition described herein.These equivalence are intended to include in following claim.

A. definition

Unless context spells out on the contrary, employed singulative in specification sheets and the appended claims " ", " a kind of " and " being somebody's turn to do " comprise that plural number refers to object.Therefore, for example, " a kind of pharmaceutical carrier " comprises the mixture of two or more these class carriers etc.

Scope can be expressed as from " approximately " concrete numerical value herein, and/or to " approximately " another concrete numerical value.When being expressed as this scope, another embodiment comprises from a described concrete numerical value and/or to described another concrete numerical value.Similarly, when by using " approximately " that numeric representation during as approximation, be should be understood that this concrete numerical value constitutes another embodiment in front.Should be understood that further that the end points of each scope is relevant with another end points and all be significant when being independent of another end points.It is to be further understood that many numerical value disclosed herein, and each numerical value is also open with " approximately " this concrete numerical value at this except numerical value itself.For example, if disclose numerical value " 10 ", " about 10 " are disclosed so also.It is to be further understood that when disclosing a numerical value, also disclose " being less than or equal to this numerical value ", possible scope between " more than or equal to this numerical value " and numerical value, this as those skilled in the art appropriate understand.For example, if disclose numerical value " 10 ", " being less than or equal to 10 " and " more than or equal to 10 " are disclosed also.Should also be understood that provides data with multiple different-format in whole the application, these are data represented end points and starting point, and the scope of any combination of these data points.For example,, should be appreciated that if disclose particular data point " 10 " and particular data point " 15 ", greater than, more than or equal to, less than, be less than or equal to, equal 10 and 15, and the numerical value between 10 and 15 is also thought and is disclosed.It will also be appreciated that each unit between two concrete units also is disclosed.For example, if disclose 10 and 15, so also disclose 11,12,13 and 14.

The reduction of " reduction " or other form refers to and reduces a certain incident or feature.It should be understood that this is usually relevant with some standard or expected value, in other words it is comparatively speaking, still is not that standard or correlation values to be referred to is necessary usually.For example, " reduce phosphorylation and " refer to the amount that reduces the phosphorylation that is taken place with respect to standard or contrast.Unless it should be understood that to specialize on the contrary that otherwise a certain compound or composition or illness can decrease with respect to another compound or composition or illness.

The inhibition of " inhibition " or other form means prevention or limits a certain concrete feature.It should be understood that this is usually relevant with some standard or expected value, in other words it is comparatively speaking, and is necessary usually but it is not standard or a correlation values to be referred to.For example " inhibition phosphorylation " refer to respect to standard or contrast stops or the amount of the phosphorylation that restriction is taken place.It should be understood that to deny non-ly to specialize on the contrary, otherwise a certain compound or composition or illness can be suppressed with respect to another compound or composition or illness.

The prevention of " prevention " or other form refers to and stops a certain concrete feature or illness.Owing to stop common ratio more absolute, so it does not need to compare with contrast as reduction or inhibition.Employed some things of this paper can be lowered but can not be suppressed or stop, but some things that can be lowered also can be suppressed or stop.Be understandable that when using reduction, suppress or stoping, opposite as if clearly not indicating, the use of two other word is also by open especially.Therefore, if disclose the inhibition phosphorylation, then also disclose and reduced and the prevention phosphorylation.

The amount that term " treatment effectively " refers to the composition that uses is to be enough to improve the one or more causes of disease of a certain disease or obstacle or the amount of symptom.This improvement only need reduce or change, and needn't eliminate.

Term " carrier " refers to, with regard to using intention or purpose, when mixing with a certain compound or composition, help described compound or preparation of compositions, storage, administration, send pass, validity, selectivity or any further feature or to its helpful compound, composition, material or structure.For example, can select, reduce to minimum and will reduce to minimum for any adverse side effect of experimenter with any degraded with activeconstituents to carrier.

In the entire chapter description and claims of this specification sheets, word " comprises " and the version of this word refers to " including but not limited to " as " comprising " and " containing ", has no intention to get rid of for example other additive, component, integer or step.

Term as used herein " cell " also refers to each microorganism cells or derives from the culture of this cell." culture " refers to the composition that comprises identical or different type isolated cell.

Term " meta-bolites " refers to is introducing a certain compound the reactive derivative that coenocorrelation produces for example in the patient time.

When referring to pharmaceutical composition and nutritive compositions, employed term " is stablized " and is understood that usually in this area: the loss of activeconstituents is less than a certain specified quantitative in designated period of time under the specific storage condition, and this amount is generally 10%.It is relevant with the purposes of every kind of product that composition is considered to stablize required time, and by produce this product, to its business practice regulation of carrying out quality control and inspection, it being flowed to the wholesale dealer or directly flowing to human consumer (carried out preservation once more this moment before its final use).The safety coefficient that comprises the some months time, the short sawn timber life-span of medicine is generally 1 year, and is preferred more than 18 months.Term as used herein " is stablized " with reference to those market truth and abilities of storing and transport this product under the envrionment conditions (for example 2 ℃ to 8 ℃ refrigerated condition) that realizes easily.

In claims of specification sheets and the end of writing in mentioned a certain composition or the article weight part of a certain concrete element or component represent: be expressed as the weight relationships between this element in the described composition of weight part or the article or component and any other element or the component.Therefore, in the compound that contains 2 parts by weight of component X and 5 parts by weight of component Y, the weight ratio of existing X and Y is 2:5, and no matter whether also contain other component in this compound, X and Y all exist with this ratio.

Unless spell out on the contrary, otherwise the weight percent of a certain component is based on the preparation that contains this component or the total amount of composition.

" isolating " and any form for example " are separated " and are referred to something and be in the situation that can be operated or be further purified form." isolating " and various synonym form thereof refer to something present located state and are different from residing state before.For example, if the ribosome-RNA(rRNA) molecule takes out, synthesizes or produce by recombination form from organism, then it is " isolating ".Usually, " separation " of a certain things is the separation with respect to other something.For example, as described hereinly can separate the eukaryote that this paper addresses in the following way:, thereby make this eukaryote under the situation that does not have other organism that to measure (can detect) amount, survive for example by cultivating eukaryote.Be understandable that unless specialize on the contrary, any disclosed composition can separate as disclosed herein.

" purifying " and any form for example " purifying " homogeneity of referring to a certain material or compound or composition state of living in than before higher state.Be understandable that, as disclosed herein, except as otherwise noted, the purity of something can be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23/24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.For example,, this means that then 90% of said composition is A, and said composition 10% is one or more things, for example molecule, compound or other material if the purity of a certain specified composition A is 90%.For example, if disclosed eukaryotic microorganisms for example produces 35%DHA, then this can be by further " purifying ", thereby makes final lipid composition have the DHA more than 90%.Except as otherwise noted, otherwise purity can determine by relative " weight " of component in the said composition.Be understandable that, unless specialize opposite, otherwise arbitrary disclosed composition purifying as disclosed.

" optional " or " randomly " refer to the incident described subsequently or situation whether occurs all can, and this narration comprises the situation and the absent variable situation thereof of wherein said incident or situation appearance.

" primer " is the such probe of a group: they can support certain fermentoid operation, and can hybridize with target nucleic acid, thereby the enzyme operation can be taken place.Primer can be by arbitrary combined preparation of not interferases operation, the obtainable nucleic acid in this area or nucleic acid derivative or analogue.

Many pieces of publications have been quoted in this part application.The disclosure of these publications is included among the application by quoting in full, and purpose is to describe more all sidedly its affiliated prior art.Disclosed reference is also included this paper in respectively and especially by quoting the content that just wherein contains, and described content is set forth in the sentence that this reference depended on.

The component that can be used for preparing disclosed composition is disclosed, and the composition self that can in disclosed method, use.These materials and other materials are disclosed in this article, and be understandable that, when disclosing the combination of these materials, subclass, interaction and group etc., although each that every kind of these compounds is different and whole combination and permutation may be open clearly, be all considered especially and described for every kind at this.For example, if disclose and discussed a certain concrete kind of thraustochytriale section, and the multiple modification that organism carried out to comprising that thraustochytriale section plants has been discussed, unless specialize on the contrary so, otherwise these kinds of thraustochytriale section and possible modification every kind and various combination and permutation are all considered especially.Therefore, if disclose molecule A, B and C, and molecule D, E and F are disclosed, the example of combination molecule A-D is disclosed simultaneously, then even without each particularize, each also is considered to significant combination, A-E separately and jointly, A-F, B-D, B-E, B-F, it is open that C-D, C-E and C-F are considered to.Equally, these any subclass or combination also are disclosed.Therefore, for example, the subgroup of A-E, B-F and C-E also is disclosed.This notion is applicable to all aspects of the application, includes but not limited to prepare and use the step in the disclosed method for compositions.Therefore,, should be understood that each step of described other steps can carry out with any specific embodiments or the combining of embodiment of disclosed method if there are a plurality of other steps to implement.

B. composition

The eukaryotic microorganisms thraustochytriale is disclosed, the genus thraustochytrium kind or the schizochytrium limacinum that preferably have the ability that produces lipid belong to (Schizochytrium) kind, described lipid is lipid acid for example, unsaturated fatty acids for example, as omega-fatty acid, as ω-6 lipid acid and ω-9 lipid acid, as the clupanodonic acid (DPA) of the docosahexenoic acid (DHA) of (n-3) series and timnodonic acid (EPA), (n-6) series and (n-9) serial palmitinic acid and stearic acid.Disclosed eukaryotic microorganisms also can produce antioxidant, such as but not limited to carotinoid compounds carotene (for example β-Hu Luobusu) and xenthophylls compound astaxanthin, zeaxanthin, canthaxanthin and myoxanthin.

The separation and the growth conditions of eukaryotic microorganisms are also disclosed.For example, the heterotrophic growth condition that produces disclosed lipid and antioxidant is disclosed respectively and jointly simultaneously.Therefore, by using the eukaryote of this uniqueness, can effectively produce (n-3) series DHA and/or (n-6) series DPA and/or the anti-oxidant compounds of carotenoid series and/or the anti-oxidant compounds of xenthophylls series, they can be used as or be used for dietetic product, foodstuff additive, medicine or industrial processes.

Disclose the composition that contains eukaryotic microorganisms, described eukaryotic microorganisms comprises genus thraustochytrium kind (example disclosed herein is the ONC-T18 bacterial strain, and its ATCC deposit number is PTA-6245) or is made up of it.

Be understandable that, also disclose the eukaryotic microorganisms that is expressed as ONC-T18 and from the isolating any clone of described organism, modified organism or gene.Disclosed organism has the ability of generation unsaturated fatty acids (as contain the DPA of the DHA of ω-3 series and EPA and ω-6 series lipid) and various antioxidant (as carotenoid, xenthophylls and aldehydes matter).

The method that produces the biomass that contains described compound is also disclosed.The method of utilizing eukaryotic microorganisms to prepare ω-3, ω-6 and carotinoid compounds is further disclosed.The method of producing the oil in microbe-derived (or unicellular source) is also disclosed.

In addition, the lipid acid and the carotenoid that are produced by disclosed eukaryotic microorganisms and any filial generation (genetically modified or otherwise modification) are disclosed, be supplemented with all feeds, dietetic product, medicine and the food of lipid and antioxidant, and with the method for these compounds as the additive of all feeds and food.

The U.S. Patent No. 5,130,242 that licenses to Barclay discloses a kind of collection and screening method, to separate the microorganism strains that produces omega-fatty acid and have following feature: 1) energy heterotrophic growth; 2) produce the high-content omega-fatty acid; 3) single celled; 4) that produce the low levels standard and ω-6 lipid acid; 5) white or the colourless cell of non-coloring; 6) heat-stable (as ability) in growth more than 30 ℃; With 7) have wide salt tolerance (as in the relative broad range salinity but the ability of preferably under low salinity, growing).

The disclosure of these ' 242 patents has also been described heterotrophism production and has been had the method for the full cell or the microbial product through extracting of high density omega-fatty acid, and described full cell or microbial product can be used for animal-derived food product or people's food subsequently.This method has been used the microorganism of identifying by the collection of this patent disclosure and screening method.These microorganisms are thraustochytriale, cultivate in ground cereal.For improving the generation of omega-fatty acid, use low temperature stress (stressing) and high-solubility oxygen, add antioxidant, somatomedin, VITAMIN and phosphorus simultaneously.The product that extraction obtains contains the omega-fatty acid (as C20:5w3, C22:5w3 and C22:6w3) of high density and ω-6 lipid acid (as C20:4w6 and C22:5w6) of lower concentration.Particularly, the ratio of C20:5w3 and C22:6w3 lipid acid is 1:1 to 1:30.To be 1:12 only be the C22:5w3 of trace to minimum to the ratio of C22:5w3 and C22:6w3 lipid acid.Equally, this microorganisms accounts for 0.6 to 0.72% DHA, 0 to 5% the DPA of total fatty acids and 0 to 18.9% EPA by weight.

The U.S. Patent No. 6,451,567 of Barclay discloses and has contained the no chlorine substratum of sodium salt (as the sodium sulfate) (＜method of cultivating thraustochytriale and schizochytrium limacinum in 3g/L).The resulting cell mass size of this no chlorine substratum is less than 150 μ m.Disclosed method can be produced microorganism and the extract that is used for used for aquiculture food.Other component of this food comprises linseed oil, Semen Brassicae campestris, soybean and avocado powder.This microorganism producible omega-fatty acid every day is the 1.08g/L substratum.Disclosure that should ' 567 has further described various substratum, and described substratum comprises that seawater, glucose (1,3,5 or 10g/L), yeast extract (0.01,0.2,0.4 and 5g/L), other nitrogenous source (for example protein hydrolysate (1g/L), LEx (1g/L), L-glutamic acid (5g/L), MSG (3g/L)) and other salt, trace VITAMIN and mineral substance are (as KH ₂PO ₄, MgSO ₄And gelatin extract).

People's such as Yokochi U.S. Patent No. 6,582,941 disclose schizochytrium limacinum belongs to kind of bacterial strain SR21 and another schizochytrium limacinum bacterial strain that belongs to same kind, and described kind has the ability that produces the fatty acid component that contains high density ω-3DHA and/or ω-6DPA and lower concentration EPA.Simultaneously, this quasi-microorganism and the method for separating this class lipid acid of cultivating disclosed.Employed substratum contains sea salt, yeast extract (0.2,1.0 or 10g/L), corn steep liquor (0.5,1.0 or 10g/L), glucose (10-120g/L) and other salt (NH for example ₄OAc, phosphoric acid salt).The DHA that this fatty acid composition contains accounts for about 15 to 20% (account for by weight total fatty acids about 28%) of biomass by weight.Said composition can be used in the food (as infant formula).

People's such as Bailey U.S. Patent No. 6,607,900 disclose the method for cultivation eukaryotic microorganisms (as schizochytrium limacinum bacterial classification ATCC No.20888), and many unsaturated lipids that described eukaryotic microorganisms can produce (especially ω-3 and-6 lipid acid) account at least 20% of its biomass.This method is included in and cultivates this microorganism in the substratum of carbonaceous sources and nitrogenous source.Use low dissolved axygen level (less than 3%) is also disclosed and low chlorine ion level (less than 3g/L) improves productive rate.The lipid productive rate that this microorganism has is 0.5g/L/h at least.The DHA of lipid composition accounts for 15% to 20% (account for by weight total fatty acids methyl esters about 35%) of biomass by weight.

People's such as Komazawa U.S. Patent Application Publication text No.2004/0161831 discloses the thraustochytriale bacterial strain (LEF1, ATCC No.FERM BP-08568) with the ability that produces DHA.By cultivating in conventional substratum, this microorganism can produce has by weight the grease of 50%DHA at least.This grease can be handled by lipase, separates DHA then.This grease can be used in food or the drink, and perhaps hydrolyzable DHA produces mountain acid.

1. lipid acid

Lipid acid is the hydrocarbon chain that has carboxylic group with end, if they contain at least one carbon-carbon double bond, then is called as undersaturatedly, then is called as polyunsaturated when they contain a plurality of so two key.Because the position of these pairs key, long chain polyunsaturated fatty acids (PUFA) or highly unsaturated fatty acids (HUFA) can be divided into (n-3) and (n-6) series.A large amount of scientific evidence show, (n-3) highly unsaturated fatty acids for example DHA cardiovascular disorder, chronic inflammatory diseases and cerebral disorders are had active effect.On the other hand, (n-6) lipid acid is considered to for example mesostate of prostaglandin(PG), leukotriene etc. of 20 acids sterol.

Polyunsaturated fatty acid can comprise 1,2,3,4,5,6,7,8,9 or 10 carbon-carbon double bond and/or carbon carbon triple bond.For example, polyunsaturated fatty acid can comprise 3-8,4-7 or 5-6 carbon-carbon double bond and/or carbon carbon triple bond.

At present, the main source of these highly unsaturated fatty acidss is fishes, has noticed that the amount of multiple mazarine fish (for example sardines and tuna) intravital DHA and EPA is respectively about 20% and 10%.Yet, if will use fish oil to remain in some shortcomings, for example taint of odour, aspect availability, exist uncontrollable fluctuation, natural fish oil content there are differences and may accumulate problem such as hostile environment pollutent as unique source of these lipids.In addition, if want to obtain highly purified (n-3) or (n-6) oil from described source, preferentially separate and purifying very difficult.Particularly, emerging high density form DHA has good prospect on newborn infant's tonic market.If fish oil is the source of described product, preferentially a large amount of DHA of separation from EPA so have to.It should be apparent that the source that needs highly purified other source and produce these specific highly unsaturated fatty acidss.

Except fish oil, various microorganisms (mainly being marine products) can produce and/or accumulate the docosahexenoic acid of (n-3) series.Especially meaningfully such fact: microorganisms producing can not be limited by by the external world and change the fluctuation that for example season, temperature and food supply etc. cause.For example, known following microorganism has the ability that produces DHA: from bacterium vibrio marinus (Vibriomarinus) (ATCC 15381), vibrios kind (Vibrio sp.) T3615, deep-sea bacterium (Photobacterium profundum) SS9, ocean mortierella (Mortierella marina) MP-1 and the Psychromonas kaikoae (ATCC BAA-363T) at deep-sea; For example Kou Shi Crypthecodinium cohnii (Crypthecodinium cohnii), concealed little ring algae (Cyclotella cryptica) and Mortierella alpina (Mortieralla alpina) be (1S-4) for little algae kind; And protobiont genus thraustochytrium kind (ATCC 20892), golden yellow thraustochytriale (Thraustochytrium aureum) (ATCC 34304) and pink thraustochytriale (Thraustochytrium roseum).But according to the method for utilizing these purified organisms, the amount of the docosahexenoic acid of every gram biomass/every liter of generation is lower, between 10 to 500mg.The organism of some examples and representational generation microbial oil is shown in Fig. 5.

Shown that omega-3 fatty acid has beneficial effect, and oil disclosed herein and composition can be used with regard to effect below it: to cystic fibrosis (Cochrane Database SystRev.3), rheumatoid arthritis (Drugs 63:845-53), asthma (Ann AllergyAsthma Immunol 90:371-7) and thrombotic apoplexy (Prev Cardiol 6:38-1), the antiinflammation of Cardioprotective aspect, and to the direct effect of atherosclerosis and irregular pulse (Prostaglandins Leukot Essent Fatty Acids.63:351-62), cancer inhibition of proliferation and reduce cancer cells (Am J Clin Nutr 77:532-43) in experimentation on animals in breast cancer cell line and prostate cancer cell line is to the antipsycholic action of schizophrenia (J NeuralTransm Suppl 64:105-17) and other psychosis (Can J Psychiatry 48:195-203); Can be used for the normal newborn growth and be used for the treatment of infection of newborn (Eur JPediatr 162:122-8) immunity nourishment additive, and it can be used for the treatment of pathological pain (Prostaglandins Leukot Essent Fatty Acids 68:219-24) by directly weakening the neurone pathology and the neuroganglion pathology that cause neuropathic pain and inflammatory pain.

2. thraustochytriale section

a)ONC-T18

Sea hydrobiont ONC-T18 disclosed herein is collected as the part of the microorganism segregating population (trip) that produces PUFA, has separated the pure growth more than 60 kinds from this colony, in detail referring to table 7.In addition, from the Advocate Harbor of Nova Scotia, Canada, also be separated to ONC-T18 in the sabkha blade of grass sheet of Bayof Fundy.By micrography and serial culture purified technology, this bacterial strain is considered to belong to the single microorganism of genus thraustochytrium.All bacterial strains and two ATCC compare culture (ATCC 20891 ﹠amp; MYA-1381) all growths in containing the seawater (SW) of 0.5% glucose, 0.2% peptone, 0.2% yeast extract, and (fatty acid methyl ester FAME) is analyzed to carry out GC.

Fig. 6 illustrates the relational system tree of inferring between ONC-T18 and other and its closely-related organism.

Use classical pine tree pollen to lure collection (pine pollen baiting) technology, on the selection substratum, cultivate then, separated ONC-T18 as single microorganism at first.Particularly, preparation contains 5g L ^-1Glucose, 2g L ^-1Peptone, 2g L ^-1The nutritional medium of yeast extract (in 1 liter of 0.2 filtering seawater of μ m).Use Bligh and Dyer extraction method and PUFA gas chromatographic technique to determine the lipid acid condition of production of ONC-T18 then.Chromatogram result confirms that this bacterial strain can produce TFA, the DHA of increasing amount and the EPA and the DPA of significant quantity.

As described herein, disclosed eukaryotic microorganisms can be used for preparing in the method for the lipid that comprises DHA, EPA and DPA or fat, but be not limited to above-mentioned ONC-T18 or PTA-6245 bacterial strain, but comprise any derivative of described bacterial strain, no matter this derivative is to obtain by genetic modification, chemomorphosis, fermentation adjusting or any alternate manner that produces the mutant of described bacterial strain, and have the feature of the heredity similar or form and function thus to eukaryotic microorganisms through the product of these modifications, include in above-mentioned open scope.

Disclose and to have produced the eukaryotic microorganisms of lipid composition, and disclose stable, the reliable and economic technical scheme of coming source problem of this lipid of keeping high functionalized and other value of having of this eukaryotic microorganisms with unique lipid category feature.Therefore, herein disclosed is and producing more (n-3) serial DHA and (n-6) wild type strain of serial DPA, and be designed at mutation that produces these polyunsaturated fatty acids to a greater extent and recombinant bacterial strain.This class mutation or recombinant microorganism comprise and be designed to have following feature: when using the same terms and culture medium culturing, those lipid contents that the more original wild type strain of the described lipid content that they have produces are higher.In addition, also comprise have following feature can be screened microorganism: they are compared with corresponding wild type strain and can produce (n-3) the serial DHA, the EPA that contain analog quantity and (n-6) lipid of serial DPA, but described microorganism can effectively be used the substrate with higher price-performance ratio.

Disclose and comprise eukaryotic microorganisms thraustochytriale purpose composition, wherein said eukaryotic microorganisms can produce unsaturated fatty acids.This polyunsaturated fatty acid can be for example ω 3 or ω 6 lipid acid, for example DHA and DPA.

Disclose the composition that comprises eukaryote, wherein said composition can produce lipid.

Also disclose a based composition, lipid wherein comprises lipid disclosed herein.

Also disclose a based composition, wherein said eukaryote comprises a plurality of thraustochytriale.

Also disclose a based composition, 18S ribosomal rna gene sequence that wherein said eukaryote has and SEQ ID NO:1 have at least 80% identity.

Be understandable that, any type of feature described herein, for example genetics of eukaryotic microorganisms, lipid feature or classification all can be used for characterizing microorganism disclosed herein.Eukaryotic microorganisms can comprise one or more microorganisms of thraustochytriale section, and their example is that ATCC is numbered 20888,20889,20890,20891 and 20892 microorganism.The relevant feature of unsaturated fatty acids much spendable, that produce with this organism and their is arranged.Be understandable that, for example, these features can any combination or spread pattern be used to limit organism, oil or the antioxidant of one or more series.For example, classification, the genetics identity of this organism, the lipid of this organism and growth conditions of the antioxidant condition of production and this organism that feature is this organism self.

B) classification

Eukaryotic microorganisms can be from net slime mould door (phylum Labyrinthulomycota).Eukaryotic microorganisms can be from net slime mould guiding principle (class Labyrinthulomycetes).Eukaryotic microorganisms can be from thraustochytriale subclass (subclass Thraustochytridae).Eukaryotic microorganisms can be from thraustochytriale (order Thraustochytriales).Eukaryotic microorganisms can be from thraustochytriale section (family Thraustochytriaceae).Eukaryotic microorganisms can be from genus thraustochytrium (genus Thraustochytrium).Eukaryotic microorganisms can be the genus thraustochytrium kind.Eukaryotic microorganisms can be golden yellow thraustochytriale (Thraustochytrium aureum).Eukaryotic microorganisms can be pink thraustochytriale (Thraustochytrium roseum).Eukaryotic microorganisms can be line shape thraustochytriale (Thraustochytrium striatum).Eukaryotic microorganisms can be schizochytrium limacinum and belongs to (genusSchizochytrium).Eukaryotic microorganisms can be the schizochytrium limacinum genus and plants.Eukaryotic microorganisms can be the modified forms of listing eukaryotic microorganisms above any.Eukaryotic microorganisms also can comprise any present the unknown or the not separated member of described prokaryotic organism guiding principle, order, section or genus.The combination of eukaryotic microorganisms can be any combination of any organism disclosed herein, comprises that one or more genus thraustochytrium kinds, schizochytrium limacinum belong to kind, golden yellow thraustochytriale, line shape thraustochytriale and pink thraustochytriale.

Eukaryotic microorganisms from thraustochytriale section can be any above those disclosed eukaryotic microorganisms.Eukaryotic microorganisms can comprise that ATCC is numbered the organism of PTA-6245.

C) genetics

Eukaryotic microorganisms can have 18S rRNA sequence SEQ ID NO:1.Eukaryotic microorganisms can have with SEQ ID NO:1 and have about 90% the homology or the 18S rRNA sequence of any other identity disclosed herein.Eukaryotic microorganisms can have the 18S rRNA sequence of hybridizing with the part of SEQ ID NO:1 or SEQ ID NO:1 under stringent condition or any other condition disclosed herein.

Sequence similarity/the identity of organism nucleic acid and nucleic acid hybridization can be as described herein.Particularly, use BLAST (basic part comparison research tool) algorithm with SEQ ID NO:1 and in genome database GenBank (American National biotechnology information center, national sanitary institute, Bei Saisida, Maryland, the U.S.) nucleotide sequence in is compared, SEQ ID NO:1 is accredited as relevant with several eucaryon genus thraustochytrium kinds (91% similarity), with genus thraustochytrium kind (thraustochytrium sp.) CHN-I[AB126669] (94.5% similarity) and thraustochytriale section bacterial classification (thraustochytriidae sp.) N1-27[AB073308] (95.5% similarity) closely related, and with line shape thraustochytriale (thraustochytrium striatum) [AF265338] the most closely related (97.5% similarity).

3. (1) sequence similarity

Be understandable that as described herein, the use of term homology and identity means with similarity to have identical implication.Therefore, for example, be understandable that then this might not indicate the evolutionary relationship between these two sequences, but be conceived to similarity or sibship between its nucleotide sequence if between two non-natural sequences, use the word homology.The method of the homology between many definite two evolution associated molecules can be applied to any two or more nucleic acid or albumen in a usual manner, and purpose is to measure sequence similarity, and no matter whether they relevant on evolving.

Generally speaking, be understandable that a kind of method of determining gene disclosed herein and proteinic any known variant and derivative, those variants that maybe may occur and derivative is by limiting variant and derivative according to the homology with specific known array.This identity of concrete sequence disclosed herein is also set forth in other place of this paper.Generally speaking, nucleic acid disclosed herein and proteinic variant usually and specified sequence or native sequences have homology at least about 50,55,60,65,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98 or 99%.Those skilled in the art will readily understand and how to determine for example homology of gene of two albumen or nucleic acid.For example, can after two sequences are compared, calculate homology, thereby make homology be in highest level.

Calculating the another kind of method of homology can be undertaken by disclosed algorithm.The best comparison that is used for the sequence of comparison can be carried out by the following method: the algorithm (Adv.Appl.Math.2:482 of local homology of Smith and Waterman, 1981), homology alignment algorithm (the J.MoI.Biol.48:443 of Needle and Qunsch, 1970), similarity searching method (the Proc.Natl.Acad.Sd.USA 85:2444 of Pearson and Lipinan, 1988), (the GAP in the Wisconsin Genetics software package is carried out in the computerize of these algorithms, BESTFIT, FASTA and TFASTA, Genetics Computer Group, 575 Seience Dr., Madison, WT) or check.

This homology of nucleic acid can be by for example disclosed algorithm acquisition: Zuker in following document, M.Science 244:48-52,1989, Jaeger et al.Proc.Natl.Acad.Sci.USA 86:7706-7710,1989, Jaeger et al.Methods Enzymol.183:281-306,1989, include these documents in this paper by reference with regard to its content relevant at least with the nucleic acid comparison.Be understandable that, any method all can be used usually, and in some cases, the possibility of result of these different methods is different, but it will be appreciated by those skilled in the art that, if this at least a method wherein of use has obtained identity, think that then this sequence has described identity, and be disclosed in this article.

For example, this paper uses the sequence that is considered to have with another sequence the particular percentile homology to refer to such sequence: it has the described homology that calculates by above-mentioned any or multiple method of calculation.For example, if use Zuker method of calculation calculate first sequence and second sequence has 80% homology, even if calculate by any other method of calculation so and find not 80% homology of described first sequence and described second sequence, described first sequence and described second sequence also have described herein 80% homology.As another example, if use Zuker method of calculation and Pearson and the Lipman method of calculation calculate first sequence and second sequence has 80% homology, even if calculate by Smith and Waterman method of calculation, Needleman and Wunsch method of calculation, Jaeger method of calculation or any other method of calculation so and find not 80% homology of described first sequence and described second sequence, described first sequence also has described herein 80% homology with described second sequence.As another example; have 80% homology (although different in practice method of calculation can calculate different percent homology usually) if use each method of calculation to calculate first sequence with second sequence, then described first sequence and described second sequence have described herein 80% homology.

(2) hybridization/selective cross

Term hybridization is often referred to for example interaction that drives of the sequence between primer or probe and the gene of at least two nucleic acid molecule.The interaction that sequence drives refers to the interaction that occurs in the special mode of Nucleotide between two Nucleotide or nucleotide analog or nucleotide derivative.For example, G and C interaction or A and T interaction is exactly the interaction that sequence drives.Usually, the interaction of sequence driving appears at the Watson-Crick interface or the Hoogsteen interface of Nucleotide.The hybridization of two nucleic acid is subjected to a plurality of conditions known to those skilled in the art and the influence of parameter.For example, whether the salt concn of reaction, pH and temperature all can have influence on two nucleic acid molecule and can hybridize.

The parameter of selective cross is well known to those skilled in the art between two nucleic acid molecule.For example, in certain embodiments, the selective cross condition can be defined as stringent hybridization condition.For example, the strict degree of hybridization is controlled by one of hybridization and washing step or both temperature and salt concn.For example, the hybridization conditions that realizes selective cross can comprise: at high inonic strength solution (among 6 * SSC or 6 * SSPE) in than T _mHybridize under the temperature that (melting temperature(Tm) is having half molecule and its hybridization companion to dissociate under this temperature) is low about 12-25 ℃, under the combination of chosen temperature and salt concn, wash then, so that wash temperature is than T _mLow 5-20 ℃.Temperature and salt condition determine by experience in preliminary experiment easily, in this preliminary experiment, is fixed on hybridizing with reference to the dna sample and the institute labeling nucleic acid of studying on the filter, and washs under different stringent conditions subsequently.For DNA-RNA and RNA-RNA hybridization, hybridization temperature is higher usually.As mentioned above, this condition can be used for realizing strict degree, perhaps is known in the art (Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, ColdSpring Harbor, New York, 1989; Kunkel et al.Methods Enzymol.154:367,1987, include it in this paper with regard to its content relevant by quoting at least) with nucleic acid hybridization.The preferred stringent condition of DNA:DNA hybridization be at about 68 ℃ (in aqueous solution) in 6 * SSC or 6 * SSPE, then 68 ℃ of washings.If need, the strict degree of hybridization and washing can the corresponding reduction with the minimizing of required complementary degree, and depends on the G-C in the searched zone of wherein any variability or the enrichment of A-T.Equally, if need, the strict degree of hybridization can the corresponding reduction with the minimizing of required homology, and depends on the wherein any G-C in high homology zone or enrichment of A-T of needing, and these are all known in the art.

The another kind of method of definition selective cross is by checking a kind of nucleic acid and another kind of nucleic acid bonded amount (per-cent).For example, in certain embodiments, the selective cross condition is meant the condition when combining with quantity-unlimiting nucleic acid at least about 60,65,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,91,98,99,100% the nucleic acid of limiting the quantity of.Usually, quantity-unlimiting primer for for example 10,100 or 1000 times excessive.This alanysis can be carried out under the following conditions: wherein limit the quantity of and quantity-unlimiting primer than its K _dLow for example 10,100 or 1000 times, perhaps wherein only a kind of nucleic acid molecule is 10,100 or 1000 times, and perhaps one or both nucleic acid molecule are higher than its K _d

The another kind of method of definition selective cross is under needs are hybridized with the condition that promotes required enzyme operation, checks the per-cent of primer realization enzyme operation.For example, in certain embodiments, the selective cross condition is the condition when realizing the enzyme operation at least about 50,55,60,65,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100% primer under the condition of promotion enzyme operation; For example, extend if enzyme operation is DNA, the selective cross condition is the condition when being extended at least about 50,55,60,65,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100% primer molecule so.Preferred condition comprises that also those conditions that manufacturers advises or this area think those conditions that enzyme is operated that are fit to.

As the situation of homology, be understandable that, herein disclosed is the multiple method of determining hybridization level between two nucleic acid molecule.Be understandable that these methods and condition can provide different hybridization per-cent between two nucleic acid molecule, but except as otherwise noted, otherwise the parameter that satisfies arbitrary method gets final product.For example, if need 80% hybridization, and, think that then it is by disclosed herein as long as hybridization occurred in the required argument scope in this any method wherein.

Be understandable that, it will be understood by those skilled in the art that if a certain composition or method satisfy together or separately these standards of determining hybridization arbitrary standard, then said composition or method are disclosed herein.

D) composition of the molecule that is produced

Be understandable that eukaryote disclosed herein can produce multiple compound and composition.These compounds and composition can be used as identifier, promptly a kind of mode of identification of organism body.For example, a kind of method that characterizes a certain organism is the condition of production that produces lipid by this organism.As described herein, these different lipid situations both can be used for characterizing this organism, also can be used for carrying out purifying, operation and collection with regard to various reasons.

(1) lipid

Be understandable that every kind of organism can produce some condition of production of unsaturated fatty acids disclosed herein.These conditions of production are the feature of this organism.Be some examples of the unsaturated lipids condition of production He other lipid production situation of this organism below.

For example, eukaryotic microorganisms can produce and account for lipid or the fatty acid component of stem cell biological amount at least about 4wt% to 6wt% (5wt% according to appointment), and described lipid or fatty acid component comprise the myristic acid (according to appointment 1wt%) of about 0wt% to about 2wt%, about 16wt% is to the palmitinic acid of about 20wt% (18wt% according to appointment), about 0wt% is to the Zoomeric acid of about 2wt% (1wt% according to appointment), about 4wt% is to the stearic acid of about 8wt% (6wt% according to appointment), about 30wt% is to the oleic acid of about 34wt% (32wt% according to appointment), about 40wt% is to the linolic acid and the n-3 EPA of about 0wt% to about 3wt% (2wt% according to appointment) of about 44wt% (42wt% according to appointment).

For example, eukaryotic microorganisms also can produce and account for lipid or the fatty acid component of stem cell biological amount at least about 1wt% to 3wt% (1.25wt% according to appointment), and described lipid or fatty acid component comprise the myristic acid of about 2wt% to about 4wt% (3wt% according to appointment), 50wt% is to the palmitinic acid of about 60wt% (55wt% according to appointment), about 2wt% is to the Zoomeric acid of about 4wt% (3wt% according to appointment), about 16wt% is to the stearic acid of about 20wt% (18wt% according to appointment), about 9wt% is to the oleic acid of about 13wt% (11wt% according to appointment), about 1wt% is to the eicosadienoic acid and the n-3 EPA of about 6wt% to about 10wt% (8wt% according to appointment) of about 3wt% (2wt% according to appointment).

Eukaryotic microorganisms for example ONC-T18 can produce and accounts for the lipid composition of stem cell biological amount at least about 30wt%, 40wt%, 50wt%, 60wt%, 70%wt% or 80wt% (80wt% according to appointment).For example, eukaryotic microorganisms can produce lipid composition, and ω-6 lipid acid that described lipid composition comprises about 25% to about 40% omega-fatty acid such as n-3DHA (for example by weight at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60%), about 0% to about 3% omega-fatty acid EPA (for example by weight at least 1% or 2%) and about 4% to about 12% is n-6DPA (for example by weight at least 4%, 5%, 6%, 7%, 8%, 9% or 10%) for example.

Be understandable that the growth conditions that can exist based on this eukaryotic microorganisms is handled the composition of the lipid that produced by eukaryotic microorganisms.Can handle composition by changing each parameter disclosed herein, to produce DHA or the DPA that for example productive rate is higher.For example, operation can not produce more actual gram number, but this operation can produce the ratio of better DHA or DPA and EPA and other required PUFA, and from the angle of purifying, this ratio is required.This paper has discussed multiple treatment condition.

Figure 10 illustrates the possible pathways metabolism of various PUFA that is produced by disclosed eukaryotic microorganisms, and this is consistent with fatty acid methyl ester meta-bolites tracking result.Use for example degenerated primer research (Metz et al. (2001) Science 293:290-3 and Kaulmann ﹠amp; Hertweck (2002) Angew.Chem.Int.Ed.41:1866-9), in approach disclosed herein, can certified protein be polyketide synthases.Use for example hybridization probe, also can identify and extend enzyme and desaturase.Use for example hybridization probe and/or degenerated primer research, also can identify fatty acid synthetase.

4. growth and cultivation

Carried out comprising phenotype microwell plate (phenotypic microplate) research of carbon, nitrogen (peptide nitrogen), p and s, osmoticum and pH.

Orthogonal array (Taguchi) method is used for determining changing conditions (the Joseph J ﹠amp of optimal medium composition and nitrogen, carbon and salt concn; Piganatiells JR (1998) IJE Trans, 20:247-254).

Stir or dO if increase ₂, then can increase biomass yield and TFA, but reduce DHA.Stir or dO if reduce ₂, then can reduce cellular biomass (g), and reduce TFA, but increase DHA, and can reduce C16:0, C16:1 and C18:1.

If elevated temperature then can increase biomass yield and TFA, but reduce DHA.If reduce temperature, then can reduce cellular biomass (g), and reduce TFA, but increase DHA, and can reduce C16:0, C16:1 and C18:1.

The cellular biomass that derives from disclosed eukaryotic microorganisms can obtain by the suitable natural or artificial seawater substratum (containing about 2% to about 100% seawater) of inoculation.This eukaryotic microorganisms can utilize the various nutrition compositions in this substratum.The example of employed carbon source is a for example glucose of carbohydrate in this substratum; fructose; dextrose (dextrose); lactulose; semi-lactosi; trisaccharide maltose; maltose; lactose; glycogen; gelatin; starch (corn or wheat); and sugar derivatives such as acetate; m-inositol (deriving from corn steep liquor); galacturonic acid (deriving from pectin); L-Fucose (deriving from semi-lactosi); gentiobiose; glycosamine; alpha-D-glucose-1-phosphoric acid salt (deriving from glucose); cellobiose (deriving from Mierocrystalline cellulose); dextrin (deriving from corn) and alpha-cylodextrin (deriving from starch); with polyvalent alcohol maltose alcohol for example; erythritol; the pure and mild oleic acid of adonose is glycerine and tween 80 and aminosugar N-ethanoyl-D-galactosamine for example for example; N-ethanoyl-D-glycosamine and N-ethanoyl-β-mannosamine.And the example of nitrogenous source is natural nitrogenous source such as peptone, yeast extract, malt extract and fish meal, perhaps is for example Sodium Glutamate of organic nitrogen source, but is not limited thereto.In addition, if it is necessary, phosphoric acid salt for example potassiumphosphate and sodium phosphate, inorganic salt for example ammonium sulfate, sodium bicarbonate, sodium orthovanadate, potassiumchromate, Sodium orthomolybdate, Sodium Selenite, single nickel salt, copper sulfate, zinc sulfate, cobalt chloride, iron(ic) chloride, Manganous chloride tetrahydrate and calcium chloride can be only with the sequestrant ethylenediamine tetraacetic acid (EDTA) as micro-nutrient, perhaps with this sequestrant and VITAMIN for example vitamin B6, VITMAIN B1, calcium pantothenate, vitamin Bx, riboflavin, nicotinic acid, vitamin H, folic acid and vitamin B12 as micro-nutrient.After preparing substratum, use acid or alkali with pH regulator to 3.0 between 10.0, when appropriate, for example be adjusted between the pH4.0 to 6.5, and substratum sterilized by for example autoclaving.In temperature is between 4 to 30 ℃, preferred 18 to 28 ℃, by ventilation shaking culture, shaking culture, static cultivation, batch culture, cultured continuously, rolling batch culture or fluctuation cultivation etc., cultivation was carried out 1 to 30 day, 1 to 21 day, 1 to 15 day, 1 to 12 day, 1 to 9 day or preferred 3 to 5 days.

Following condition is the example that can produce the condition of a series of lipids, and described lipid can be used as commodity aspect output.To studies show that of the culture condition of ONC-T18, eukaryotic microorganisms disclosed herein is at natural or artificial seawater or containing well-grown in the substratum that concentration is low to moderate 5% natural or artificial seawater most.As mentioned above, carbon source and the nitrogenous source that adds in this substratum can be normally used those carbon sources and nitrogenous source.Natural nitrogenous source or organic nitrogen source are equivalent comparatively speaking, and the total nitrogen concentration in this substratum keeps constant.These carbon sources or nitrogenous source are added in the substratum with normal concentration.As disclosed herein, if satisfy these conditions, then can be influential slightly to ratio or the amount of lipid content, accumulation DHA, DPA and EPA.

For high gravity fermentation ONC-T18, can use several method to increase cellular biomass and lipid productive rate.These methods comprise that respectively both are respectively from 5g L with the concentration of carbon in the substratum and nitrogen (both ratios are that temperature is between 4 to 30 ℃, preferred 18 to 28 ℃ between 6:1 to 15:1, the preferred 6:1 to 13:1) ^-1To 60g L ^-1Be increased to 100g L ^-1To 160g L ^-1With from 4g L ^-1To 10g L ^-1Be increased to 40g L ^-1To 60g L ^-1Make in this way, the biomass that is produced and the ratio of lipid also increase with corresponding horizontal.In addition, by with carbon source from 5g L ^-1To 60g L ^-1Be increased to 100g L ^-1To 160g L ^-1And nitrogenous source remains unchanged, and can increase lipid output.In addition, by with the amount of nitrogenous source from 10g L ^-1Be increased to 60g L ^-1And carbon source remains unchanged, and can increase biomass yield, keeps lipid content simultaneously.In addition, experiment shows biomass and lipid output, and (scope is between 100 to 1000rpm with stirring, better between 350 to 600rpm, and best between 350 to 450rpm) quickening and improve greatly, and lipid content only has MIN reduction, the lipid acid condition of production does not reduce, and it is particularly useful in early days to be stirred in heterotrophic fermentation.Experiment shows that also the dissolved oxygen content in the substratum can obtain optimum lipid output between 1 to 10%, preferably at 5% o'clock.At last, acetate, trace element, metal and VITAMIN added to produce substratum (as mentioned above) and increased DHA, EPA and DPA output, and do not reduce the TL value with respect to other lipid acid.

By carrying out above-mentioned heterotrophic fermentation, can continue to produce cellular biomass, described cellular biomass can produce the lipid that contains (n-3) serial DHA, and described DHA is higher in the concentration of substratum, is not less than 5g/ and rises substratum, more preferably is not less than 20g/ and rises substratum.In addition, experiment shows after reaching the maximum biomass level, have major part to accumulate in these lipids during the logarithm late period/transitional period of cultivating.Yet during the fermentation, lipid content can not be lower than 25% of total biomass usually, is about 80% usually to the maximum.Cultivation under the above-mentioned condition can use conventional stirred fermentor to carry out.Also can use bubble column fermentor (cultivating in batch or cultured continuously) or fluctuation fermentor tank.

Carry out the lipid separation collection of cellular biomass before and can use various ordinary methods for example centrifugal (as centrifugal solid-liquid separating machine (solid-ejecting centrifuge)) or filtration (for example cross-flow filtration) to carry out, and comprise the precipitation agent (for example sodium phosphate, sodium-chlor or polyacrylamide (polyacridamide)) that uses acceleration collecting cell biomass.

5. the separation of lipid

Fig. 7 illustrates lipid and the DHA condition of production figure that changes with each parameter.All these data can be used for obtaining the concrete feature about the ONC-T18 eukaryotic microorganisms.Figure 13 illustrates the lipid acid condition of production figure of disclosed Eukaryotic cardinal principle.

Contain (n-3) serial DHA and (n-6) fat of serial DPA can obtain as follows: for example by mill, supersound process is destroyed or the cellular biomass that breaks and collect, uses solvent for example chloroform, hexane, methyl alcohol, ethanol or extract by supercutical fluid extracting mode then.Resulting containing (n-3) serial DHA and (n-6) fat of serial DPA preferably be higher than 0.25g at the content of every gram stem cell biological amount, more preferably be higher than 0.6g.

For example ONC-T18 can produce disclosed eukaryotic microorganisms as the lipid of above-mentioned acquisition, and the various mutation of this eukaryotic microorganisms and the combination of each mutation can produce the lipid with following composition situation.The per-cent of neutral lipid is at least 95% of TL by weight.Eukaryotic microorganisms is lipid acid typical composed as follows in the neutral lipid that produces of ONC-T18 for example: 15% myristic acid, 8% pentadecanoic acid, 35% palmitinic acid, 7% Zoomeric acid, 1% stearic acid, 2% oleic acid, 1% timnodonic acid, 6% clupanodonic acid and 25% docosahexenoic acid (GC composes and is shown in Fig. 1).

For example ONC-T18 can produce disclosed eukaryotic microorganisms as the lipid of above-mentioned acquisition, and the various mutation of this eukaryotic microorganisms and the combination of each mutation can produce the lipid with following composition situation.The per-cent of monoglyceride, triglyceride and triglyceride level is respectively 0% to about 2%, 0 to about 2% and 96 to about 100% in the neutral lipid component of ONC-T18.Comprise and combining with neutral lipid and not bonded phosphatidylcholine, phosphatidylserine and phosphatidic acid (phosphotidic acid) with it and account for the polar lipid component of lipid composition 5% to about 10%.

Be understandable that, these lipids can any combination or arrangement be present in these organisms.Will also be appreciated that as mentioned above the concentration of these lipids can be controlled by changing growth conditions and culture medium condition.

(1) lipid concentration

Eukaryotic microorganisms can produce and contain more than or equal to about 4.0g L ^-1N-3 DHA, the EPA of substratum and the lipid composition of n-6 DPA.Eukaryotic microorganisms can produce and contain more than or equal to about 20.0g L ^-1N-3 DHA, the EPA of substratum and the lipid composition of n-6 DPA.Eukaryotic microorganisms can produce and contain more than or equal to about 14.0g L ^-1N-3 DHA, the EPA of substratum and the lipid composition of n-6 DPA.Eukaryotic microorganisms can produce about 1.5g L ^-1To about 5.0g L ^-1(4.6g L according to appointment ^-1) n-3 DHA, about 0.5g L ^-1To about 1.5g L ^-1(0.22g L according to appointment ^-1) n-3 EPA and about 0.5g L ^-1To about 1.5g L ^-1N-6 DPA.In addition, eukaryotic microorganisms can produce between 301.2 to 360.3mg g ^-1Even be up to 790mg g ^-1Cellular biomass, contain the lipid composition of following material: myristic acid, Semen Myristicae diluted acid, pentadecanoic acid, palmitinic acid, Zoomeric acid, stearic acid, oleic acid, linolic acid, eicosadienoic acid, arachidonic acid, timnodonic acid, docosahexenoic acid and clupanodonic acid.Eukaryotic microorganisms also can produce the component that contains following material: 44.3 to 57mg g ^-1Myristic acid (equals 1134.5 to 1458.1mg L ^-1), 0.5 to 0.65mg g ^-1The Semen Myristicae diluted acid (equals 13.3 to 16.63mg L ^-1), 33.5 to 34.6mgg ^-1Pentadecanoic acid (equals 856.9 to 885.1mg L ^-1), 121.9 to 165.1mg g ^-1Palmitinic acid (equals 3118.2 to 4223.3mg L ^-1-), 7.9 to 28.5mg g ^-1Zoomeric acid (equals 202.1 to 729mg L ^-1), 4.38 to 5.9mg g ^-1Stearic acid (equals 112 to 151mg L ^-1), 6.94 to 9.9mg g ^-1Oleic acid (equals 177.5 to 253.2mg L ^-1), 0.4 to 1.3mg g ^-1Linolic acid (equals 11.26 to 33.3mg L ^-1), 0.5 to 1.0mg g ^-1Eicosadienoic acid (equals 12.8 to 25.6mg L ^-1), 0.4 to 0.5mg g ^-1Arachidonic acid (equals 10.2 to 13mg L ^-1), 75 to 100mg g ^-1Docosahexenoic acid (equals 1918 to 2560mg L ^-1), 1.9 to 6mg g ^-1Timnodonic acid (equals 48.6 to 153.5mg L ^-1) and 17.1 to 33.7mg g ^-1Clupanodonic acid (equals 437.4 to 862.1mg L ^-1), total fatty acid content is between 301 to 790mg g in the cellular biomass ^-1Between (equal 7700 to 20,209mg L ^-1).

(2) other molecule

Eukaryotic microorganisms can further produce carotenoid and xenthophylls.The example of this class carotenoid and xenthophylls comprises β-Hu Luobusu, Lyeopene, astaxanthin, canthaxanthin, phoenicoxanthin, zeaxanthin, myoxanthin, β-cryptoxanthin, capsanthin, lutein (lutin), bixin, β-apo-8-carotenal and β-apo-8-carotenal-ester.

Can put together mutually with the various PUFA that produce by described disclosed eukaryotic microorganisms equally by the xenthophylls that disclosed eukaryotic microorganisms produces.

(a) antioxidant

Usually, antioxidant is for oxygen reaction and usually by compound that oxygen consumed.Because antioxidant is common and the oxygen reaction, so antioxidant is also common and free radical resultant and free radical reaction (" The Antioxidants-The Nutrients that Guard Your Body " byRichard A.Passwater, Ph.D., 1985, Keats Publishing Inc. includes this paper with regard to its content relevant with antioxidant in by quoting at least).Composition can comprise any antioxidant, limiting examples can include but not limited to: non-flavonoid antioxidant and the nutrition that can directly remove free radical, comprise comprehensive carotene (multicarotene), β-Hu Luobusu, alpha-carotene, gamma carotene, Lyeopene, lutein and zeaxanthin, selenium, vitamin-E, comprise α-, β-and the succinate of γ (tocopherol, especially alpha-tocopherol etc.) vitamin-E and trolox (a kind of soluble vitamin-E analogue) vitamins C (anti-bad thrombus) and Niacin (vitamin B3, nicotinic acid and niacinamide), vitamin A, the 13-cis-retinoic acid, N-ethanoyl-L-halfcystine (NAC), sodium ascorbate, tetramethyleneimine-dithio-carbamate and Coenzyme Q10 99.0; Catalysis destroys the enzyme of free radical, comprises that peroxidase for example acts on H ₂O ₂For example the Selenoperoxidase of organo-peroxide (GSHPX) comprises acting on H ₂O ₂Catalase (CAT), disproportionation O ₂H ₂O ₂Superoxide dismutase (SOD), Thiadiazolidine isomerase (GSHTx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD) (G6PD) and their stand-in, analogue and polymkeric substance (analogue of antioxidant reductase and polymkeric substance for example SOD are described in for example United States Patent (USP) series number No.5,171,680, include it in this paper with regard to its content relevant by quoting at least) with antioxidant and antioxidant reductase; Gsh; Ceruloplasmin; Halfcystine and cysteamine (Mercamine Cysteamine) and flavonoid and flavonoid sample molecule be folic acid and folate for example.Antioxidase and stand-in thereof and anti-oxidant nutraceutical summary can be referring to Kumaret al, Pharmac.Ther.39:301,1988 and Machlin L.J.and Bendich, FASEB Journal 1:441-445,1987, include these two pieces of documents in this paper with regard to its content relevant by quoting with antioxidant.

Flavonoid is also referred to as " benzochromone ", is the water-soluble cpds that is present in nature, has oxidation-resistance.Flavonoid is distributed widely in the vascular plant, and is present in a large amount of vegetables, fruit and beverage such as tea and the wine (particularly red wine).Flavonoid is the conjugated aromatics.Having the most widely flavonoid is that (for example myricetin (3,5,7,3 ' for flavones and flavonol, 4 ', 5 ' ,-quercetagetin), oak Beijing opera ketone (3,5,7,3 ', 4 '-Xanthaurine), campherol (3,5,7,4 '-Luteolin) and flavones apigenin (5,7,4 '-trihydroxyflavone) and digicitrine (5,7,3 ', 4 '-Luteolin) and glucosides and oak Beijing opera ketone).

Carotenoid is the important natural pigment that is produced by multiple microorganism and plant, is generally red, orange or yellow.Carotenoid has been used to feed, food and dietetic product industry in the past.Known they be that plant-growth and photosynthesis are necessary, and be the main meals source of philtrum vitamin A.Meals antioxidant for example carotenoid (β-Hu Luobusu, Lyeopene, astaxanthin, canthaxanthin, zeaxanthin, capsanthin, lutein, bixin, β-apo-8-carotenal and β-apo-8-carotenal-ester) shows significant anti-cancer activity, and plays an important role in the prevention chronic disease.Carotenoid is effective biology antioxidant, the excitation energy on the singlet oxygen can be absorbed on the carotenoid chain, thereby cause the degraded of carotenoid molecule, but can prevent that other molecule or tissue are compromised.

Oxygen is that metabolic function is necessary, but it also can attack cells.Human body has a large amount of metabolic enzyme and antioxidants that remove the molecule of deoxidation generation from its cell.Oxidative stress is considered to the influence factor of many illnesss, and described illness is rheumatoid arthritis, ischemic heart disease and apoplexy, Alzheimer, cancer and aging etc. for example.Therefore, antioxidant has the potential of the multiple disease of prevention.From the marine organisms source, several anti-oxidant compounds have been separated; These anti-oxidant compounds comprise astaxanthin, β-Hu Luobusu and other carotenoid.

Carotenoid is naturally occurring component cloth pigment widely, has the natural fat-soluble pigment more than 700 kinds mainly to be produced by little algae, big algae, bacterium and fungi kind, and astaxanthin and derivative thereof are subjected to special concern commercial.Astaxanthin is extremely effective anti-oxidant protective agent.Yet different with β-Hu Luobusu, astaxanthin passes hemato encephalic barrier/blood retina barrier easily, therefore also has the potential of prevention encephalopathic and illness in eye.Preclinical study has shown the various beneficial effects of taking astaxanthin, and for example: (i) cancer that suppresses in bladder, colon, liver, mammary gland and the oral cavity forms and growth; (ii) make the retina of eyes avoid oxidative damage, therefore have the effect of opposing age related macula lutea disease; (iii) promote immunocompetence to improve; The protection that avoids ultraviolet injury (iv) is provided; And (v) provide better muscular endurance.

B) separation of microorganism

Disclose the microorganism from thraustochytriale section that is obtained by the following method, described method comprises: lure nourishing body (vegetative) sample in the collection salt solution (natural sea-water or artificial salt solution) and cultivate with pollen granule; Separate pollen granule and be transferred to it in heterotrophism substratum and cultivate; Identify to produce the strain isolated of lipid acid, the strain isolated that obtains from evaluation separates the microorganism from thraustochytriale section.The separation of other form comprises and is aided with suitable antibiotic substratum and by aforesaid micro-mode or by using the evaluation of 18S rRNA gene primer or probe.The heterotrophism substratum can be as mentioned below.

5. lipid and other molecule that produces by eukaryotic microorganisms

Disclose lipid composition, described lipid composition comprises the n-3 DHA of about 25wt% to about 40wt%, and about 6wt% is to the n-6 DPA and the n-3EPA of about 0wt% to about 3wt% of about 10wt%.

Lipid composition can further comprise about 11wt% to the myristic acid of about 15wt% (13wt% according to appointment), about 7wt% to the pentadecanoic acid of about 11wt% (9wt% according to appointment), about 37wt% the palmitinic acid of about 41wt% (39wt% according to appointment), the about 3wt% stearic acid of Zoomeric acid, about 0 to about 3wt% (1wt% according to appointment) of about 7wt% (5wt% according to appointment) or the about 1wt% oleic acid of about 4wt% (2wt% according to appointment) extremely extremely extremely.

Lipid composition can contain the n-3 DHA of concentration greater than about 400mg biomass, and concentration is greater than the n-6 DPA of 100mg biomass.

Lipid composition also can contain carotenoid.The example of this class carotenoid comprises β-Hu Luobusu, Lyeopene, astaxanthin, zeaxanthin, canthaxanthin, myoxanthin, phoenicoxanthin, capsanthin, lutein, bixin, β-apo-8-carotenal and β-apo-8-carotenal-ester.

On the one hand, said composition can contain at least about the n-6 DPA of the n-3 DPA of the n-3 DHA of 24wt%, about 1wt%, about 6wt% and the n-3 EPA of about 1wt%.

6. the composition that contains the molecule that produces by eukaryotic microorganisms

Food, additive, the pharmaceutical composition of humans and animals (comprising marine animal) usefulness can comprise said composition (lipid, the lipid with antioxidant and antioxidant self).

The infant formulas that comprises said composition (lipid, the lipid with antioxidant and antioxidant self) is also disclosed.

C. method

1. lipid preparation method

Disclose the method for preparing lipid composition, described method comprises: cultivate the eukaryotic microorganisms that comprises from one or more microorganisms of thraustochytriale section, and separate this lipid composition.

Can adopt several different methods to be recovered in the cellular biomass that fermentation obtains in the multiple substratum, for example by filtration or centrifugal.Washing, freezing, lyophilize or spraying drying cell then, and under oxygen-free environment, store, to eliminate existing of oxygen, then it is mixed in the food or feeds product of processing.

Contain (n-3) DHA, EPA and (n-6) PUFA such as DPA cytolipin also can by such as method such as supercutical fluid extracting or by adopt solvent for example the extraction process of chloroform, hexane, methyl alcohol, methylene dichloride or methyl alcohol extract from cellular biomass, and resulting extract evaporates under condition of negative pressure, to produce through spissated lipid matter sample.ω-3 and ω-6 PUFA can further concentrate as follows: this lipid of hydrolysis, and by adopting ordinary method for example urea adduct method (urea adduction) or fractionation, column chromatography or the concentrated highly unsaturated component of the supercutical fluid fractional separation method of passing through.Also can break or lysing cell and with the lipid extracting to vegetables oil or animal oil (as fish oil).The oil that extracting obtains can refine by the known method (for example refining by chemistry or the physics refinement) that is generally used for refining vegetables oil.Before the oil that extracting obtains used as edible oil or sells, these process for extractings can be with impurity from wherein removing.After the refinement, this oil can directly be used as feed or foodstuff additive are rich in ω-3 and/or ω-6 with generation product.Perhaps, this oil can further be processed and purifying as described below, is used for above application then, and can be used for pharmacy.

Producing in the another kind of method of spissated ω-3 or ω-6 oil, the collected cellular biomass that obtains (fresh or drying) can by known technology for example the enzymic digestion of ultrasonication, liquid shear disruption method, bead grinding, high-pressure extrusion, freeze thawing or cell walls break or ooze and split.Lipid in the ruptured cell can use certain solvent or solvent, and for example hexane, chloroform, ether or methanol mixture extract.Remove and to desolvate, and by using any known method (comprising alkaline hydrolysis, acidic hydrolysis or enzymolysis) that triglyceride level is converted into the ester of free fatty acids or lipid acid to come hydrolyze lipids.After hydrolysis was finished, nonsaponifiable compound was extracted into solvent for example in ether, hexane or the chloroform and be removed.Then remaining solution is come acidifying by adding acid, and free fatty acids is extracted to solvent for example in hexane, ether or the chloroform.The solvent solution that will contain this free fatty acids then is cooled to low to the temperature that is enough to make non-PUFA compound crystal, described subsequently non-PUFA compound can by filter, centrifugal or precipitate and remove.The result be the PUFA compound that is left obtained concentrating and be used as people's nutritional additive, as foodstuff additive or be used for medicinal application.

Simultaneously, disclose by above disclosing the lipid composition of method preparation.

Microorganism from thraustochytriale section can be any microorganism disclosed above.

A) substratum

The heterotrophism substratum can comprise (artificial or natural) sea salt, one or more carbon sources and one or more nitrogenous sources.The amount that this sea salt exists can be about 2.0 to about 40.0g L ^-1The concentration range of employed carbon source and nitrogenous source is respectively 5g L under the type culture condition (be not at high gravity fermentation, but ferment at low cost) ^-1To 60g L ^-1With 4g L ^-1To 10g L ^-1For high gravity fermentation, the concentration range of employed carbon source and nitrogenous source is respectively to be respectively 100g L under the type culture condition ^-1To 160g L ^-1With 40g L ^-1To 60g L ^-1The trend of oil and fat accumulation is: eukaryotic microorganisms is grown in (aforesaid those) substratum, and wherein providing of nitrogen is restricted after about 24 to about 48 hours, and providing still of carbon is flush with money.This eukaryotic microorganisms continues to absorb carbon (monose form), but no longer carries out cell fission owing to lack the nitrogen that forms related protein and nucleic acid.The result is that these sugar are converted to the deposit grease, and this and the described mode of RatledgeC. (Lipid Tech.16:34-39,2004) are closely similar, and Fig. 9 provides peculiar this phenomenon of this organism.

Nitrogenous source can be one or more in peptone, yeast extract, malt extract and the Sodium Glutamate.Nitrogenous source also can be corn steep liquor or cottonseed extract.Nitrogenous source can contain yeast extract and/or peptone or msg powder type.For example, nitrogenous source can include but not limited to EMD ^TMYE-MSG, EMD ^TMYE, EMD ^TMPeptone-MSG, Sigma ^TMYE-MSG, Sigma ^TMYE, Fermtech ^TMYE-MSG, Fermtech ^TMYE or Fish meal (62% protein).The existing amount of yeast extract can be about 2g L ^-1The amount that msg powder type exists can be about 8g L ^-1

Carbon source can be one or more in the following material: the D-trehalose; glycerine; the D-glyconic acid; L-lactic acid; D, L MALIC ACID; D-ribose; Tween 20; D-fructose; acetate; acetate; alpha-D-glucose; maltose; thymidine; altheine; the D-wood sugar; Tween 40; α-ketone group-pentanedioic acid; sucrose; L-glutaminate; Tween 80; Beta-methyl-D-glucoside; trisaccharide maltose; adenosine; fumaric acid; bromosuccinic acid; the L-Serine; the D-cellobiose; L-alanyl-glycine; Pyruvic Acid Methyl ester; L MALIC ACID; glycyl-L-proline(Pro); D-palcose; the L-lyxose; pyruvic acid; α-D-lactose; dextrin; the D-pectinose; the 2-deoxy-D-ribose; gelatin; dextrose; starch; 3-0-β-D-galactopyranose base-D-pectinose; the D-tagatose; 5-ketone group-D-glyconic acid; oxalyl oxysuccinic acid (oxalomalic acid); Sorbic Acid; L-ornithine and dihydroxy acetic acid ester.On the one hand, carbon source can be D, L MALIC ACID, D-fructose, D-wood sugar, fumaric acid, D-cellobiose, 5-ketone group-D-glyconic acid, pyruvic acid, α-D-lactose, corn dextrin, gelatin, W-Gum or wheat starch.The amount that carbon source exists can be about 1g L ^-1To 60gL ^-1And be up to about 200g L ^-1

In an example, substratum contains the 5g D-glucose of having an appointment, about 2g peptone and about 2g yeast extract/rise salt solution (natural or artificial).In another example, substratum contains the 60g D-glucose of having an appointment, about 10g yeast extract/rise salt solution (natural or artificial).In another example, substratum can contain the 8g yeast extract of having an appointment, 32g MSG, 24g sea salt (natural and artificial) and 300g D-glucose/liter.

Substratum can further contain phosphoric acid salt (as potassiumphosphate and sodium phosphate).Substratum can further contain inorganic salt (as ammonium sulfate, sodium bicarbonate, sodium orthovanadate, potassiumchromate, Sodium orthomolybdate, selenous acid, single nickel salt, copper sulfate, zinc sulfate, cobalt chloride, iron(ic) chloride, magnesium chloride).Substratum can further contain inner complex (as EDTA).Substratum can further contain VITAMIN (as vitamin B6, VITMAIN B1, calcium pantothenate, para-amino benzoic acid, riboflavin, nicotinic acid, vitamin H, folic acid and vitamin B12).The pH of substratum is about 4.0 to about 6.5.

Cultivation can be carried out about 1 to about 9 days (3 days to about 5 days according to appointment).Cultivation can be carried out at about 18 to about 30 ℃ (18-25 ℃ according to appointment).Cultivation can further comprise vibration or ventilation.

The process of separating lipid can comprise this microorganism is contacted with extraction solvent.This solvent can comprise and is selected from chloroform, hexane, methyl alcohol or ethanol or supercritical CO ₂One or more solvents.

This method can produce any composition disclosed herein.

This eukaryotic microorganisms can produce and contain more than or equal to about 20g L ^-1The lipid composition of the n-3 DHA of substratum.This eukaryotic microorganisms can produce and contain the L more than or equal to 40g ^-1The lipid composition of the n-3 DHA of substratum.This eukaryotic microorganisms can produce and contain the L more than or equal to 80g ^-1The lipid composition of the n-3DHA of substratum.

2. screen and authentication method

Eukaryotic microorganisms disclosed herein can produce and contain (n-3) serial docosahexenoic acid and timnodonic acid and (n-6) lipid of serial DPA.These eukaryotic microorganisms can use for example following screening method screening.The nourishing body sample can be placed and 10mL aseptic (0.2 μ m filtration) natural sea-water is housed (contains concentration and be respectively 300 and 500mg L ^-1Penicillin and Streptomycin sulphate) the 20mL bottle in.Lure the collection bottle with aseptic pollen granule then, and cultivated 48 hours at 18 to 25 ℃.Then pollen granule is transferred on the agar plate that contains microbiotic (as mentioned above), and cultivates under the same conditions.Select single random transparent colony and the atypical yeast or the bacterium colony of sphere or limaciform cell, and the cultivation of under substratum same as described above and identical condition, going down to posterity.Use Nutrient broth according to growing state and lipid acid production these strain isolateds to be screened then, described Nutrient broth contains 5gL by the filtering natural sea-water preparation of 0.2 μ m ^-1Glucose, 2g L ^-1Peptone and 2g L ^-1Yeast extract, the result obtains cellular biomass by carrying out centrifugal to liquid nutrient medium or precipitating to collect.Can use ordinary method directly lipid acid to be carried out transesterify, and the fatty acid methyl ester composition can be by gas chromatographic analysis, the bacterial strain that screening produces appropriate amount n-3 series DHA and n-6 series DPA is used for further research.

Disclose the method for identifying eukaryotic microorganisms, described method comprises: with the nourishing body sample in the pollen granule luring and collecting sea water (natural sea-water or artificial seawater) and cultivate; Be transferred to this pollen granule in the heterotrophism substratum and cultivate; And evaluation produces the strain isolated of lipid acid.

The lipid composition that eukaryotic microorganisms produced that is obtained by top evaluation is also disclosed.

The lipid composition that is produced by the method for using disclosed eukaryotic microorganisms and method disclosed herein is also disclosed.

The eukaryotic microorganisms (ONC-T18) that ATCC is numbered PTA-6245 is also disclosed.

Also disclose and belong to thraustochytriale purpose eukaryotic microorganisms (ONC-T18), they have for example 18S rRNA shown in the SEQ ID NO:1, and are accredited as the genus thraustochytrium kind.

Also disclose and to have produced concentration and be higher than 400mg L respectively ^-1With 100mg L ^-1DHA and the eukaryotic microorganisms genus thraustochytrium kind of DPA.

Also disclose and to have produced scope at 50 to 1250mg kg by above-mentioned heterotrophic fermentation ^-1Carotenoid and can produce 1 to 20mg kg ^-1Astaxanthin, 0.25 to 10mg kg ^-1Zeaxanthin, 1 to 20mg kg ^-1Keratin, 1 to 20mg kg ^-1Myoxanthin and 1 to 200mg kg ^-1The eukaryotic microorganisms genus thraustochytrium kind of β-Hu Luobusu.

Also disclose the method for cultivating eukaryotic microorganisms, this method comprises: cultivate eukaryotic microorganisms under certain condition, wherein said condition comprises the substratum that contains following composition: artificial sea salt (trophicity seawater) form, 2.0 to 15.0g L ^-1Sodium-chlor, yeast extract form and msg powder type form, concentration are respectively 2.0 and 8.0g L ^-1Nitrogenous source and glucose form, be up to 130g L ^-1Carbon.

Disclosed method can be cultivated for example ONC-T18, make at least 24% weight of total fatty acids be DHA, at least 6% be DPA by weight, and at least 1% is EPA.

Disclosed cultural method also can be cultivated for example ONC-T18, thereby make by weight at least 1% to be the carotenoid material, wherein 1 to 2% and at least 1.2% is astaxanthin, 0.25 to 1% and at least 0.45% being zeaxanthin, 5 to 16% and at least 9% is keratin, and 1 to 2% and at least 1.2% is myoxanthin and 12 to 16% and 14% β-Hu Luobusu by weight.

3. nucleic acid

Multiple molecule disclosed herein is the molecule based on nucleic acid, comprises for example encode for example rRNA and any other proteinic nucleic acid disclosed herein, and various functional nucleic acid.Disclosed nucleic acid is made of for example Nucleotide, nucleotide analog or nucleotide substitution.The limiting examples of these and other molecule is stated in this article.Be understandable that when for example carrier was expressed, expressed mRNA was made of A, C, G and U/T usually in cell.Be understandable that equally for example if antisense molecule send to pass by for example external source and is introduced into cell or cellular environment, then advantageously, antisense molecule constitutes by can reduce the nucleotide analog that antisense molecule degrades in cellular environment.

A) Nucleotide and associated molecule

Nucleotide is the molecule that contains base portion, sugar moieties and phosphoric acid part.Nucleotide can link together by its phosphoric acid part and sugar moieties that forms bonding between nucleosides.The base portion of Nucleotide can be VITAMIN B4-9-base (A), cytosine(Cyt)-1-base (C), guanine-9-base (G), uridylic-1-base (U) and thymus pyrimidine-1-base (T).The sugar moieties of Nucleotide is ribose or ribodesose.The phosphoric acid of Nucleotide partly is pentavalent phosphoric acid.The limiting examples of Nucleotide is 3 '-AMP (3 '-single adenosine phosphate) or 5 '-GMP (5 '-Guanosine 5'-Monophosphate).

Nucleotide analog is to contain base, sugar or phosphoric acid are partly made the Nucleotide that certain class is modified.Known in the art to being modified to of Nucleotide, comprise for example 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, xanthoglobulin and 2-aminoadenine, and the modification of partly making at sugar or phosphoric acid.

Nucleotide substitution is for to have identity function with Nucleotide, but the not molecule of phosphoric acid part, for example peptide nucleic acid(PNA) (PNA).The molecule that the part of nucleotide substitution for discerning nucleic acid with Watson-Crick or Hoogsteen mode but by non-phosphoric acid part links together.Nucleotide substitution also can form the double helical form structure when interacting with appropriate target nucleic acid.

The molecule (conjugate) of other form can also be coupled together to strengthen for example cell absorption with Nucleotide or nucleotide analog.Conjugate can connect Nucleotide or nucleotide analog by chemical mode.This class conjugate includes but not limited to lipid part, for example cholesterol moiety (Letsingeret al., Proc.Natl.Acad.Sd.USA, 86:6553-6556,1989).

Watson-Crick interact for at least a interaction at the Watson-Crick interface of Nucleotide, nucleotide analog or nucleotide substitution.The Watson-Crick interface of Nucleotide, nucleotide analog or nucleotide substitution comprises C1, the N1 of Nucleotide, nucleotide analog or nucleotide substitution based on purine and C6 position and based on C2, N3 and the C4 position of Nucleotide, nucleotide analog or the nucleotide substitution of pyrimidine.

Hoogsteen interacts and is the interaction at the Hoogsteen interface that occurs in Nucleotide or nucleotide analog, and this interface is exposed in the major groove of duplex DNA.The Hoogsteen interface comprises the active group (NH of purine nucleotides C6 position ₂Or O) and the N7 position.

B) sequence

Have a plurality of sequences and for example SEQ ID NO:1 and any other nucleic acid disclosed herein relevant, and these sequences and other sequence and each subsequence of wherein being contained are all included this paper in by quoting with protein (open) at Genbank.

This paper provides a plurality of sequences, and these sequences can be referring to Genbank (www.pubmed.gov) with other sequence.Those skilled in the art understands how to differentiate ordering bias and difference, and understands how to adjust composition and the method relevant with a certain concrete sequence, makes it adapt to other correlated series.According to information disclosed herein and information known in the art, can design the primer and/or the probe of any sequence.

C) primer and probe

The composition that comprises primer and probe is disclosed, they can with gene interaction disclosed herein.In certain embodiments, primer is used to support dna amplification reaction.Usually primer can be extended in the sequence specific mode.The primer of sequence specific mode prolongs and comprises any method, wherein with the sequence of primer hybridization or otherwise relevant nucleic acid molecule and/or composition instructs or composition or the sequence of influence by the primer prolongation product that obtains that produces.Therefore, the prolongation of the primer of sequence specific mode includes but not limited to PCR, dna sequencing, DNA prolongation, DNA polymerization, rna transcription or reverse transcription.Preferably with the technology and the condition of sequence specific mode amplimer.In some aspects, primer can be used as the specific specificity of thraustochytriale that this paper mentions or bacillus or belongs to specific probe.In this case, primer can design specificity at eukaryotic microorganisms, and carries out the PCR reaction subsequently.Can determine the existence of target kind by successfully forming the PCR product then.In some aspects, primer also can be used for dna amplification reaction, for example PCR or directly order-checking.Be understandable that in some aspects, primer also can use non-enzymatic technology to prolong, the Nucleotide or the oligonucleotide that wherein for example are used to prolong primer are modified, and prolong primer thereby make them that chemical reaction can take place in the sequence specific mode.Usually, disclosed primer can with a certain area hybridization of nucleic acid or nucleic acid, perhaps they can with the complementary sequence of nucleic acid or the complementary sequence hybridization in a certain zone of nucleic acid.

D) nucleic acid send and passs

Such just as one of ordinary skill in the understanding, comprise foreign DNA given and the experimenter's that is ingested intracellular aforesaid method (being gene transfer or transfection) in, disclosed nucleic acid can be naked DNA or rna form, perhaps this nucleic acid can be present in this nucleic acid sent and passs to the carrier of cell, and the dna fragmentation of encoding antibody is subjected to the transcriptional regulatory of promotor by this.Carrier can be and is purchased preparation, for example adenovirus carrier (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada).Nucleic acid or carrier are passed to sending of cell can pass through number of mechanisms.As an example, send and pass and to be undertaken by liposome, can use to be purchased for example LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc. of Liposomal formulation, Gaithersburg, MD), SUPERFECT (Qiagen, Inc.Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, WT), and other liposome that develops according to the standard method of this area.In addition, disclosed nucleic acid or carrier can pass through electroporation ((ImaRxPharmaceutical Corp., Tucson AZ) carry out sending in the body and pass by Genetronics company (San Diego, the technology that CA) provides) and by the SONOPORATION machine.

As an example, carrier send to be passed and can for example can pack the genomic retroviral vector of recombinant retrovirus system and carry out (referring to for example Pastan et al., Proc.Natl.Acad.ScL U.S.A.85:4486,1988 by viral system; Miller et al., Mol.Cell.Biol.6:2895,1986).This recombinant retrovirus can be used for cells infected subsequently, and the nucleic acid of the main neutralizing antibody (or its active fragments) of will encoding thus send and passs to this cells infected.Certainly, the nucleic acid that changes is to some extent introduced mammalian cell really blanking method be not limited to use retrovirus.For this method, also extensively there is other technology, comprise and use adenovirus carrier (Mitani et al., Hum.Gene Ther.5:941-948,1994), adeno associated virus (AAV) carrier (Goodman et al., Blood 84:1492-1500,1994), lentiviral vectors (Naidini et al., Science 272:263-267,1996) and pseudo-type retrovirus vector (Agrawal et al., Exper.Hematol.24:738-747,1996).Also can use physics transduction technology, for example send to pass and pass and other endocytosis mechanism is carried out (referring to for example Schwartzenberger et al., Blood 87:472-478,1996) with receptor-mediated sending by liposome.The composition of the disclosure and method can be used with any of these or other normally used gene transfer method.

4. expression system

Sent the nucleic acid of passing to cell to contain the expression regulation system usually.For example, the insertion gene in virus and the retrovirus system contains promotor and/or enhanser usually, to help the expression of the required gene product of regulation and control.Promotor is generally the one or more dna sequence dnas that work when the relatively-stationary position of distance transcription initiation site.Promotor contains the necessary core parts of generation basic interaction between RNA polymerase and the transcription factor, and can contain upstream element and response element.

Be understandable that except the general-purpose system of hereinafter discussing, to also have the multiple transcriptional control system that can be used in the organism disclosed herein.Be understandable that organism disclosed herein can be with several genes that this paper set forth marker gene or have that other required attribute for example strengthens or the gene of unique growth characteristics carries out transfection and conversion for example.

A) viral promotors and enhanser

The preferred promoter that control is transcribed from carrier in the mammalian host cell can be available from multiple source for example such as the genome of following virus: polyomavirus, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and cytomegalovirus most preferably is perhaps from allos mammalian promoter such as beta-actin promotor.Early stage and the late promoter of SV40 virus obtains (Fiers et al., Nature, 273:113,1978) with the SV40 restriction fragment form that also contains SV40 virus replication starting point usually.The immediate early promoter of human cytomegalic inclusion disease virus obtains (Greenway, PJ.etal, Gene 18:355-360,1982) with HindIIIE restriction fragment form usually.Certainly, also can be used for the application from host cell or relevant promotor of planting.

Enhanser is often referred at the distance transcription initiation site dna sequence dna that works of fixed range not, and can be at 5 ' end (Laimins of transcription unit, L.et al., Proc.Natl.Acad.Sci.78:993,1981) or 3 ' end (Lusky, MX., et al., Mol Cell Bio.3:1108,1983).In addition, enhanser can be positioned in intron (Banerji, J.L.et al., Cell33:729,1983) and the encoding sequence self (Osborne, T.F., et al., Mol CellBio.4:1293,1984).They grow 10 usually to 300bp, and they work in the cis mode.Enhanser plays raising from transcribing that promotor-proximal begins.Enhanser also contains the response element that mediates transcriptional regulatory usually.Promotor also contains the response element that mediates transcriptional regulatory.Enhanser plays a decisive role to the regulation and control of genetic expression usually.Although known a lot of enhancer sequence can be used from the enhanser of eukaryotic cell virus usually and totally express from mammalian genes (globin, Proteinase, bone marrow serine, albumin, alpha-fetoprotein and Regular Insulin) now.Preferred examples is the sub-enhanser of SV40 enhanser, cytomegalovirus early promoter of replication orgin (100-270bp) side in late period, the polyomavirus enhanser and the adenovirus enhanser of replication orgin side in late period.

Can the be excited particular chemical incident or the light of its function of promotor and/or enhanser activates specifically.System can be subjected to regulate such as reagent such as tsiklomitsin and dexamethasone.Also exist by being exposed to for example γ irradiation or the alkanisation chemotherapeutic agents method of coming the enhanced virus vector gene to express of irradiation.

In certain embodiments, promotor and/or enhanser zone can be used as constitutive promoter and/or enhanser, to express transcription unit zone to be transcribed to greatest extent.In some construct, even if promotor and/or enhanser zone only in the cell of specified time, express in particular type, it in all eukaryotic cell types also all be have active.Such preferred promoter is CMV promotor (650bp).Other preferred promotor is SV40 promotor, cytomegalovirus (total length promotor) and retroviral vector LTE.

Show that selective expressions' such as particular cell types such as melanoma cells expression vector all can be cloned and be used for being structured in to all particular adjustments elements.Glial fibrillary acidic protein (GFAP) promotor has been used to selective expression's gene in the cell in colloid source.

The expression vector that uses in eukaryotic host cell (yeast, fungi, insect, plant, animal, people or karyocyte) also can contain the necessary sequence of transcribing that stops influencing the mRNA expression.These zones are transcribed into the polyadenylation section in the untranslated part of mRNA coding tissue factor protein.3 ' untranslated zone also can comprise the Transcription Termination site.Polyadenylation region is also contained in preferred transcription unit.The advantage in this zone is that it has increased the probability of transcription unit's processed and transportation as mRNA.Polyadenylation signal identifying and using for known in this field in the expression construct.Preferably the homology polyadenylation signal is used for transgenic constructs.In some transcription unit, polyadenylation region derives from the early stage polyadenylation signal of SV40, and by about 400 based compositions.Other standard sequence is only contained in also preferred transcription unit, perhaps contains above-mentioned sequence simultaneously, to improve the expression or the stability of construct.

B) mark

Virus vector can comprise the nucleotide sequence of coded markings product.This marked product is used for determining whether this gene is sent passs to cell, in case and quilt sent and pass then expressed.Preferred marker gene is escherichia coli lacz gene (coding beta-galactosidase) and green fluorescent protein.

In certain embodiments, mark can be selected marker.The example of the suitable selected marker that is used for mammalian cell is Tetrahydrofolate dehydrogenase (DHFR), thymidine kinase, Xin Meisu, neomycin analog G418, Totomycin and tetracycline.When this class selected marker successfully is transferred in the mammalian host cell, if will mammalian cell place through transforming selective pressure next it can survive.There is the different widely used selection scheme of two classes.The first kind is based on the use of cellular metabolism and mutational cell line, and described mutational cell line lacks the ability that does not rely on supplemented medium and grow.Two examples are CHO DHRF cell and mouse LTK cell.These cells lack the ability of growing under not adding such as nutraceutical situations such as thymidine or xanthoglobulin.Because these cells lack complete necessary some gene of Nucleotide route of synthesis, therefore if do not provide the Nucleotide that is lacked in supplemented medium, then they can not be survived.Another approach of supply substratum is that complete DHFR or TK gene are introduced in the cell that lacks corresponding gene, changes its growth demand thus.Fail in non-supplemented medium, can not survive by each cell of DHFR or TK gene transformation.

Second class is that dominance is selected, the selection scheme that described dominance selection refers to be used for arbitrary cell type and do not need to use mutational cell line.These schemes use medicine to stop the growth of host cell usually.These cells with new gene can be expressed the protein that transmits drug resistance, and can survival after experience is selected.The example that this dominance is selected uses medicine Xin Meisu (Southern P.and Berg, P., J Molec.Appl.Genet.1:327,1982), Mycophenolic Acid (Mulligan, R.C.and Berg, P.Science 209:1422,1980) or Totomycin (Sugden, B.etal., Mol.Cell.Biol.5:410-413,1985).These three examples adopt the bacterial gene that is subjected to the control of eukaryote matrix, to give the resistance to suitable medicine G418 or Xin Meisu (Geneticin), xgpt (Mycophenolic Acid) or Totomycin respectively.Other example comprises neomycin analog G418 and tetracycline.

5. peptide class

A) protein variant

As described herein, the proteic a plurality of variants of organism disclosed herein all are known and are considered in this article.In addition, for the known thraustochytriale purpose of function bacterial strain, there is the proteic derivative of thraustochytriale that much can be used for disclosed method and composition equally.Protein variant and derivative are known by those skilled in the art, and can comprise the amino acid sequence modifications thing.For example, the amino acid sequence modifications thing generally includes a class or the multiclass in following three classes: displacement variant, insertion variant or disappearance variant.Insertion comprise that aminoterminal and/or carboxyl terminal merge and the sequence of single or multiple amino-acid residues between insert.Insert normally than the littler insertion of those insertions of aminoterminal or carboxyl terminal fusion, for example the order of magnitude is the insertion of one to four residue.The immunogenicity solvent protein derivative, those immunogenicity solvent protein derivatives of describing in an embodiment for example, can prepare by the following method:, will merge even as big as giving immunogenic polypeptide and target sequence by external crosslinked or have the reconstitution cell of the DNA of coding fusions to cultivate by conversion.Disappearance is characterised in that removes one or more amino-acid residues from protein sequence.Usually, arbitrary position has and is no more than about 2 to 6 residue disappearance in the protein molecular.These variants can prepare by the site-specific mutagenesis of Nucleotide among the DNA of coded protein usually, produce the DNA of coding variant thus, and at expressible dna in the reconstitution cell culture subsequently.To carry out the technology of replacement mutation be known to predetermined site in the known DNA of sequence, for example mutagenesis of M13 primer and PCR mutagenesis.Amino-acid substitution is generally a residue, but also can occur at a plurality of different positionss simultaneously; The order of magnitude that inserts is generally about 1 to 10 amino-acid residue; And the scope of disappearance is about 1 to 30 residue.Disappearance or insert preferably in adjacent residues carrying out promptly lacks 2 residues or inserts 2 residues.Displacement, disappearance, insertion or their arbitrary combination can join together to obtain final construct.Sudden change can not place sequence outside the reading frame, and preferably can not form the complementary region that can produce the mRNA secondary structure.The displacement variant is removed at least one residue wherein and different residue are inserted into those variants of this position.This class displacement can be carried out according to following table 1 and 2 usually, and is considered to conservative substitution.

The remarkable change of function or immunity aspect can realize by selecting conservative property to replace not as those metathetical of table 2, promptly being chosen in influences the more significant residue of aspect difference to what keep following structure or character: (a) structure of replacement areas polypeptide backbone, for example as lamella or helical conformation, (b) electric charge of the molecule of target site or hydrophobicity or (c) side chain size.Usually by expectation meeting these following displacements that are replaced into: (a) with hydrophilic residue such as seryl or Threonyl displacement hydrophobic residue such as leucyl in the variation maximum that produces aspect the property of protein; isoleucyl; phenylalanyl; valyl or alanyl; (b) with halfcystine or any other residue of proline(Pro) displacement; (c) with the residue such as the lysyl that have the positive electricity side chain; arginyl or histidyl-displacement negative electricity residue such as glutamyl or aspartyl displacement; perhaps (d) do not have the residue such as the glycine of side chain with residue with bulky side chain such as phenylalanine displacement; in this case, (e) by increasing the number of sulfation and/or glycosylation site.

Table 1: amino acid abbreviations

Amino acid	Abbreviation
		L-Ala	Ala A
Arginine	Arg R
		L-asparagine	Asn N
Aspartic Acid	Asp D
		Halfcystine	Cys C
Glutamine	GIn Q
		L-glutamic acid	GIu E
Glycine	GIy G
		Histidine	His H

Isoleucine	lie I
		Leucine	Leu L
Methionin	Lys K
		Methionine(Met)	Met M
Phenylalanine	Phe F
		Proline(Pro)	Pro P
Serine	Ser S
		Threonine	Thr T
Tryptophane	Trp W
		Tyrosine	Tyr Y
Xie Ansuan	VaI V

Table 2: amino-acid substitution

Original residue	Exemplary conservative substitution *
		Ala	Ser
Arg	Lys；Gln
		Asn	Gln；His
Asp	Glu
		Cys	Ser
Gln	Asn；Lys
		Glu	Asp
Gly	Pro
		His	Asn；Gln
Ile	Leu；Val
		Leu	Ile；Val
Lys	Arg；Gln
		Met	Leu；Ile
Phe	Met；Leu；Tyr
		Pro	Gly
Ser	Thr
		Thr	Ser
Trp	Tyr
		Tyr	Trp；Phe
Val	IIe；Leu

* other is known in the art

For example, an amino-acid residue is by being called conservative substitution at biology and/or chemically similar another amino-acid residue replacement by those skilled in the art.For example, conservative substitution will be replaced another hydrophobic residue with a hydrophobic residue, perhaps replace another polar residues with a polar residues.Above-mentioned displacement comprises following combination, Gly for example, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg and Phe, Try.Each clearly openly these conservative substitution variants of sequence be included in chimeric (mosaic) polypeptide scope that this paper provides.

Can adopt displacement mutagenesis or deletion mutagenesis to insert N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr) site.The disappearance of halfcystine or other unstable residue also may be required.The disappearance of potential proteolysis site such as Arg or displacement realize by for example making one of alkaline residue disappearance, or realize by replacing a residue with glutaminyl or histidyl-residue.

Derivatization after some translation be since recombinant host cell to due to the effect of expressed polypeptide.Glutaminyl and asparagyl residue usually after translation deamidate become corresponding glutamyl and aspartyl residue.Perhaps, these residues deamidate under the sour environment of gentleness.Other posttranslational modification comprises the hydroxylation of proline(Pro) and Methionin, the phosphorylation of the oh group of seryl or Threonyl residue, (the T.E.Creighton that methylates of the o-amino group of Methionin, arginine and Histidine side chain, Proteins:Structure and MolecularProperties, W.H.Freeman ﹠amp; Co., San Francisco pp 79-86,1983), acetylize that the N end is amino and the amidation of C end carboxyl in some cases.

Be understandable that a kind of method that defines proteinic variant disclosed herein and derivative is by defining this variant and derivative according to its homology/identity with concrete known array.The disclosed especially this paper of being discloses proteinic variant, and described variant and described sequence have at least 60%, 70% or 75% or 80% or 85% or 90% or 95% homology.Those skilled in the art will readily understand and how to determine two kinds of proteinic homologys.For example, homology can calculate after these two sequences of comparison, thereby makes this homology be in highest level.

Calculating the another kind of method of homology can be undertaken by the algorithm of having announced.The best comparison of comparative sequences can be carried out by the following method: local homology's algorithm (Smith and Waterman, 1981, Adv.Appl.Math 2:482), homology alignment algorithm (Needleman and Wunsch, 1970, J.Mol.Bio.48:443), similarity method for searching (Pearson and Lipman, 1988, Proc.Natl.Acad.Sci.USA 85:2444), these algorithms of being finished by computer are (as the GAP in the Wisconsin genetics software package, BESTFIT, FASTA and TFASTA, Genetics Computer Group, Madison, WI, U.S.A) or hand inspection.

The nucleic acid homology of same type can by for example in following document disclosed algorithm obtain: Zuker, M.Science 244:48-52,1989, Jaeger et al.Proc.Natl.Acad.Sci USA 86:7706-7710,1989, Jaeger et al.Methods Enzymol.183:281-306,1989, include these documents in this paper with regard to its content relevant by quoting at least with the nucleic acid comparison.

Be understandable that, the description of conservative sudden change and homology can any built up section be got up for example have the specific examples of at least 70% homology with concrete sequence, wherein said variant suddenlys change for conservative.

Because this specification sheets has been set forth various albumen and protein sequence, it being understood that therefore the nucleic acid of these protein sequences of coding is disclosed equally.This will comprise all degenerate sequences relevant with the specific protein sequence, promptly have all nucleic acid of the sequence of a concrete protein sequence of coding, and all nucleic acid of the variant of disclosed this protein sequence of encoding and derivative (comprising degeneracy nucleic acid).Therefore, although each concrete nucleotide sequence does not write out in this article, what it should be understood that is that in fact each sequence all is disclosed in this article and is described by disclosed protein sequence.What it is also understood that is, when disclosing disclosed proteic concrete variant herein, which kind of concrete dna sequence dna is this albumen of encoding in vivo although do not have aminoacid sequence to show to be, but the proteic known nucleic acid sequence of this kind is known equally in this proteic concrete bacterial strain of coding generation, and is able to disclosure and description in this article.

Be understandable that, have many amino acid and peptide analogs that are introduced into disclosed composition.For example, there are many D-amino acid or have the amino acid of the sense substituent different with amino acid shown in table 1 and the table 2.The corresponding steric isomer of naturally occurring peptide and the steric isomer of peptide analogs are disclosed.These amino acid can be introduced into polypeptide chain at an easy rate in the following manner: make the tRNA molecule charged with selected amino acid, and for example utilize amber codon amino acid analogue to be inserted peptide chain and come genetic constructs is carried out genetic engineering modified (Thorson etal. in the site-specific mode, Methods in MoI.Biol.77:43-73,1991, Zoller, Curr.Opin.Biotech., 3:348-354,1992; Ibba, Biotechnol.Genet.Eng.13:197-216,1995, Cahill et al., Trends Biochem.Sci, 14:400-403,1989; Benner, Trends.Biotechnol., 12:158-163,1994) include all these documents in this paper with regard to its content relevant by quoting at least) with amino acid analogue.

Can produce molecule similar with peptide but that be not connected by the native peptides key.For example, the key of connection amino acid or amino acid analogue can comprise CH ₂NH--,--CH ₂S--,--CH ₂-CH ₂--,--CH=CH--(cis and trans),--COCH ₂--,--CH (OH) CH ₂-and--CHH ₂SO--(these keys and other key can be referring to Spatola, A.F.in Chemistry and Biochemistry of Amino Acids, and Peptides, and Proteins, B.Weinstein, eds., Marcel Dekker, New York, p.267,1983; Spatola, A.F., Vega Data (March 1983), Vol.1, Issue3, Peptide Backbone Modifications (general introduction); Morley, Trends Pharm.Sci.463-468,1980; Hudson, D.et al., Int.J.Pept.Prot.Res.14:177-185,1979 (--CH ₂NH--, CH ₂CH ₂-); Spatola et al.Life Sci38:1243-1249,1986 (--CH H ₂-S); Hann J.Chem.Soc.Perkin Trans.I307-314,1982 (--CH--CH--, cis and trans); Almquist et al.J.Med.Chem.23:1392-1398,1980 (--COCH ₂--); Jennings-White et al.Tetrahedron Lett., 23:2533,1982 (--COCH ₂-); Szelke et al.EuropeanAppln., EP 45665 CA:97:39405,1982 (--CH (OH) CH ₂--); Holladay et al.Tetrahedron Lett.24:4401-4404,1983 (--C (OH) CH ₂--) and Hruby LifeSci.31:189-199,1982 (--CH ₂--S--), each piece document is all included this paper by reference in.Particularly preferred non-peptide bond is--CH ₂NH--.Be understandable that peptide analogs can have more than one atom, for example b-L-Ala, g-aminobutyric acid etc. between the atom at above-mentioned key two ends.

Amino acid analogue and peptide analogs have usually and strengthen or required attribute, for example produce more that economy, chemical stability are higher, pharmacological properties (transformation period, absorbing state, tire, effectiveness etc.) strengthens, specificity changes (wider as biological activity), antigenicity reduction etc.

D-amino acid can be used for forming more stable peptide, reason be D-amino acid not can discern by peptase etc.Come that with the D-amino acid of same type one or more amino acid of conserved sequence are replaced (for example replacing L-Methionin with D-Methionin) comprehensively can be used to form more stable peptide.Cysteine residues can be used to cyclisation or connects two or more peptides.This is useful (Rizo and Gierasch Ann.Rev.Biochem.61:387,1992, include this paper in by quoting) for forcing peptide to form specific conformation.

6. additive

This paper also discloses nutritional additive.Nutritional additive is can be by giving the experimenter or being taken by the experimenter, to provide, to supply with or the material that has additional nutrients (as VITAMIN, mineral substance, essential trace element, amino acid, peptide, nucleic acid, oligonucleotide, lipid, cholesterol, steroid, carbohydrate etc.).On the one hand, herein disclosed is the nutritional additive that contains any compound disclosed herein.For example, nutritional additive can comprise any lipid disclosed herein.The fatty acid residue of these lipids can be any lipid acid disclosed herein (as unsaturated or saturated fatty acid residues).

Nutritional additive can contain any amount compound disclosed herein, determines can offer the Benzenediol derivative of experimenter's required dosage (as CoQ but contain usually ₁₀) and/or the appropriate amount of lipid acid.The exact amount of needed compound is different because of the experimenter in the nutritional additive, and this depends on experimenter's species, age, body weight and integral status, the severity of the dietary deficiency of being treated, concrete mode of administration etc.Therefore, can not stipulate the concrete amount of each nutritional additive.But appropriate vol can only use normal experiment just can determine under the situation of the instruction of considering this paper by those of ordinary skills.In a concrete example, nutritional additive can contain about by weight 0.05 to about 20%, about 1 to about 7.5% or about 3 to about 5% compound.In another example, nutritional additive can contain about by weight 0.05,0.10,0.15,0.20,0.25,0.30,0.35,0.40,0.45,0.50,0.55,0.60,0.65,0.70,0.75,0.80,0.85,0.90,0.95,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10,10.5,11.0,11.5,12.0,12.5,13.0,13.5,14.0,14.5,15.0,15.5,16.0,16.5,17.0,17.5,18.0,18.5,19.0,19.5 or 20.0% compound, arbitrary when appropriate described numerical value can form terminal point or starting point.On the other hand, when being nutritional additive, this additive can be made of the additive that is up to 100%.

Nutritional additive also can contain other nutritive substances such as VITAMIN, trace element, mineral substance etc.In addition, nutritional additive can contain other components for example sanitas, biocide, antioxidant, sequestrant, thickening material, seasonings, thinner, emulsifying agent, dispersing auxiliary and/or tackiness agent.

Nutritional additive usually can be oral, and can be any form that is suitable for oral administration.For example, nutritional additive can be tablet, gel capsule, capsule, liquid, wafer or syrup form usually.

7. delivery device

Any compound described herein all can mix delivery device.The example of delivery device includes but not limited to micro-capsule, microballoon, nanometer ball or nano particle, liposome, noisome, nanometer rhodoplast, solid-liquid nano particle, gel, gel capsule, tablet, lotion, paste, sprays or emulsion.Other examples that are fit to the delivery device of non-oral administration comprise small porous particle pulmosphere.The example of concrete delivery device that is used for this paper is as described below.

Disclosed compound can be mixed in the liposome.As known in the art, liposome derives from phosphatide or other lipid matters usually.Liposome is by brilliant formation of single or multiple lift aqueous fluid that is scattered in the aqueous medium.Can accept on any nontoxic, physiology that can form liposome and metabolizable lipid all can use.The disclosed composition of liposome form also contains stablizer, sanitas, vehicle etc. except that containing compound disclosed herein.The example of suitable lipid is natural and synthetic phosphatide and phosphatidylcholine (Yelkin TTS).The method for preparing liposome is known in the art.Referring to for example Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, et seq. p.33,1976, include this paper with regard to its instruction in by quoting to liposome and preparation method thereof.

In other examples, liposome can be cationic liposome (as DOTMA, DOPE, DC cholesterol) or anionic liposome.If need, liposome can further include the protein that helps the concrete cell of target.Thereby the administration that contains the composition of compound and cationic liposome can give to the blood that flows into target organ or be sucked into the cell of respiratory tract target respiratory tract.About liposome, referring to for example Brigham, et al, Am J Resp Cell Mol Biol 1:95-100,1989, Feigner, et al., Proc Natl Acad Sci USA 84:7413-7,1987 and U.S. Patent No. 4,897,355, include this paper by reference in regard to its instruction to liposome.As an example, send and pass and to carry out via liposome, and can use and be purchased for example LIPOFECITN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg of Liposomal formulation, MD), SUPERFECT (Qiagen, Inc.Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison is WI) and according to other liposome of the standard method of this area exploitation etc.Also can use compound wherein from the diffusion of liposome or send that to pass be the liposome that designs at special speed or dosage.

Noisome as herein described can be used to send the delivery device of passing the open composition of this paper.Noisome is multicell or the unilamellar vesicle that comprises nonionic surface active agent.The aqueous solution of solute is wrapped up by the bilayer that is formed by the assembling of tensio-active agent macromole.Similar with liposome, noisome also is used for target and send and pass for example anticarcinogen, comprises methotrexate, Dx and immunological adjuvant.It has been generally acknowledged that they and transferosome, by the vesica of amphiphilic carbohydrate preparation with contain the polymkeric substance such as the chitin and inequality of amino group.

Nanometer rhodoplast as herein described is to can be used for sending the delivery device of passing the open composition of this paper.The nanometer rhodoplast is the nano vesicle that is prepared via the dialysis of the filter by determining the aperture by red corpuscle.These vesicas can be loaded the array of various bioactive moleculess, and described bioactive molecules comprises protein disclosed herein and composition.They are usually as the antineoplastic agent ideal carrier of bleomycin, dactinomycin etc. for example, but also can be used for sending passs steroid and other lipids etc.

Artificial red blood cells as herein described can be used for sending the another kind of delivery device of passing the open composition of this paper.The artificial red blood cells can form by interfacial polymerization and composite emulsifying method.Usually, " cell " wall is made of polyphtaloyl L-Methionin polymkeric substance/polystyrene, and inside is made of the hemoglobin solutions from the sheep red blood cell (SRBC) lysate.The granular size of microballoon institute load oxyphorase is generally about 1 to about 10mm.Their size, suppleness and oxygen carrying capacity are similar to red corpuscle.

Solid-liquid nano particle described herein is to can be used for sending the another kind of delivery device of passing the present composition.The solid-liquid nano particle is a nano particle, and they are scattered in the water phase surfactant mixture.They are made of the solid hydrophobic core with unimolecular layer phosphatide encrusting substance, are prepared by high-pressure homogenization usually.Immunostimulating complex (ISCOMS) is the example of solid-liquid nano particle.They are 40nm cage type supramolecule devices, contain lipid, cholesterol and hydrophobic antigen, mainly as immunological adjuvant.For example, ISCOM can be used for prolonging the blood plasma level of hypodermic ring spore mould A.

Microballoon as herein described and micro-capsule are to can be used for sending the another kind of again delivery device of passing the open composition of this paper.Send delivery system different with liposome, microballoon and micro-capsule do not have the water-based core usually, but have solid polymer substrate or film.These delivery devices obtain by the following method: the control precipitator method of polymkeric substance, the crosslinking of soluble polymer and two kinds of monomeric interfacial polymerizations or high-pressure homogenization.These entrapped compounds are separated by erosion or are discharged gradually from bank from the particle diffusion.Successfully develop fugitive peptide class preparation, for example such as the LHRH agonist of Leuprolide and music score of Chinese operas Rayleigh (triptoreline) etc.Poly-(lactide-co-glycolide) (PLGA) microballoon once was used for the treatment of advanced prostate cancer, endometriosis and other hormones response illness with every month dosage form of three times with every month at present.Leuprolide is a kind of LHRH super-agonist, uses solvent extration/method of evaporation it can be mixed in the multiple PLGA matrix.Just as described, all these delivery devices all can be used in the method disclosed herein.

Pulmosphere is other examples of another class that can be used for delivery device of the present invention.Pulmosphere is that density is lower (less than about 0.1m mL ^-1) the hollow porous particle.Pulmosphere has good redispersibility usually, concentrates the preparation of (supercritical fluid condensation) technology by supercritical liq usually.The protein that adopts the synchronous spraying desiccating method of some matrix such as carbohydrate, human serum albumin etc. to improve to be used for lung to send to pass and the stability of peptide (as Regular Insulin) and other biological molecule.This class is sent and is passed also and can be finished by microemulsion and liplid emulsions, and this two classes emulsion is to need not to import in a large number ultra-fine, ultra-thin, transparent oil-in-water (o/w) emulsion that mechanical energy can spontaneous formation.In this technology, emulsion can prepare under a certain temperature, and this temperature must want high than the phase inversion temperature of system.Under higher temperature conditions, this emulsion is water-in-oil (w/o) form, and when cooling off under phase inversion temperature, this emulsion then is reversed to o/w.Since very little mutually in it, so they are extremely stable, can be used for the slowly-releasing of steroid and vaccine.Liplid emulsions comprises neutral lipid core (being triglyceride level), and described core is by tensio-active agent such as Yelkin TTS triglyceride level and miglyol and by the unimolecular layer stabilization of amphiphilic lipids (being phosphatide).They are fit to passive target and active target.

Also studied other oral delivery system that send, described system is based on density and floating system (density and floating), the adhesivity (mucoadhesiveness) etc. of osmotic pressure adjustment, pH adjustment, swelling adjustment, change.The current rule round the clock according to disease of using or studying is sent these preparations of drug delivery and sustained release preparation bacterium to can be used to send and is passed composition disclosed herein.

One disclosed herein concrete aspect, disclosed compound (comprising nutritional additive and pharmaceutical preparation thereof) can as described hereinly be impregnated in the micro-capsule.

Aspect one disclosed herein, disclosed compound can be impregnated in the micro-capsule.On the one hand, micro-capsule contains the aggregation (agglomeration) and the chromium cpd as herein described of (primary) micro-capsule substantially, each basic micro-capsule individuality has a main casing (primary shell), wherein said chromium cpd is sealed by described main casing, and wherein said aggregation is by shell enclosure.These micro-capsules are called as " multinuclear micro-capsule " in this article.

On the other hand, this paper has described the micro-capsule that comprises chromium cpd, main casing and auxilliary shell, and wherein said main casing is sealed chromium cpd, and described auxilliary shell is sealed load material and main casing.These micro-capsules are called as " monokaryon micro-capsule " in this article.

Randomly, other load material can be encapsulated with chromium cpd.The load material can be the incomplete any material of dissolved in aqueous mixture.On the one hand, the load material is the mixture of solid, hydrophobic liquid or solid and hydrophobic liquid.On the other hand, the load material contains fat, oils, lipid, medicine (as small molecules), biologically active substance, nutritional additive (as VITAMIN), perfume compound or their mixture.The example of oils includes but not limited to animal oil (as fish oil, the living Mammals wet goods in sea), vegetables oil (as rapeseed oil or rapeseed oil), mineral oil, its derivative or their mixture.The load material can be oily matter purifying or partially purified such as lipid acid or its ester, triglyceride level or their mixture.On the other hand, the load material can be carotenoid (as Lyeopene), weighting agent (satiety agent), perfume compound, medicine (as water-insoluble medicine), particle, agricultural compound (as weedicide, sterilant, fertilizer) or aquaculture composition (as feed, pigment).

On the one hand, the load material can be omega-fatty acid.The example of omega-fatty acid include but not limited to alpha-linolenic acid (18:3n3), therapic acid (18:4n3), timnodonic acid (20:5n3) (EPA), docosahexenoic acid (22:6n3) (DHA), clupanodonic acid (22:5n3) (DPA), eicosatetraenoic acid (20:4n3), 21 carbon 5 alkene acids (21:5n3), clupanodonic acid (22:5n3), and their derivative and their mixture.The multiclass derivative of omega-fatty acid is known in the art.The example of suitable derivative includes but not limited to ester class for example plant sterol ester, side chain or straight chain C ₁-C ₃₀Alkyl ester, side chain or straight chain C ₂-C ₃₀Alkenyl esters or side chain or straight chain C ₃-C ₃₀Cycloalkyl ester (as plant sterol ester) and C ₁-C ₆Alkyl ester.Oil sources can be from aquatic organism (as sardines, capelin, Atlantic cod, oceanic herring, Atlantic Ocean mackerel, Atlantic Ocean catfish, salmon, sardines, shark, yaito tuna etc.) and plant (as flax, vegetables etc.) and microorganism (as fungi and algae).

On the one hand, the load material can contain antioxidant.The example of antioxidant includes but not limited to vitamin-E, CoQ ₁₀, tocopherol, antioxidant that polarity is stronger fat-soluble derivant such as ascorbic acid fatty acid ester (as Quicifal), plant milk extract (as rosemary oil, sage oil and wild marjoram oil), Seaweed Extract and synthetized oxidation preventive agent (as BHT, TBHQ, Santoflex, alkyl gallic acid ester, quinhydrones, tocotrienol).

Many different polymkeric substance can be used for producing the shell of monokaryon micro-capsule and multinuclear micro-capsule.The example of this base polymer includes but not limited to protein, poly phosphate, polysaccharide or their mixture.On the other hand, the shell material that is used to prepare monokaryon micro-capsule and multinuclear micro-capsule further comprises A type gelatin, the Type B gelatin, poly phosphate, gum arabic, alginate, chitin, carrageenin, pectin, starch, modified starch, alpha-lactalbumin, beta-lactoglobulin (lactoglobumin), ovalbumin, polysorbate (polysorbiton), maltodextrin, cyclodextrin, Mierocrystalline cellulose, methylcellulose gum, ethyl cellulose, HPMC (hydropropylmethylcellulose), carboxymethyl cellulose, milk protein, whey-protein, soybean protein, rapeseed protein, albumin, chitin, polylactide, poly--lactide-co-glycolide, the deutero-chitin, chitin, polylysine, various inorganic-organic composite materials, or their any mixture.What take into account equally is that the derivative of these polymkeric substance also can use.On the other hand, polymkeric substance can be kosher gelatin, non-kosher gelatin, Halal gelatin or non-Halal gelatin.

On the one hand, one or more layers shell contains the Bloom value less than 50 gelatin in monokaryon micro-capsule and the multinuclear micro-capsule.This gelatin is called as " low Bloom gelatin " in this article.The Bloom value has been described the gel-strength that 6.67% solution forms in the time of 18 hours 10 ℃ of gels.On the one hand, the Bloom value of low Bloom gelatin is lower than 40, is lower than 30, is lower than 20 or be lower than 10.On the other hand, the Bloom value of gelatin is 45,40,35,30,25,20,15,10,9,8,7,6,5,4,3,2,1 or 0, and wherein any two numerical value can be used to form a scope.On the other hand, low Bloom gelatin is present among the main casing and shell of multinuclear micro-capsule.On the one hand, low Bloom gelatin is an A type gelatin.On the other hand, low Bloom gelatin is Kenney ﹠amp; Ross Ltd., R.R.#3Shelburne, the A type gelatin that NS Canada produces.On the other hand, all having the Bloom value among the main casing of multinuclear micro-capsule and shell is zero gelatin.

On the one hand, be used to prepare the material of shell of monokaryon micro-capsule or multinuclear micro-capsule for by the preparation-obtained two-component system of the different mixture of polymers of two classes.On the one hand, this material is the complex coacervate between the polymeric constituent.The complex coacervation effect is because due to the interaction between two kinds of polymkeric substance that have an opposite charges.On the one hand, the shell material that is used to produce monokaryon micro-capsule and multinuclear micro-capsule is made of following material: (1) low Bloom gelatin and (2) Type B gelatin, poly phosphate, gum arabic, alginate, chitin, carrageenin, pectin, carboxymethyl cellulose, whey-protein, soybean protein, rapeseed protein, albumin or their any mixture.The mol ratio of different polymkeric substance can change to some extent.For example, the mol ratio of low Bloom gelatin and other polymeric constituent is 1:5 to 15:1.For example, when using low Bloom gelatin and poly phosphate, the mol ratio of low Bloom gelatin and poly phosphate is that about 8:1 is to about 12:1; When using low Bloom gelatin and Type B gelatin, mol ratio is 2:1 to 1:2; And when using low Bloom gelatin and alginate, mol ratio is 3:1 to 8:1.

Can include processing material in the shell material (as main casing or shell).The use of processing material can have multiple reason.For example, they can be used for promoting cohesion, the stable emulsion system of basic micro-capsule, the character of improving shell, control micro-capsule size and/or are used as antioxidant.On the one hand, processing material can be emulsifying agent, lipid acid, lipid, wax, microorganism cells (as yeast cell system), clay or mineral compound (as yellow soda ash).Do not wish to be entangled in theory, but these processing materials can promote the barrier of micro-capsule.On the one hand, one or more antioxidants can be added in the shell material.Oxidation-resistance all is useful (promptly prolonging the shell life-span etc.) in (as during cohesion and/or spraying drying) and the micro-capsule after forming during processing.The preferred small number of operations auxiliary agent that has used multiple effect.On the one hand, antioxidant can be phenolate thing, plant milk extract or sulfur-containing amino acid.On the one hand, xitix (or its salt for example sodium ascorbate or potassium ascorbate) can be used to promote the cohesion, control micro-capsule size of basic micro-capsule and as antioxidant.The spendable amount of antioxidant for about 100ppm to about 12,000ppm, perhaps about 1,000ppm is extremely about 5,000ppm.Other processing material for example metallo-chelate also can use.For example, ethylenediamine tetraacetic acid (EDTA) can be used to bind metal ion, and this metal ion can reduce the catalysed oxidn of load material.

On the one hand, the mean diameter of basic micro-capsule (main casing) for about 40nm to about 10 μ m, 0.1 μ m to about 10 μ m, 1 μ m to about 10 μ m, 1 μ m to about 8 μ m, 1 μ m to about 6 μ m, 1 μ m to about 4 μ m or 1 μ m extremely about 2 μ m, perhaps 1 μ m.On the other hand, the mean diameter of multinuclear micro-capsule be about 1 μ m to about 2000 μ m, 20 μ m to about 1000 μ m, about 20 μ m to about 100 μ m or about 30 μ m to about 80 μ m.On the other hand, the external diameter of monokaryon micro-capsule is about 1 μ m to 2,000 μ m.

Micro-capsule as herein described has high workload and structural strength usually simultaneously.For example, the workload of load material account for by weight monokaryon micro-capsule or multinuclear micro-capsule 20% to 90%, 50% to 70% or account for 60% by weight.

On the one hand, the disclosed method of U.S. Patent Application Publication text No.2003/0193102 (including in by quoting in full) can be used to seal chromium cpd described herein.What consider equally is can shelve one or more layers extra shell on the shell of monokaryon micro-capsule or multinuclear micro-capsule.On the one hand, the described technology of open text No.WO 2004/041251 A1 of international application (including in by quoting in full) can be used for extra shell is attached to monokaryon micro-capsule and multinuclear micro-capsule.

A) pharmaceutical composition and nutritive compositions

These lipids and antioxidant are intended to be used for animal-feed, pharmaceutical preparation, dietetic product (especially infant formulas) and are used for industrial production.What this comprised dietetic product too send the form of passing such as gel capsule and common microencapsulation etc.

As mentioned above, composition also can be used for vivo medicine-feeding with pharmaceutically acceptable carrier form." pharmaceutically acceptable " refer to certain material can't biologically or others cause untoward reaction, promptly this material can give the experimenter with nucleic acid or carrier, and can not cause any bad biological impact or with harmful mode and any other component interaction that contains the pharmaceutical composition of this material.As well known by the skilled person in the art, will inevitably select, thereby make that the activeconstituents degraded is minimum, and its adverse side effect for the experimenter is minimized carrier.

Composition can be by oral, parenteral (as intravenously) approach, intramuscularly, peritoneal injection, through administrations such as skin, external, local approach, comprises local intranasal administration or by the sucker administration.This paper employed " local intranasal administration " refers to by one or two nostril composition to be sent and passs to nose or nasal meatus, can comprise by spray system or the mechanism or send by atomizing nucleic acid or carrier and to pass of instiling.Giving composition by sucker can be via sending to pass by nose or mouth and is undertaken by spraying or the mechanism of instiling.Send and pass any zone that also can directly arrive respiratory system (as lung) via intubation.The exact amount of needed composition is different because of the experimenter, and this depends on experimenter's species, age, body weight and integral status, by severity, employed concrete nucleic acid or carrier, its mode of administration etc. of the abnormalism disorder of being treated.Therefore, can not stipulate the exact amount of every kind of composition.But appropriate vol can only use normal experiment just can determine under the situation of the instruction of considering this paper by those of ordinary skills.

If use the parenteral admin of composition, its feature is to pass through drug administration by injection usually so.Injection can be prepared as conventionally form (liquor or suspension), be mixed with solid form or the emulsion form that is suspended in the solution in the liquid before being adapted at injecting.Recently the parenteral admin method that makes improvements comprises uses slow-released system to keep constant dosage.Referring to for example U.S. Patent No. 3,610,795, include it in this paper by quoting.

This material can be present in solution, the suspension (for example being incorporated in particulate, liposome or the cell).These materials can be via antibody, acceptor or the specific cell type of receptors ligand target.Below with reference to document is to utilize example (Senter, et al., Bioconjugate Chem., 2:447-451,1991 of this technology with specific protein target tumor tissue; Bagshawe, K.D., Br.J.Cancer, 60:275-281,1989; Bagshawe, et al., Br.J.Cancer, 58:700-703,1988; Senter, et al., Bioconjugate Chem., 4:3-9,1993; Battelli, et al., Cancer Immunol.Immunother., 35:421-425,1992; Pietersz andMcKenzie, Immunolog.Reviews, 129:57-80,1992 and Roffler, et al., Biochem.Pharmacol., 42:2062-2065,1991).Vehicle is the high degree of specificity therapeutic retrovirus target of glioma (glicoma) cell in the lymphocyte of the liposome (lipid that comprises mediation drug targeting colorectal carcinoma) puted together of " Stealth " and other antibody, the acceptor by cell specific ligand mediated dna target, guiding cancer target and the mouse body for example.Below with reference to document is to utilize the example (Hughes et al., CancerResearch, 49:6214-6220,1989) of this technology with specific protein target tumor tissue; With Litzinger and Huang, Biochimica etBiophysica Acta, 1104:179-187,1992).In a word, acceptor participates in the endocytosis approach with composing type mode or the part mode of inducing.These acceptors are intensive in clathrin-coated pits, enter cell via the vesicle of clathrin parcel, pass acidifying endosome (acceptor is therein by sorting), subsequently or be recycled to cell surface and be stored in the cell, perhaps are degraded in lysosome.The internalization approach plays multiple effect, and for example the opportunistic of the removing of the absorption of nutritive substance, activated protein, macromolecular removing, virus and toxin enters, the dissociating and the adjusting of degraded and receptor level of part.A lot of acceptors are followed more than one the interior approach of born of the same parents, and this depends on cell type, acceptor density, part type, part is tired and ligand concentration.The existing summary of the molecule of receptor-mediated endocytosis and cell mechanism (Brown and Greene, DNA and Cell Biology 10:399-409,1991).

(1) pharmaceutically acceptable carrier

The composition that comprises antibody can be united with pharmaceutically acceptable carrier and is used for the treatment of.

Suitable carriers and preparation thereof are described in Remington:The Science and Practiceof Pharmacy (19th ed.) ed.A.R.Gennaro, Mack Publishing Company, and Easton, PA 1995.Usually, can in preparation, use proper normal pharmacologically acceptable salt to ooze to guarantee preparation etc.The example of pharmaceutically acceptable carrier includes but not limited to salt solution, Ringer ' s solution and glucose solution.The pH of solution is preferably about 5 to about 8, and more preferably from about 7 to about 7.5.Other carrier comprises that sustained release preparation for example contains the semipermeability matrix of the solid-state hydrophobic polymer of antibody, and this matrix is the particle form with definite shape, for example thin slice, liposome or particulate form etc.It is obvious to the skilled person that some carrier is more preferred, this for example depends on route of administration and by the concentration of administration composition.

Pharmaceutical carrier is known to those skilled in the art.They are generally the standard vector with the medicine administration of human most, comprise for example buffered soln of sterilized water, salt solution and physiological pH of solution.Composition can be through intramuscular or subcutaneous route administration.Other compound can give according to the employed standard method of those skilled in the art.

Except selected molecule, pharmaceutical composition also can comprise carrier, thickening material, thinner, buffer reagent, sanitas, tensio-active agent etc.Pharmaceutical composition also can comprise one or more activeconstituentss for example biocide, anti-inflammatory agent, narcotic etc.

Pharmaceutical composition is administration in many ways, and this depends on needs topical therapeutic or whole body therapeutic and zone to be treated.Can be by local approach (comprising eye approach, vaginal approach, rectum approach), oral route, inhalation route or parenteral route administration, for example by intravenous drip, subcutaneous injection, peritoneal injection or intramuscularly administration.Disclosed antibody can pass through in intravenous route, intraperitoneal approach, intramuscular approach, subcutaneous route, the chamber or through the skin administration.

The parenteral admin preparation comprises aseptic aqueous solution or non-aqueous solution, suspension and emulsion.Examples of non-aqueous is propylene glycol, polyoxyethylene glycol, vegetables oil (as sweet oil) and injectable organic ester (as ethyl oleate).Aqueous carrier comprises water, spirituous solution/aqueous solution, emulsion or suspension, comprises salt solution and buffering medium.The parenteral vehicle comprises sodium chloride solution, Ringer ' s glucose, glucose and sodium-chlor, lactic acid Ringer ' s or fixed oil.The intravenously vehicle comprises fluid and nutritious supplementary, electrolyte replenisher (for example based on those electrolyte replenishers of Ringer ' the s glucose) etc.Also can there be sanitas and other additive, for example biocide, antioxidant, sequestrant and rare gas element etc.

Local administration preparation can comprise ointment, lotion, paste, gel, drops, suppository, sprays, liquid and pulvis.Conventional medicine carrier, aqueous matrix, pulvis matrix or oleaginous base, thickening material etc. may be absolutely necessary or need.

Composition for oral administration comprises pulvis or granule, suspension or liquid, capsule, wafer or the tablet in water-soluble or the non-aqueous media.Thickening material, seasonings, thinner, emulsifying agent, dispersing auxiliary or tackiness agent may need.

Some compositions might be effective with pharmaceutically acceptable acid additive salt or base addition salt form administration, described additive salt by with mineral acid (example hydrochloric acid, Hydrogen bromide, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid etc.) and organic acid (as formic acid, acetate, propionic acid, hydroxyethanoic acid, lactic acid, pyruvic acid, oxalic acid, propanedioic acid, succsinic acid, toxilic acid and fumaric acid etc.) reaction forms, or by with mineral alkali (as sodium hydroxide, ammonium hydroxide, potassium hydroxide etc.) and organic bases (as monoalkylamine, dialkylamine, the thanomins of trialkylamine and arylamines and replacement etc.) reaction forms.

(2) therepic use

The dosage regimen of effective dose and composition can be determined according to experience, and make this class decision in those skilled in the art's limit of power.The dosage range of composition administration is for even as big as producing those dosage ranges of the required curative effect that wherein symptom disorder obtains medical treatment.Dosage should be greatly to causing adverse side effect, for example bad cross reaction, anaphylaxis etc.Usually, dosage can be different with following factor: whether patient age, illness, sex and disease severity, route of administration perhaps comprise other medicines, and can be determined by those skilled in the art in the dosage regimen.If any contraindication occurs, then this dosage can be adjusted by each doctor.Dosage can change to some extent, but and give with single agent or multi-agent form of medication every day, continue one day or several days.The guide of the suitable dose of appointment classification medicament can be referring to document.For example, select the guide of suitable antibody dosage can be referring to document, as Handbook of Monoclonal Antibodies about the therepic use of antibody, Ferrone et al., eds., Noges Publications, Park Ridge, NJ., (1985) ch.22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber etal., eds., Raven Press, New York (1977) pp.365-389.The antibody that uses separately typical every day dosage as every day about 1 μ g/kg to being up to 100mg/kg body weight or more, this depends on above-mentioned factor.

B) target send and passs

Disclosed liposome and micro-capsule can be via antibody, acceptor or receptors ligand by target to particular cell types such as islet cells.Following reference is for utilizing this technology to come the example of target particular organization (Senter, et al, Bioconjugate Chem 2:447-51,1991; Bagshawe, Br JCancer 60:275-81,1989; Bagshawe, et al, Br J Cancer 58:700-3,1988; Senter, et al, Bioconjugate Chem 4:3-9,1993; Battelli, et al, Cancer Immunol Immunother 35:421-5,1992; Pietersz and McKenzie, Immunolog Reviews 129:57-80,1992 and Roffler, et al, BiochemPharmacol 42:2062-5,1991).These technology can be used for various other specific cell types.

8. food

This paper also discloses the food that contains arbitrary micro-capsule disclosed herein and emulsion." food " refers to any material that can be taken (as eat, drink or absorb) by the experimenter.On the one hand, micro-capsule can be used as the nutritional additive of food.For example, micro-capsule and emulsion can be mounted with VITAMIN, omega-fatty acid and to other healthy and helpful compound.On the one hand, food can be bakery, wheaten food, meat product, frozen dairy product, milk-product, cheese product, egg-products, seasonings, powder, dessert, nut goods, plant protein preparation, hard candy, soft sweets, poultry prod, fruit juice, granulated sugar (as white sugar or brown sugar (Saccharum Sinensis Roxb.)), soy sauce, gravy, syrup, nourishing beverage, beverage, drink dried powder, jam or jelly, fish product or pet companion food through processing.On the other hand, food is bread, corn-dodger, cereal food, sausage, chicken, ice-creams, sour milk, milk, salad food flavouring, rice bran, fruit juice, drink dried powder, swiss roll, biscuit, crispbread, fast food, fruit pies or cake.

9. chip and microarray

Disclose that wherein to have a part of address at least be the chip of the part of sequence listed in arbitrary nucleotide sequence disclosed herein or sequence.Disclose also that wherein to have a part of address at least be the chip of the part of sequence listed in arbitrary peptide sequence disclosed herein or sequence.

Disclose also that wherein to have a part of address at least be the chip of variant of the part of sequence listed in arbitrary nucleotide sequence disclosed herein or sequence.Disclose also that wherein to have a part of address at least be the chip of variant of the part of sequence listed in arbitrary peptide sequence disclosed herein or sequence.

10. computer-readable medium

Be understandable that disclosed nucleic acid and protein can be expressed as the sequence of being made up of amino acid whose Nucleotide.Exist several different methods to show these sequences, for example, the Nucleotide guanosine can be expressed as G or g.Equally, the amino acid Xie Ansuan can be expressed as Val or V.Those skilled in the art understand as how any mode (every kind of mode all is considered to open in this article) in the existing multiple mode show and express arbitrary nucleic acid or protein sequence.Special consideration is that these sequences are shown in computer-readable medium in this article, for example commercially available floppy disk, tape, chip, hard disk, CD and optic disk or other computer-readable medium.The binary code that also discloses disclosed sequence is represented method.It is that those skilled in the art understands for which kind of computer-readable medium.Therefore, this computer-readable medium writes down, stores or preserved nucleic acid or protein sequence.

The computer-readable medium that records listed sequence of this paper and the information relevant with these sequences is disclosed.

11. test kit

Herein disclosed is the test kit that can be used for implementing method disclosed herein or can be used for using or preserving the reagent of composition disclosed herein is housed.This test kit can comprise discussed in this article or be considered to implement disclosed method requisite or useful any reagent or combination of agents.For example, this test kit can comprise one or more eukaryotic microorganisms disclosed herein and the substratum that for example is used to cultivate them.This test kit also comprises for example lipid or antioxidant and use or gives the instrument of these materials.

12. have the composition of identity function

Be understandable that composition disclosed herein has some function, for example produce the lipid of certain ratio.Herein disclosed is the requirement of some configuration aspects, genetic aspect and the function aspects that realize disclosed function, and be understandable that, exist multiple can realize with the structure of the identical function of disclosed structurally associated, genetic background and the function background, and these structures finally can obtain identical result, for example produce the lipid of certain ratio.

D. prepare method for compositions

Unless clearly indicate in addition, otherwise composition disclosed herein and carry out the requisite composition of disclosed method can adopt well known by persons skilled in the art any method of this concrete reagent or compound to prepare by using.

1. nucleic acid is synthetic

For example, the oligonucleotide that nucleic acid for example prepares as primer can use the standard chemical synthesis method to prepare, and perhaps uses enzymatic method or any other currently known methods to produce.These class methods are included in the enzymic digestion of standard and carry out the nucleotide fragments separation afterwards (referring to for example Sambrook et al, MolecularCloning:A Laboratory Manual, 2nd Edition (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters5,6) and pure synthesis method, for example (for example the model of Milligen-Biosearch is 8700 automatic DNA synthesizer DNA by adopting Milligen or Beckman System 1Plus dna synthesizer, Burlington, MA or ABI 380B type) the cyanoethyl phosphoramidite method.The synthesis method that is used to prepare oligonucleotide is described in Ikuta et al., Ann.Rev.Biochem.53:323-356,1984 equally, (phosphotriester method and tris phosphite method) and Narang et al., Methods Enzymol., 65:610-620,1980, (phosphotriester method).The protein nucleic acid molecule can use currently known methods for example by Nielsen et al., Bioconjug.Chem.5:3-7, and 1994 described those methods prepare.

2. peptide is synthetic

Producing disclosed proteinic a kind of method is by the albumen chemical technology two or more peptides or polypeptide to be coupled together.For example, peptide or polypeptide can use current experiments equipment, adopt Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tertbutyloxycarbonyl) chemical preparations (Applied Biosystems, Inc., Foster City, CA) chemosynthesis.Those skilled in the art will readily understand, can synthesize by for example standard chemical reaction with corresponding peptide of disclosed protein or polypeptide.

For example, can synthesize certain peptide or polypeptide and it is not cut down from its synthetic resins, and can synthesize other peptide or protein fragments and it is cut down from resin, expose the end group that function is blocked on other fragment thus subsequently.By the peptide condensation reaction, these two fragments can be covalently bound at its C-terminal and N-terminal respectively via peptide bond, thereby form antibody or its fragment (Grant GA (1992) Synthetic Peptides:A User Guide.W.H.Freemanand Co., N.Y. (1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis.Springer-Verlag Inc., NY (including it in this paper with regard to the synthetic relevant content of itself and peptide at least)).Perhaps, peptide or polypeptide are as described herein independent in vivo synthetic.After separation, peptide that these are separate or polypeptide can be connected via similar peptide condensation reaction and form peptide or its fragment.

For example, clone obtains or the zymetology of synthetic peptide section connects and makes relatively short peptide fragment to be connected to produce bigger peptide fragment, polypeptide or whole protein structure domains (Abrahmsen L etal., Biochemistry, 30:4151,1991).Perhaps, can utilize the native chemical connection of synthetic peptide to make up big peptide or polypeptide by synthesizing than short peptide fragment.This method is formed (Dawson et al.Science, 266:776-779,1994) by two step chemical reactions.The first step is between the peptide section that contains aminoterminal Cys residue of synthetic peptide-thioesters and another kind of not protection of not protection chemical selective reaction to take place, thereby produces as initial covalency product, the intermediate product that is connected with thioesters.Under the situation that does not change reaction conditions, spontaneous, intramolecular reaction fast that this intermediate product can experience, thus form natural peptide bond (Baggiolini M et al.FEBS Lett.307:97-101,1992 in connection site; Clark-Lewis I et al., J.Biol.Chem., 269:16075,1994; Clark-Lewis I et al., Biochemistry, 30:3128,1991; Rajarathnam K et al., Biochemistry 33:6623-30,1994).

Perhaps, do not have the peptide section of protection to connect by chemical mode, wherein the key that forms between the peptide section because chemistry connects is non-natural (non-peptide) key (Schnolzer, M et al.Science, 256:221,1992).This technology be used to the analogue of synthetic proteins structural domain and purer relatively in a large number, have a complete bioactive protein (deLisle Milton-RC-et in ProteinChemistry IVT Academic Press, New York, pp.257-267,1992).

3. preparation method for compositions

The method that discloses the preparation composition and prepared the intermediate product of said composition.For example, the eukaryotic microorganisms that can produce required lipid and antioxidant and separating and the method for required lipid of purifying and antioxidant is disclosed.There are multiple these method for compositions that can be used for preparing, for example synthetic chemistry method and standard molecular biology method.Be understandable that, prepare these and other disclosed method for compositions by specifically open.

Disclose by method and produced the cell that obtains with arbitrary nucleic acid transformant.The method that discloses by the disclosed nucleic acid transformant that exists with arbitrary non-natural produces the cell that obtains.

Disclose by disclosed eukaryotic microorganisms and produced any lipid that obtains.The method that discloses by expression of peptides in disclosed organism produces any peptide that obtains.The use method for compositions is disclosed

4. with the method for composition as research tool

Disclosed composition can be used as research tool in every way, and is used to produce for example lipid and antioxidant.

As described herein, disclosed composition both can be used as the reagent in the microarray, also can be used as research or analyzed the reagent that has microarray now.Disclosed composition can be used for separating or identifying in any currently known methods of single nucleotide polymorphism.Composition also can be used for determining in any allelotrope analytical procedure of organism bacterial strain for example disclosed herein, especially allelotrope analysis, and reason is that it is relevant with the generation of lipid and antioxidant.Composition also can be used in any known screening assays relevant with chip/microarray.Composition also can be used for using any currently known methods of the computer readable device of disclosed composition, thereby for example studies dependency or carry out the molecule modeling analysis relevant with disclosed composition.

5. genetic modification and gene disruption method

Disclosed composition and method are used for the gene disruption and the genetic modification of target in can experiencing any animal of these incidents.Genetic modification and gene disruption refer to selectivity and remove or change gene or one section method, technology and composition that karyomit(e) is purpose in the organism eukaryote for example disclosed herein, and its mode is to continue this modification by duplicating of organism.Usually, for example, with being designed to and can transforming cell for example as herein described with the carrier of a certain regional homologous recombination of the concrete karyomit(e) that comprised in the cell or nucleic acid.This homologous recombination incident can produce and contain the foreign DNA that is fed to reading frame and have the karyomit(e) of DNA on every side.In the genome that is comprised in feasible sudden change (for example point mutation) transfered cell that can specificity is very high of such experimental program.The method of carrying out such homologous recombination is also open at this paper.

Embodiment

Herein disclosed is the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ IDNO:1 list has at least 94% identity.This eukaryotic microorganisms can produce the unsaturated fatty acids with condition of production shown in Figure 2.This eukaryotic microorganisms is from net slime mould door, net slime mould guiding principle, thraustochytriale subclass, thraustochytriale, thraustochytriale section and/or genus thraustochytrium.This eukaryotic microorganisms can be genus thraustochytrium kind, golden yellow thraustochytriale, pink thraustochytriale or line shape thraustochytriale.This eukaryotic microorganisms also can and have ATCC numbering 20888,20889,20890,20891 or 20892 from thraustochytriale section.

This paper also discloses the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQID NO:1 list has at least 94% identity, and wherein said microorganism belongs to from schizochytrium limacinum.This eukaryotic microorganisms can be the schizochytrium limacinum genus and plants.

This paper also discloses the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQID NO:1 list has at least 94% identity, and wherein this eukaryotic microorganisms contains ω-3 or ω-6 lipid acid.This eukaryotic microorganisms also can contain DHA or DPA.

This paper also discloses the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQID NO:1 list has at least 94% identity, and wherein the lipid or the fatty acid component of this Institute of Micro-biology's generation are at least about 4wt% to 6wt%.Described lipid can contain DHA.Lipid composition also can contain account for about 25wt% fatty acid component to the n-3DHA of about 40wt% fatty acid component, account for about 6wt% fatty acid component extremely about 10wt% fatty acid component n-6DPA and account for the extremely n-3EPA of about 3wt% fatty acid component of about 0wt% fatty acid component.

Also disclose the composition that comprises the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.Said composition can further include substratum and/or nutritive substance.

Also disclose the composition that comprises the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, and wherein said composition is a biomass.Eukaryotic microorganisms in the composition can be from net slime mould door, net slime mould guiding principle, thraustochytriale subclass, thraustochytriale, thraustochytriale section or genus thraustochytrium.This eukaryotic microorganisms can be genus thraustochytrium kind, golden yellow thraustochytriale, pink thraustochytriale or line shape thraustochytriale.This eukaryotic microorganisms also can and have ATCC numbering 20888,20889,20890,20891 or 20892 from thraustochytriale section.

Also disclose the composition that comprises the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:I list has at least 94% identity, and wherein said eukaryotic microorganisms belongs to from schizochytrium limacinum.This eukaryotic microorganisms can be the schizochytrium limacinum genus and plants.

Also disclose the composition that comprises the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, and this eukaryotic microorganisms can produce the unsaturated fatty acids with condition of production shown in Figure 2.This unsaturated fatty acids contains ω-3 or ω-6 lipid acid.This unsaturated fatty acids also contains DHA or DPA.

The composition that comprises the eukaryotic microorganisms with 18S sequence is also disclosed, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, and wherein lipid that this eukaryotic microorganisms produced or fatty acid component are at least about 4wt% to 6wt%.Described lipid can contain DHA.Lipid composition also can contain account for about 25wt% fatty acid component to the n-3DHA of about 40wt% fatty acid component, account for about 6wt% fatty acid component extremely about 10wt% fatty acid component n-6DPA and account for the extremely n-3EPA of about 3wt% fatty acid component of about 0wt% fatty acid component.

Also disclose the composition that comprises the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 80% identity.

Composition is also disclosed, described composition contain account for about 25wt% fatty acid component to the n-3 DHA of about 40wt% fatty acid component, account for about 6wt% fatty acid component extremely about 10wt% fatty acid component n-6 DPA and account for the extremely n-3 EPA of about 3wt% fatty acid component of about 0wt% fatty acid component.

Also disclose the method for preparing lipid composition, described method comprises: cultivate eukaryotic microorganisms as herein described in different oxygen substratum, and separate this lipid composition.The lipid composition for preparing according to this method is also disclosed.

The delivery device that includes above-mentioned arbitrary composition is also disclosed.For example, disclose a kind of delivery device, described delivery device includes the composition of the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.This delivery device can comprise micro-capsule, microballoon, nanometer ball or nano particle, liposome, noisome, nanometer rhodoplast, solid-liquid nano particle, Leuprolide, gel, gel capsule, tablet, lotion, paste, sprays, emulsion or pulvis.

Also disclose the aggregation that comprises basic micro-capsule and the micro-capsule of load material, each basic micro-capsule individuality all has main casing, and wherein said load material contains arbitrary above-mentioned composition, and is sealed by main casing, and wherein said aggregation is by shell enclosure.Main casing and/or shell can comprise tensio-active agent, gelatin, poly phosphate, polysaccharide or their mixture.Main casing and/or shell also can comprise the Type B gelatin, poly phosphate, gum arabic, alginate, chitin, carrageenin, pectin, starch, modified starch, alpha-lactalbumin, beta-lactoglobulin (lactoglobumin), ovalbumin, polysorbate (polysorbiton), maltodextrin, cyclodextrin, Mierocrystalline cellulose, methylcellulose gum, ethyl cellulose, HPMC (hydropropylmethylcellulose), carboxymethyl cellulose, milk protein, whey-protein, soybean protein, rapeseed protein, albumin, the kosher gelatin, the non-kosher gelatin, Halal gelatin or non-Halal gelatin, or their mixture.It is that about 51 to about 300 gelatin, Bloom value are coacervate about 0, about 210, about 220 or about 240 gelatin, gelatin and poly phosphate for about 0 to about 300 gelatin, Bloom value for about 0 to about 50 gelatin, Bloom value that main casing and/or shell also can comprise complex coacervate, A type gelatin, isinglass, Bloom value.

The loading substance of disclosed micro-capsule can comprise the oil from thraustochytriale, schizochytrium limacinum or their mixture.This loading substance is about 20% to about 90% or 50% to about 70% of micro-capsule by weight.

Its mean diameter of the shell of disclosed micro-capsule be about 1 μ m to about 2000 μ m, about 20 μ m to about 1000 μ m, about 30 μ m to about 80 μ m, about 40 μ m to about 10 μ m or about 0.1 μ m to about 5 μ m.

The nutritional additive that comprises arbitrary above-mentioned composition, delivery device or micro-capsule is also disclosed.Disclosed nutritional additive is tablet, gel capsule, capsule, liquid or syrup form.

The food that comprises arbitrary above-mentioned composition, delivery device or micro-capsule is also disclosed.This food can be bakery, wheaten food, meat product, frozen dairy product, milk-product, cheese product, egg-products, seasonings, powder, dessert, nut goods, plant protein preparation, hard candy, soft sweets, poultry prod, fruit juice, granulated sugar, soy sauce, gravy, syrup, nourishing beverage, beverage, drink dried powder, jam or jelly, infant formulas or infant food through processing.This food also can be fish product, pet companion food, cattle food or aquaculture feed.Food also can be bread, corn-dodger, cereal food, sausage, chicken, ice-creams, sour milk, milk, salad food flavouring, rice bran, fruit juice, drink dried powder, swiss roll, biscuit, crispbread, fast food, fruit pies or cake.

Also disclose and sent the method for passing the experimenter, comprised giving the arbitrary above-mentioned composition of this experimenter, delivery device, micro-capsule or food composition.This experimenter can be Mammals.This experimenter can also be the people.

Also disclose arbitrary above-mentioned micro-capsule and be used to prepare the purposes of the load material being sent the medicine of passing the experimenter.

Also disclose the method that reduces subject inner cholesterol level, triglyceride levels or their combination, comprised the step of the arbitrary above-mentioned composition, delivery device, micro-capsule, nutritional additive or the food that give this experimenter's significant quantity.

The method of essential trace element in the additional subject is also disclosed, described method comprises the step of the arbitrary above-mentioned composition, delivery device, micro-capsule, nutritional additive or the food that give this experimenter's significant quantity, and wherein said composition, delivery device, micro-capsule, additive and food contain essential trace element.

Also disclose the method that improves insulin sensitivity in the subject, comprised the step of the arbitrary above-mentioned composition, delivery device, micro-capsule, nutritional additive or the food that give this experimenter's significant quantity.

Also disclose the method that alleviates experimenter's hyperglycemia, comprised the step of arbitrary above-mentioned composition, delivery device, micro-capsule, nutritional additive or the food of this experimenter's significant quantity.

Also disclose the method that alleviates experimenter's hypercholesterolemia, comprised the step of arbitrary above-mentioned composition, delivery device, micro-capsule, nutritional additive or the food of this experimenter's significant quantity.

Also disclose the method that reduces experimenter's body fat, comprised the step of arbitrary above-mentioned composition, delivery device, micro-capsule, nutritional additive or the food of this experimenter's significant quantity.

Also disclose promotion experimenter ways of preventing obesity, comprised the step of arbitrary above-mentioned composition, delivery device, micro-capsule, nutritional additive or the food of this experimenter's significant quantity.

Also disclose the method for treatment or prevention experimenter diabetes, comprised the step of arbitrary above-mentioned composition, delivery device, micro-capsule, nutritional additive or the food of this experimenter's significant quantity.

Also disclose a kind of pharmaceutical preparation, comprised arbitrary above-mentioned composition, delivery device or micro-capsule and pharmaceutical carrier.

Also disclose the food that includes certain composition, described composition includes the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.This food can be bakery, wheaten food, meat product, frozen dairy product, milk-product, cheese product, egg-products, seasonings, powder, dessert, nut goods, plant protein preparation, hard candy, soft sweets, poultry prod, fruit juice, granulated sugar, soy sauce, gravy, syrup, nourishing beverage, beverage, drink dried powder, jam or jelly, infant formulas or infant food through processing.This food also can be fish product, pet companion food, cattle food or aquaculture feed.Food also can be bread, corn-dodger, cereal food, sausage, chicken, ice-creams, sour milk, milk, salad food flavouring, rice bran, fruit juice, drink dried powder, swiss roll, biscuit, crispbread, fast food, fruit pies or cake.

Reduction subject inner cholesterol level is also disclosed, the method of triglyceride level or their combination, comprise the composition that gives significant quantity, nutritional additive, the step of delivery device or food, described composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ IDNO:1 list has at least 94% identity, described nutritional additive contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described delivery device contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described food contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.

The method of essential trace element in the additional subject is also disclosed, described method comprises the composition that gives significant quantity, nutritional additive, the step of delivery device or food, described composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described nutritional additive contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described delivery device contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described food contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.

The method that improves insulin sensitivity in the subject is also disclosed, described method comprises the composition that gives significant quantity, nutritional additive, the step of delivery device or food, described composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described nutritional additive contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described delivery device contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described food contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.

The method that alleviates experimenter's hyperglycemia is also disclosed, described method comprises the composition that gives significant quantity, nutritional additive, the step of delivery device or food, described composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described nutritional additive contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described delivery device contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described food contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.

The method that alleviates experimenter's hypercholesterolemia is also disclosed, described method comprises the composition that gives significant quantity, nutritional additive, the step of delivery device or food, described composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described nutritional additive contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described delivery device contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described food contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.

The method that reduces experimenter's body fat is also disclosed, described method comprises the composition that gives significant quantity, nutritional additive, the step of delivery device or food, described composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described nutritional additive contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described delivery device contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described food contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.

Promotion experimenter ways of preventing obesity is also disclosed, described method comprises the composition that gives significant quantity, nutritional additive, the step of delivery device or food, described composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described nutritional additive contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described delivery device contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described food contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.

The method of treatment or prevention experimenter diabetes is also disclosed, described method comprises the composition that gives significant quantity, nutritional additive, the step of delivery device or food, described composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described nutritional additive contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described delivery device contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity, described food contains composition, said composition contains the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.

Also disclose the pharmaceutical preparation that contains composition, described composition contains the eukaryotic microorganisms with 18S sequence, and the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.

VI embodiment

Provide following embodiment the complete disclosure and description that provides the application's claimed compounds, composition, article, device and/or method how to prepare and assess for those skilled in the art is provided; be to illustrate for example purely, and have no intention disclosure is limited.Endeavoured to ensure the accuracy of numeral (as amount, temperature etc.) aspect, can some sum of errors deviation of existence but still should consider.Except as otherwise noted, otherwise umber is parts by weight, and temperature is ℃ or room temperature, and pressure is normal atmosphere or near normal atmosphere.

1. the separation of embodiment 1:ONC-T18 genus thraustochytrium kind bacterial strain

Adopted classical bacteriology bacterial strain purification technique, purpose is that from Advocate Harbor the mangrove leaf that Nova Scotia collects separates ONC-T18.(contain 5g L in the filtering seawater of 1 L, 0.2 μ m at nutritional medium ^-1Glucose, 2g L ^-1Peptone, 2g L ^-1Yeast extract and 15.0g L ^-1Agar) in 25 ℃ of cultured continuously ONC-T18, until obtaining enough purity.Subsequently, preparation contains 15% artificial seawater (nutritional type seawater), is added with nitrogenous source and carbon source (is respectively 60g L ^-1Glucose and 10g L ^-1Yeast extract) liquid nutrient medium.Inoculate this substratum (in the 250ml flask 50ml being housed) with ONC-T18,, and inflate by vibrating in 120rpm then 25 ℃ of cultivations.

Separate ONC-T18 by centrifugal from substratum, then washed cell biomass, recentrifuge and thoroughly lyophilize.The pair cell biomass is weighed then, and purpose is to pass through the numerical value of the biomass that write down/rise substratum and determines culture efficiency.Use Bligh ﹠amp; The Dyer method is extracted lipid composition from biomass, subsequently the separation of fatty acids methyl esters.Carry out transesterify by following steps: cryodesiccated cellular material is transferred to 10ml has in the test tube of screw-cap, and 10% methyl alcohol HCl and methylene dichloride are added this test tube, make this mixture 90 ℃ of reactions 2 hours.Then by adding hexane: chloroform extracts fatty acid methyl ester, and measures the methyl esters component by gas-chromatography (FID), and purpose is to determine the lipid acid condition of production of various microorganisms and symbiosis group (ONC-T18).By relatively in the beginning (C23:0) of transesterify process and when finishing (C19:0) to mark the GC peak area of (C19:0 and C23:0) in determine that amount adds two, measured the concentration of various fatty acid methyl esters (C14:0 to C22:6).Make the total fatty acids amount/gram stem cell biological amount that calculates in this way and the percentage composition of every kind of lipid acid be shown in Fig. 2.

By to these results' analysis and in conjunction with those results that are shown among Fig. 1, can find that ONC-T18 shows the DHA of generation increasing amount and the ability of significant quantity EPA and DPA.ONC-T18 has produced about 25%DHA, 8.0% (n-6) DPA and 1.0%EPA in this non-Optimal compositions of fermentation medium.Select ONC-T18:(1 based on the required combination of features of following economy subsequently) can farthest carry out heterotrophic growth (comparing) with control strain; (2) contain ω-3 highly unsaturated fatty acids of high per-cent; (3) can in cheap nutritive substance, grow; (4) has thermotolerance and (5) have wide salt tolerance.

In addition, multiple different strains and the ONC-T18 with oleaginous microorganism compares.This every kind of microorganism wherein all is considered to include thraustochytriale, and produces the oil of the amount of Table 3.

Table 3, []=mg/g

Be understandable that ONC-T18 as described herein is the same,, disclose and used by the represented a series of oleaginous microorganisms of produce oil power disclosed herein by the per-cent of for example DHA and oily ultimate production or by the DHA ultimate production.

2. embodiment 2: use genetic technique to identify eucaryon genus thraustochytrium kind ONC-T18

Use polymerase chain reaction (PCR) technology and target in the primer (universal primers of all eukaryote species) of 18S ribosomal RNA gene, can produce the PCR product (as described in embodiment 1) that separates the eukaryotic microorganisms structure gene that obtains from ONC-T18.Then the PCR product is checked order, and will be somebody's turn to do sequence called after SEQ ID NO:1 (referring to Fig. 2) for the eukaryote species.

Use BLAST (basic part comparison research tool) algorithm, with SEQ ID NO:1 and in genome database GenBank (American National biotechnology information center, national sanitary institute, Bei Saisida, Maryland, the U.S.) nucleotide sequence in is compared, and identifies SEQ IDNO:1 and line shape thraustochytriale [AF265338] relation (97.5% similarity) the most closely found.

The BLAST result of ONC-T18 genus thraustochytrium kind is as follows.

Sequences producing significant alignments： (bits)Value

gi|14279326|gb|AF265338.1|Thraustochytrium striatum small subun... 2126 0.0

gi|50508012|dbj|AB183657.1|Thraustochytriidae sp.MBIC110 72 gen... 2121 0.0

gi|54778780|gb|AY773276.1|Thraustochytriidae sp.FJN-10 18S rib... 1857 0.0

gi|50508019|dbj|AB183664.1|Thraustochytriidae sp.MBIC11093 gen... 1828 0.0

gi|38524571|dbj|AB126669.1|Thraustochytrium sp.CHN-1 gene for... 1748 0.0

gi|24817740|dbj|AB073308.2|Thraustochytriidae sp.N1-27 gene fo... 1628 0.0

gi|50508018|dbj|AB183663.1|Thraustochytriidae sp.MBIC11092 gen... 1257 0.0

gi|50508017|dbj|AB183662.1|Thraustochytriidae sp.MBIC11091 gen... 1257 0.0

gi|50508015|dbj|AB183660.1|Thraustochytriidae sp.MBIC11084 gen... 1255 0.0

gi|50508011|dbj|AB183656.1|Thraustochytriidae sp.MBIC11070 gen... 1255 0.0

gi|50508016|dbj|AB183661.1|Thraustochytriidae sp.MBIC11086 gen... 1249 0.0

gi|15823623|dbj|AB052555.1|Schizochytrium sp.KH105 gene for 18... 1245 0.0

gi|50508013|dbj|AB183658.1|Thraustochytriidae sp.MBIC11075 gen... 1227 0.0

gi|50508010|dbj|AB183655.1|Thraustochytriidae sp.MBIC11067 gen... 1213 0.0

gi|54303872|gb|AY758384.1|Schizochytrium sp.FJU-512 18S riboso... 1158 0.0

gi|14279326|gb|AF265338.1|AF265338 Thraustochytrium striatum sma... 1106 0.0

gi|6492308|gb|AF155209.1|AF155209 Labyrinthulid quahog parasite... 765 0.0

gi|16209570|gb|AY052644.1|Labyrinthulid quahog parasite QPX sma... 757 0.0

gi|9755031|gb|AF261664.1|AF261664 Labyrinthulid quahog parasite... 757 0.0

gi|58176547|gb|AY870336.1|Thraustochytriidae sp.Fngl 18S ribos... 735 0.0

gi|67624914|dbj|AB191425.1|Uncultured eukaryote gene for small... 724 0.0

gi|5509891|dbj|AB022112.1|Thraustochytrium striatum gene for 18... 724 0.0

gi|561884|gb|L34054.1|ULKRRE Ulkenia profunda 18S ribosomal RNA... 702 0.0

gi|50508014|dbj|AB183659.1|Thraustochytriidae sp.MBIC11077 gen... 686 0.0

gi|50508008|dbj|AB183653.1|Thraustochytriidae sp.MBIC11060 gen... 686 0.0

gi|50508009|dbj|AB183654.1|Thraustochytriidae sp.MBIC11063 gen... 658 0.0

gi|41391986|emb|AJ535188.1|Pleurosira cf.laevis 18S rRNA gene，... 634 e-178

gi|28316562|gb|AF525670.1|Pleurosira laevis small subunit ribos... 634 e-178

gi|5509889|dbj|AB022110.1|Thraustochytrium aureum gene for 18S... 634 e-178

gi|561883|gb|L34668.1|TUKRRE Thraustochytrium kinnei 18S ribosom... 628 e-176

gi|5509894|dbj|AB022115.1|Ulkenia radiata gene for 18S rRNA 624 e-175

gi|5509893|dbj|AB022114.1|Ulkenia profunda gene for 18S rRNA 624 e-175

gi|5509895|dbj|AB022116.1|Ulkenia Misurgensis gene for 18S rRNA 603 e-169

gi|9027563|gb|AF257315.2|Thraustochytriidae sp.BS2 18S ribosom... 589 e-164

gi|5509886|dbj|AB022107.1|Schizochytrium limacinum gene for 18S... 581 e-162

gi|48727879|gb|AY620254.1|Metromonas simplex clone TC-S small s... 571 e-159

gi|33309650|gb|AF411282.1|Unidentified cercozoan 18S ribosomal... 569 e-158

gi|28076844|gb|AF530543.1|Uncultured eukaryote clone AT4-68 18S... 531 e-147

gi|30144485|gb|AY256273.1|Uncultured eukaryote isolate E170 sma... 517 e-143

gi|30144529|gb|AY256317.1|Uncultured eukaryote isolate D107 sma... 507 e-140

gi|14579477|gb|AF363207.1|Eukaryote marine clone ME1-24 18S rib... 505 e-139

gi|39578677|gb|AY426906.1|Uncultured marine eukaryote clone BL0... 504 e-139

gi|39981869|gb|AY381216.1|Uncultured eukaryote clone BL010625.3... 504 e-139

gi|73533408|gb|DQ103811.1|Uncultured marine eukaryote clone M4_... 504 e-139

gi|73533402|gb|DQ103805.1|Uncultured marine eukaryote clone M3_... 504 e-139

gi|73533389|gb|DQ103792.1|Uncultured marine eukaryote clone M2_... 504 e-139

gi|73533382|gb|DQ103785.1|Uncultured marine eukaryote clone M1_... 504 e-139

gi|30144534|gb|AY256322.1|Uncultured eukaryote isolate D179 sma... 504 e-139

gi|24817738|dbj|AB073305.2|Thraustochytriidae sp.H1-14 gene fo... 504 e-139

gi|30268157|emb|AJ519935.1|AST519935Aplanochytrium stocchinoi p... 504 e-139

gi|58531881|gb|AY882527.1|Uncultured marine eukaryote clone T41... 504 e-139

gi|463127|gb|L27634.1|LADDLRRNA Labyrinthuloides minuta 16S-like... 504 e-139

gi|39981839|gb|AY381186.1|Uncultured eukaryote clone OR000415.1... 502 e-138

gi|39981824|gb|AY381171.1|Uncultured eukaryote clone HE001005.1... 502 e-138

gi|18026024|gb|AY046848.1|Uncultured eukaryote isolate C3_E019... 502 e-138

gi|18026022|gb|AY046846.1|Uncultured eukaryote isolate C3_E017... 502 e-138

gi|18026014|gb|AY046838.1|Uncultured eukaryote isolate C3_E008... 502 e-138

gi|18026008|gb|AY046832.1|Uncultured eukaryote isolate C3_E002... 502 e-138

gi|18025980|gb|AY046804.1|Uncultured eukaryote isolate C2_E014... 502 e-138

gi|18025969|gb|AY046793.1|Uncultured eukaryote isolate C2_E002... 502 e-138

gi|18025801|gb|AY046625.1|Uncultured eukaryote isolate C1_E024... 502 e-138

gi|67624915|dbj|AB191426.1|Uncultured eukaryote genefor small... 502 e-138

gi|67624913|dbj|AB191424.1|Uncultured eukaryote gene for small... 502 e-138

gi|67624912|dbj|AB191423.1|Uncultured eukaryote gene for small... 502 e-138

gi|39981861|gb|AY381208.1|Uncultured eukaryote clone BL010320.1... 500 e-138

gi|14349249|dbj|AB052556.1|Thraustochytrium sp.KK17-3 gene for... 500 e-138

gi|20218962|dbj|AB073307.1|Thraustochytriidae sp.M4-103 gene f... 498 e-137

gi|59709960|gb|AY916582.1|Uncultured eukaryote clone Zeuk76 18S... 496 e-136

gi|18025960|gb|AY046784.1|Uncultured eukaryote isolate A3_E043... 496 e-136

gi|18025789|gb|AY046613.1|Uncultured eukaryote isolate C1_E009... 496 e-136

gi|30144548|gb|AY256336.1|Uncultured eukaryote isolate D278 sma... 496 e-136

gi|2138106|gb|U59933.1|U59933 Scybalium jamaicense 18S ribosomal... 496 e-136

gi|53828186|gb|AY744948.1|Phytophthora palmivora isolate 88108... 494 e-136

gi|60687349|gb|AY821976.1|Uncultured oomycete clone CV1_B2_5 sm... 494 e-136

gi|60687347|gb|AY821974.1|Uncultured Phytophthora-like oomycete... 494 e-136

gi|60687342|gb|AY821969.1|Uncultured oomycete clone CV1_B1_49 s... 494 e-136

gi|39981870|gb|AY381217.1|Uncultured eukaryote clone BL010625.3... 494 e-136

gi|39981864|gb|AY381211.1|Uncultured eukaryote clone BL010320.2... 494 e-136

gi|39981860|gb|AY381207.1|Uncultured eukaryote clone BL010320.6... 494 e-136

gi|39981844|gb|AY381191.1|Uncultured eukaryote clone BL000921.1... 494 e-136

gi|18026046|gb|AY046870.1|Uncultured eukaryote isolate C3_E044... 494 e-136

gi|18026039|gb|AY046863.1|Uncultured eukaryote isolate C3_E035... 494 e-136

gi|18026031|gb|AY046855.1|Uncultured eukaryote isolate C3_E026... 494 e-136

gi|42412527|gb|AY486144.1|Pythium insidiosum 18S ribosomal RNA... 494 e-136

gi|73533425|gb|DQ103828.1|Uncultured marine eukaryote clone M2_... 494 e-136

gi|34576227|gb|AY129064.1|Uncultured marine eukaryote UEPAC45p4... 494 e-136

gi|30144522|gb|AY256310.1|Uncultured eukaryote isolate D85 smal... 494 e-136

gi|30144521|gb|AY256309.1|Uncultured eukaryote isolate D84 smal... 494 e-136

gi|30144518|gb|AY256306.1|Uncultured eukaryote isolate D79 smal... 494 e-136

gi|30144475|gb|AY256263.1|Uncultured eukaryote isolate E106 sma... 494 e-136

gi|30144473|gb|AY256261.1|Uncultured eukaryote isolate E94s mal... 494 e-136

gi|21954246|gb|AY116220.1|Uncultured eukaryote clone ANT12-26 1... 494 e-136

gi|41393027|emb|AJ535176.1|LMI535176 Leptocylindrus minimum 18S... 494 e-136

gi|53693111|gb|AY742743.1|Phytophthora tropicalis isolate.129F-... 494 e-136

gi|53693108|gb|AY742759.1|Pythium vexans isolate Pyv6-2 18S rib... 494 e-136

gi|53693105|gb|AY742756.1|Pythium splendens isolate 117 18S rib... 494 e-136

gi|53693104|gb|AY742755.1|Pythium aphanidermatum 18S ribosomal... 494 e-136

gi|53693097|gb|AY742748.1|Phytophthora capsici isolate 98110 18... 494 e-136

gi|53693096|gb|AY742747.1|Phytophthora tropicalis isolate 23047... 494 e-136

gi|53693094|gb|AY742745.1|Phytophthora palmivora isolate 8829 1... 494 e-136

gi|58531862|gb|AY882508.1|Uncultured marine eukaryote clone T53... 494 e-136

3. embodiment 3: use the production of bacterial strain ONC-T18 optimization biomass

A plurality of process variables are depended in the production that derives from (or single celled) oil of microorganism, for example initial inoculation thing level, substrate type, substratum composition, temperature and pH.Particularly, use the thraustochytriale bacterial strain, show between biomass and the lipid acid production that based on the production of the highly unsaturated fatty acids of microorganism direct relation is arranged.Therefore, be the important factor that obtains maximum production to the understanding of primary demand or to the optimization of parameter.Therefore, for determining to produce the optimal medium of the lipid acid amount that increases, carried out initial biomass optimization experiment.Particularly, use (the Joseph J and Piganatiells JR of the Taguchi method based on orthogonal array of exploitation recently, UE Trans20:247-254,1998), purpose is to determine to make the optimal medium of optical density(OD) (producing directly related with biomass) increase to form.In this case, use the Taguchi method to understand the storage effect that biomass is produced influential variable.The change of nitrogen (yeast extract, peptone, L-glutaminate), carbon (glucose) and salt concn (artificial sea salt) produces influential to biomass.Therefore, prepared multiple liquid nutrient medium, contain in the described substratum various amounts yeast extract, peptone and L-glutaminate (0,4,10,20,50gL ^-1) and the g/s solution of various amounts (be respectively 5,40,100,160,200g L ^-1With 0,6,20,30,40g L ^-1).According to L ²⁵Orthogonal array is calculating concentration in the following manner: the formula below utilizing, distinguish selected nitrogen substratum by using the signal to noise ratio (SNL) when 48 hours and 120 hours:

$\mathrm{SNL}=-10\log \left[\frac{1}{n}\underset{i=1}{\overset{n}{\mathrm{\Σ}}}\frac{1}{y_i^2}\right]$

Wherein, the horizontal number of n=, y=output is (from the OD of three parallel laboratory tests ₆₀₀Mean value).

Result's (as shown in Figure 2) of these experiments (especially at the Consideration biomass) shows, with optical density(OD) (OD ₆₀₀) nitrogen use efficiency of maximally related ONC-T18 is peptone, is yeast extract then, follow by the L-glutaminate.But to be grown to the basis the soonest, the optimum nitrogen source that then improves biomass yield is a yeast, is peptone then, follows by the L-glutaminate.In addition, by being the similar experiment of variable with glucose and salinity (sea salt concentration), the best and the most cheap substratum that are used to produce the ONC-T18 biomass consist of: substratum contains 2g L ^-1Yeast extract, 8g L ^-1MSG, 60g L ^-1Glucose and 6g L ^-1Sea salt.

4. embodiment 4: with the production of bacterial strain ONC-T18 optimization docosahexenoic acid (DHA)

The substratum that preparation is made up of nitrogenous source (peptone, yeast extract, L-glutaminate (MSG) or their combination) and carbon source (glucose) (being dissolved in salt solution (artificial seawater) solution) is determined the optimal medium composition (being shown in table 4) that energy optimization biomass and DHA produce in the mode similar to 3 describing modes of embodiment.After 25 ℃ and 130rpm cultivate three days, according to embodiment 1 and method described herein, the amount of DHA the results are shown in table 4 in weight percentage by the total fatty acids in gas Chromatographic Determination biomass, the every liter of substratum, lipid acid, the percentage composition that DHA accounts for total fatty acids and the every liter of substratum.

In this case,, compare, measured DHA with known DHA standard substance by using gas-chromatography associating mass spectrum and peak value locking (peaklocking) method.Experimentalists and technicians (experimental package)

Result's (wherein the variation of the nitrogen of natural and organic two kinds of forms all being studied) show that for the best production of biomass and DHA, the yeast extract that optimal medium should contain in forming and the amount of L-glutaminate are 4.0 to 6.0g L ^-1On the other hand, experimentalists and technicians

(having studied the variation of the sodium component that adds to substratum) show, but use the biomass and the DHA of artificial sea salt output the best.In addition, experimentalists and technicians (wherein the concentration of sodium is different in the substratum) shows, using 5 to 15% artificial seawater L ^-1DH ₂The maximum output that occurs DHA and biomass during O.Experimentalists and technicians

The result of (wherein the variation of glucose level being assessed) shows that 40 to being less than 160g L ^-1Glucose range can obtain best biomass and the production of DHA.At last, experimentalists and technicians

The result show that during as carbon source, cellular biomass that ONC-T18 produced and DHA concentration numerical value are of equal value in glucose or glycerol.

Table 4: substratum is formed under the different situations, DHA production optimization result of experiment.

5. embodiment 5:ONC-T18 produces the best collection time of maximum DHA

Under substratum composition identical with condition and condition, cultivate ONC-T18 with those substratum compositions shown in the embodiment 1.In this particular case, what studied is for obtaining maximum DHA, DPA and EPA, the time that ONC-T18 should be collected, also having considered to obtain the essential time of described amount (referring to Fig. 3) simultaneously.

The time-histories experimental result shows that the best collection time of ONC-T18 production optimization DHA in flask and bio-reactor is respectively between 3 to 5 days.

6. embodiment 6: the analysis that the lipid that derives from ONC-T18 is carried out

Use improved Bligh ﹠amp; The Dyer method is extracted the TL component of ONC-T18.Particularly, 4 ℃ of rehydrations of in 8ml distilled water, 2.0g stem cell biological amount being spent the night.With 30ml methyl alcohol: chloroform (2:1 volume/volume) adds in the mixture and at the gentle vibration of 120rpm 20min, decants resulting supernatant.Then precipitation is resuspended in methyl alcohol: chloroform: H ₂O (2:1:0.8 volume/volume/volume) repeats this process, collects supernatant and it is transferred in the separatory funnel.Then with 5ml chloroform and 5ml H ₂O adds funnel, thereby forms two liquid system.In separatory funnel, after the thorough mixing, discard chloroform layer, concentrate under the condition of nitrogen gas, be resuspended in the chloroform and in-20 ℃ of preservations, until analysis.The TL component point sample of about 1 μ l on a plurality of chromatobars, is used Iatroscan MK6 TLC/FID instrument to separate and analyzes.

Interpretation of result shows that the fatty acid component that is produced by ONC-T18 under the heterotrophic fermentation condition almost all is triglyceride level (at least 95%) in essence.Except above-mentioned neutral fat acid constituents, ONC-T18 also produces recognizable carotenoid and phospholipid fraction.To the separation (at first pass through 50%Burn, pass through 75%Burn subsequently) of phospholipid fraction, can determine to exist the phospholipid fraction of large amount of complex by subsequently and then by separation based on solvent.The result shows, has phosphatidylcholine, phosphatidylserine and phosphatidic acid component in sample.

7. embodiment 7: use bacterial strain ONC-T18 to produce antioxidant

Use aforementioned condition and substratum to cultivate eukaryote ONC-T18.After the heterotrophic fermentation resulting cellular biomass via centrifugal, filter or precipitation is collected.Cell passes through in the centrifugal collection of 3800 * g, and washs with phosphate buffered saline.Cellular biomass (just having produced that obtain or cryodesiccated) is suspended in the acetone of 10 * volume, and 200rpm stirred 5 minutes, centrifugal 5 minutes of 3800 * g, and evaporation concentration is extremely dry under condition of nitrogen gas.Then this pigment is resuspended in immediately 10% acetone (being dissolved in hexane) of minute quantity, and, analyzes until carrying out HPLC-20 ℃ of preservations.(CA USA) goes up evaluation carotenoid extract for Agilent, PaloAlto at Agilent 1100 HPLC that are equipped with the variable-wavelenght detector that is set in 470nm then.(MA USA) is injected to Bondclone C18 reversed-phase column (Phenomenex, Torrance, CA, USA to sample for Waters, Milford by Symmetry C18 guard column; Particle is 10 μ m; Internal diameter is 3.9 x 300mm).Volume injected is 10 μ l, and employed moving phase is 10% acetone (being dissolved in hexane), and flow velocity is 1.00ml/min, carries out 25 minutes.With the peak area and the known standard substance of carotenoid (is astaxanthin, keratin, β-zeaxanthin, zeaxanthin, myoxanthin and β-Hu Luobusu in the case; ChromaDex, Santa Ana, CA USA) compares, and obtains the quantitative data of carotenoid.Do not exist the known standard product for example under the situation of carotenoid, phoenicoxanthin, calculating its concentration with the peak area of astaxanthin.The further affirmation of carotenoid feature by use be equipped with introduce Micromass ESI-Q-Tof mass spectrograph (Waters, Milford, MA, the Waters HPLC of photodiode arrangement USA) (Waters model 996) carries out via HPLC-MS.To the HPLC analysis revealed of ONC-T18, in cellular biomass, there are several anti-oxidant compounds (50 to 1250mg/kg) subsequently.These compounds comprise that scope is respectively 1 to 20mg/kg, 0.25 to 10mg/kg, 1 to 20mg/kg, 1 antioxidant carotenoid astaxanthin, zeaxanthin, keratin, myoxanthin and β-Hu Luobusu to 20mg/kg and 1 to 200mg/kg, and the not certified as yet flavonoid polyphenolic substance of several character (flavenoid polyphenoliccompound).

8. embodiment 8: with the comparison of known microorganisms

The ability that ONC-T18 is produced DHA, EPA and DPA is compared with this ability of known microorganisms.By cultivating golden yellow thraustochytriale ATCC 34304, genus thraustochytrium kind ATCC 20891, genus thraustochytrium kind ATCC 20892, pink thraustochytriale ATCC 28210, genus thraustochytrium kind ATCC 26185, schizochytrium limacinum belongs to kind of an ATCC 20888, collection is given birth to schizochytrium limacinum (Schizochytriumaggregatum) ATCC 28209 and Schizochytrium limacinum MYA-1381, measured the amount of cellular biomass in the every liter of substratum that produces when cultivating above-mentioned bacterial strains, the percentage composition of fat or lipid acid in every gram stem cell biological amount, DHA in the total fatty acids, the percentage composition of EPA and DPA, and DHA, the amount of EPA and DPA has also been measured the DHA that is obtained when cultivating ONC-T18 of the present invention, the output of EPA and DPA.

The lipid production of table 5. pair several representative thraustochytriale bacterial strains and the comparison of biomass feature

Microorganism	The amount of cellular biomass (g l -1)	Lipid percentage composition (%g -1)	DHA EPA DPA percentage percentage percentage composition content content (%g -1)(％g -1) (％g -1)	The total total DPA of the total EPA of DHA (g l -1) (mg l -1) (mg l -1)
					Genus thraustochytrium kind ATCC 20891 genus thraustochytrium kind ATCC 20892 genus thraustochytrium kind ATCC 26185 golden yellow thraustochytriale ATCC 34304 pink thraustochytriale ATCC 28210 schizochytrium limacinums belong to kind of ATCC 20888 collection and give birth to schizochytrium limacinum ATCC 28209S.limacinum SR21 MYA-1381	1.832.34-58-1710.51.423-40	No data 7 No data 8-2018-25501.740-53.5	12 No data No data, 35 No data No data, 41.9 3.1 1024-51 3.6-9.3 No data, 50 No data No data 25-37 No data No data, 6.0 6.1 No data 29.7-34 0.2-0.4 No data	No data No data No data 0.07 No data No data No data No data No data 0.1-0.5 0.0001 No data 0.6-2.1 No data No data 1.95 No data No data 11 No data 3.0-7.2 0.08 No data
ONC-T18	25-55	45-80	24-34.2 0.1-2 6-10	4.6-13 0.2-0.8 0.9-3.8

As shown in table 5, it should be apparent that when using ONC-T18 of the present invention to cultivate, compare with other tested bacterial strain, the cell biological numerical quantity is high in every liter of substratum.In addition, according to the present invention, ONC-T18 compares the lipid with very high percentage composition with above-mentioned other bacterial strain.In addition, according to the present invention, the percentage composition of DHA and DPA is high in the ONC-T18, and the EPA water-glass reveals suitable with all screened bacterial strain.Therefore, obviously ONC-T18 has the ability that produces a large amount of DHA, EPA and DPA under embodiment 1 described fermentation condition.

9. embodiment 9: the information that substitutes carbon source

Shown that ONC-T18 preferentially grows on a kind of like this substratum, wherein major nitrogen source is yeast extract, Sodium Glutamate and/or peptone, and main carbon source is a D-glucose.Based on the detailed metabolism distributed intelligence of ONC-T18, notice that glycerine (carbon source) is feasible substitute materials equally.In addition, also fish oil processing-waste (containing glycerine) is tested as the applicability of nutrition substitute materials with low cost.Situation to glycerine has been carried out following experiment: use the 200ml substratum be loaded in the 500ml flask, 25 ℃, 120rpm growth 3 days.Volume), and with 6% glycerine adding 200ml substratum (weight: volume) in contrast the glycerol content of two kinds of fish oil processing refuse GWW (glycerine water lotion) and GAW (glycerine acid wash liquid) accounts for 40% (volume: of 200ml substratum (being adjusted to pH6.5).

Table 6. substitutes lipid acid, biomass and the glycerol content of carbon source research

These results' analysis is confirmed, when for example byproduct glycerine is as the carbon source of the large scale fermentation of ONC-T18 with fish oil waste liquid component, although cause the total fatty acids amount in the microorganism cells to reduce, DHA content wherein remain unchanged (Figure 10).

10. embodiment 10: the enlarged culturing of dry cell weight

Can be in maximum to 100, cultivate genus thraustochytrium kind ONC-T18 in the multiple structure of reactor of 000L and produce ω-3 oil.All fermenting processs all start from preparing the inoculum of the 10-20% that accounts for final volume, and it can be used for forming fermenting culture.Initial medium is formed to include and is up to 6g/L sea salt, 10g/L nitrogenous source and 60g/L carbon source, and carries out feed supplement in initial fermentation after 24 to 36 hours, adds other 75g/L carbon source in batches, continue 72 to 96 hours again, and temperature range is 18-25 ℃.For example, use contains the substratum of 6g/L sea salt, 2g/L yeast extract, 8g/L L-glutaminate and 60g/L D-glucose (adding 75g/L after 36 hours again) 25 ℃ of cultivations 96 hours, and ONC-T18 can produce 40g/L dry cell weight (dcw), account for total fatty acids (the TFA)/lipid composition (between between the C14:0 to C24:0) of dry cell weight (dcw) 80% and (TFA) DHA of 30%.Equally, under the situation that does not influence TFA or DHA content, can make biomass that the increase of similar multiple also be arranged, thereby increase dry cell weight by the content of be multiplied nitrogen and carbon nutrient media components.For example, use contains the substratum of 24g/L sea salt, 8g/L yeast extract, 32g/L L-glutaminate and 300g/L D-glucose 25 ℃ of cultivations 312 hours, and ONC-T18 can produce the dry cell weight (dcw) of 80g/L, total fatty acids (the TFA)/lipid composition (between between the C14:0 to C24:0) that accounts for dry cell weight (dcw) 60% and (TFA) DHA of 38%.

11. embodiment 11: genus thraustochytrium kind ONC-T18 in various alternate carbon (C) source and nitrogen (N) source growing state and the influence of pair cell dry weight and lipid

Genus thraustochytrium kind ONC-T18 is studied at various nitrogenous sources with low cost and the growing state in the carbon source.50ml ONC-T18 in being housed, was cultivated 72 hours in 25 ℃ by the 250ml flask of the artificial sea salt of 6g/L.Carbon source and nitrogen concentration are as follows, unite listed each nitrogenous source that uses with 8g/L L-glutaminate and are 2g/L (use the situation exception of fish meal, this fish meal consumption is 4g).Carbon source changes as shown.All test parallel carrying out three times; All that are used for the fatty acid methyl ester analysis are extracted all parallel carrying out three times, simultaneously parallelly carry out three GC injections.

The result shows, when genus thraustochytrium kind ONC-T18 grows in nitrogenous source EMD yeast extract and fish meal, can produce the cell dry biomass (promptly than two kinds of control medium height) of optimization.On the contrary, when using corn steep liquor and EMD peptone, find the lipid content comparison according to low, and DHA content is optimized.At last, find that the carbon source dextrose can increase lipid content, and fructose and dextrose can produce comparison according to higher DHA content.

Table 7: the growing state of genus thraustochytrium kind ONC-T18

Abbreviation:

MSG=L-glutaminate (sodium) YE=yeast extract

12. embodiment 12: the extractive technique that is used to separate TL and each component

Method to multiple separation selected ω-3 oil is tested, and purpose is to determine best separation efficiency.These methods comprise: the Bligh ﹠amp of standard; Dyer method (Bligh ﹠amp; Dyer, Can J.Biochem.Physiol., 37:912-917,1959), be specifically designed to that uniting of thraustochytriale kind extracts and ester-interchange method (can process sample and be used for fast GC FAME analysis) (Lewis et al., J.Microbiol.Methods, 43:107-116,2000), adopt saponified extraction method (Cartens et al. simultaneously, J.Am.Oil Chem.Soc.73:1025-1031,1996) and use alternative separation of glycerin three esters, the solid-phase extraction method of the silicagel column of triglyceride and monoglyceride (Pinkart et al., J.Microbiol.Methods, 34:9-15,1998; Bateman ﹠amp; Jenkins, J.Agric.Food Chem., 45:132-135,1997).

Particularly, the 40g dry cell weight genus thraustochytrium kind biomass that will produce in single fermentation operation (referring to embodiment 1) is divided into a plurality of groups of 0.44g, and uses various technology to separate.All technology are all parallel carries out three times, and uses by the fatty acid methyl ester of FID-GC and determine that method comes analysis efficiency, same parallel carrying out three times, and each sample is parallel carries out three times.The result shows that the total fatty acid content that the whole bag of tricks obtained is different, and this fluctuation is most likely because solvent: the saturated situation of compound, biomass destroy situation and other physical condition Considerations such as (as temperature and times).

Table 8: the extractive technique of separating TL and each component

Bligh & Dyer

DHA EPA C14:0 C14:1 C15:0 C16:0 C16:1 C18:1 C20:0 C20:4 C22:5 TFA

Mg omega-3/ restrains biomass (mg/g)

1 104.394.25 36.28 5.82 113.94 77.27 4.92 44.01 1.13 1.67 28.86 430.99

2 136.755.45 46.51 7.40 142.96 98.38 6.07 56.49 1.40 2.19 37.92 552.98

3 134.594.78 42.51 6.91 128.54 87.01 5.20 51.10 1.30 2.10 35.98 532.91

Av.125.24 4.83 41.77 6.71 128.48 87.55 5.40 50.53 1.28 1.99 34.25 505.63

Directly transesterify

DHA EPA C14:0 C15:0 C16:0 C16:1 C18:0 C18:1 C20:0 C20:4 C22:5 TFA

Mg omega-3/ restrains biomass (mg/g)

1 104.39 4.24 36.39 5.42 112.94 75.27 5.42 44.01 1.13 1.67 28.86 420.99

2 89.83 4.54 34.81 5.60 103.04 73.43 5.56 42.85 0.98 1.87 25.35 392.88

3 101.64 4.25 37.16 5.98 106.94 75.98 5.35 43.95 1.11 1.78 26.46 410.65

Av. 98.65 4.34 36.12 5.67 107.64 74.89 5.44 43.60 1.07 1.77 26.89 408.17

Saponification simultaneously

DHA EPA C14:0 C15:0 C16:0 C16:1 C18:0 C18:1 C20:0 C20:4 C22:5 TFA

Mg omega-3/ restrains biomass (mg/g)

1 204.85 6.75 46.55 8.56 182.26 134.81 8.73 105.04 2.16 3.20 66.68 785.25

2 188.51 6.17 47.64 9.32 208.29 121.25 10.35 95.80 2.53 2.89 61.41 770.14

3 198.25 6.12 47.21 9.65 207.71 136.51 9.58 98.50 2.41 3.10 63.58 782.54

Av. 197.20 6.35 47.13 9.18 199.42 130.86 9.55 99.78 2.37 3.06 63.89 779.31

Solid phase extractions

DHA EPA C14:0 C15:0 C16:0 C16:1 C18:0 C18:1 C20:0 C20:4 C22:5 TFA

Mg omega-3/ restrains biomass (mg/g)

1 169.17 0.42 68.41 10.83 204.43 140.14 8.09 76.97 1.75 3.00 47.05 748.33

2 172.26 0.44 69.59 11.01 207.01 143.74 8.11 78.72 1.74 3.27 47.86 819.04

3 173.65 0.43 69.21 11.31 208.97 146.64 8.16 77.64 1.73 3.64 46.98 785.64

Av. 171.69 0.43 69.07 11.05 206.80 143.51 8.12 77.78 1.74 3.30 47.30 784.34

N.B: each numerical value listed above is the mean value of three parallel laboratory tests of the FID-DC that is used for FAME analysis

13. embodiment 13:

A) material and method

(1) separation of thraustochytriale and cultivation

During in July, 2002 and August, at Nova Scotia, Prince Edward Island, New Brunswick, 70 ocean samples have been collected in the Canadian coastal region in east China of Newfoundland and Labrador, comprising: spartina alterniflora loisel (Spartina alterniflora), sea tangle (Zostera marina) and silt.Sample is placed the 20mL bottle that following material is housed: 10mL aseptic filtering natural sea-water of 0.2 μ m and 300mg L ^-1Penicillin and 500mg L ^-1Streptomycin sulphate.According to Bremer, Marine Mycology-A Practical Approach, FungalDiversity Press, Hong Kong, pp 49-61 (2000) is described, suspension is lured collection with aseptic pollen (maple (Acer sp.)), and hatched 48 hours at 18 ℃.(the 1L natural sea-water contains 1g L by transfering loop transfer pollen granule and containing antibiotic B1 agar plate then ^-1Yeast extract, 1g L ^-1Peptone, 10g L ^-1Agar) go up line and cultivation.Select single random transparent colony and the atypical yeast or the bacterium colony of sphere or limaciform cell, and on the B1 plate, go down to posterity at least and cultivate three times to carry out purifying.

(2) be used for the biomass production that lipid acid screens

For screening is used to cultivate the strain isolated of producing with lipid acid, use the filtering natural sea-water obtaining liq of 0.2 μ m substratum, this liquid nutrient medium contains 2g L ^-1(NJ is USA) with 2g L for BD, Franklin Lakes for peptone ^-1(NJ USA), to medium sterilization, adds 5g L by autoclaving to yeast extract then for BD, Franklin Lanes ^-10.2 the glucose of μ m filter filtration sterilization (Sigma-Aldrich, St.Louis, MO, USA) (Bowles et al., J Biotechnol70:193-202 (1999)).Is the culture of 30mL by transfering loop from agar plate inoculation volume, and is being set on the vibrator of 100RPM in 18 ℃ of cultivations 4 days.Then this culture of 5mL is inoculated in the 95ml substratum, and cultivates again 4 days (stationary phase).By 4, the 500RPM centrifugal collecting cell is with 5mL distilled water wash and recentrifuge.With the cell precipitation lyophilize, weigh and, carry out the derivatization analysis of lipid acid then-80 ℃ of preservations.

(3) preparation of fatty acid methyl ester (FAME)

Can improve the method that gets by direct ester exchange method and carry out the extraction of fatty acid methyl ester (FAME) from people such as Lewis (J Microbiol Meth.43:107-116 (2000)).Particularly, add cryodesiccated material of 20mg and 3ml transesterification reaction mixture (methyl alcohol: hydrochloric acid: chloroform (10:1:1 volume/volume)).The cell vortex was vibrated for 10 seconds, guaranteeing the biomass homodisperse, and placed 120 minutes in 90 ℃.After finishing transesterify, take out sample and it is cooled to room temperature.Adding entry (1ml) and vortex then vibrated for 10 seconds.Extract FAME then as follows: the hexane that adds 3 * 2ml equal portions: chloroform (4:1), vortex vibrated for 10 seconds, left standstill, until obtaining clarifying liquid separation thing.

(4) gas-chromatography of FAME (GC) is analyzed

The GC that mark in using two (each 200 μ l) carries out FAME analyzes.One is that hydroxyhexacosanoic acid (hexacosaenoic acid) (C23:0), adds between transesterify; Another is nonadecanoic acid (C19:0), directly adds before analyzing.Use Agilent 6890 GC (AgilentTechnologies, Palo Alto, CA, USA) analyze, it is OMEGAWAX 320 fused quartz capillary column (Sigma-Aldrich, St.Louis, the MO of 30m * 0.32m that this apparatus preparation has internal diameter, USA) and flame ionization detector (volume injected is 1 μ l, and carrier gas is H ₂, constant flow rate is 5.0ml min ^-1, be set in 250 ℃, the splitting ratio of fid detector is 50:1 in the time of 275 ℃).(MA USA) and with the residence time of experimental standard product compares, and carries out the affirmation of FAME feature for Thermo Electron, Boston by using Trace GC-DSQ mass spectrograph.

(5) Genetic identification

(CA USA), extracts genomic dna according to the specification sheets of manufacturers for MoBioLaboratories, Carlsbad to use MoBio UltraClean Microbial DNA Isolation Kit.The Oligonucleolide primers of 18S rRNA gene of being used to increase is to improve from the used primer of people such as Honda (JEukaryot Microbiol.46:631-641 (1999)), i.e. T18S1F 5 '-CAACCTGGTTGATCCTGCCAGTA-3 ' and T18S5R5 '-TCACTACGGAAACCTTGTTACGAC-3 '.The PCR reaction mixture of 20 μ l contains 2UBiolase ^TMArchaeal dna polymerase (Bioline, Boston, MA, USA), 1 * NH ₄Reaction buffer, 3mM MgCl ₂, the 1M trimethyl-glycine (Sigma-Aldrich, St Louis, MO, USA), 200 μ M blended PCR Nucleotide (Promega, Madison, WI, USA), forward primer and reverse primer (the MWG Biotech. of each 1 μ M, High Point, NC, USA) and 100ng genomic dna template.At first after 94 ℃ of sex change 3 minutes, use Eppendorf Master CycleGradient gene-amplificative instrament (Eppendorf, Westbury, NY, USA) carry out pcr amplification, its program is: 94 ℃ 45 seconds, 64 ℃ 30 seconds, 72 ℃ 2 minutes, this program is carried out 30 circulations, prolongs 10 minutes at 72 ℃ at last.Use MoBio UltraClean PCR Clean-upKit (MoBio Laboratories Inc, Carlsbad, CA, USA) come purified pcr product, (MWG Biotech., High Point so that use primers F A2, FA3, RA1, R (Mo et al., Mar Biol 140:883-8892002), T18S1F and T18S5R directly check order, NC, USA).Use DS Gene (Accelrys, San Diego, CA USA) comes resulting sequence is compared, and itself and the nucleotide sequence that is kept at the similar microorganism of GenBank (Benson et al., Nucleic Acids Res33:D34-38 (2005)) compared.Use adjacent method (Neighbor-Joining method) (Saito and Nei then, Mol Biol Evol 4:406-425 (1987)) forms genealogical tree, and use 1000 bootstrapping analog sample samples (Felsenstein, Evolution 39:783-791 (1985)) to assess statistical significance.

(6) evaluation of carotenoid

By coming collecting cell in that 3800 * g is centrifugal, and wash with phosphate buffered saline.Be resuspended in then 10 * volume acetone (Sigma-Aldrich, St Louis, MO, USA) in, 200 RPM stirred 5 minutes, centrifugal 5 minutes of 3,800 * g, and pass through N ₂Evaporation concentration is to doing.Be resuspended in afterwards in 10% acetone (being dissolved in hexane) of minute quantity, carry out HPLC subsequently and analyze.(CA identifies on USA) for Agilent, Palo Alto at Agilent 1100 HPLC that are equipped with the variable-wavelenght detector that is set in 470nm.(MA USA) is injected to Bondclone C18 reversed-phase column (Phenomenex, Torrance, CA, USA to sample for Waters, Milford by Symmetry C18 guard column; Particle is 10 μ m; Internal diameter is 3.9 * 300mm).Volume injected is 10 μ l, and employed flow velocity is the acetone (being dissolved in hexane) of 1ml/min 10%, carries out 25 minutes.The further affirmation of carotenoid feature by mass spectrometry (Micromass ESI-QTof MS, Waters, Milford, MA USA) carries out.The quantitative data of various types of carotene forms calibration curve and peak area is compared comparatively basis with definite concentration to use standard substance (astaxanthin, zeaxanthin, keratin, myoxanthin and β-Hu Luobusu).

(7) fermentation optimization

Detect the influence of carbon, nitrogen and sea salt by in 250ml Erlenmeyer bottle, carrying out batch culture (130 RPM vibration was cultivated 3 days in 25 ℃) to lipid acid and DHA production.Use

(PA USA) carries out further culture studies to Bplus Twin 5L bio-reactor for Sartorius BBI Systems Inc., Betlehem.The inoculum of 100ml is inoculated in the 4.9L substratum that is contained in the bio-reactor.(MO USA), comes measure glucose concentration according to the specification sheets of manufacturers for Sigma-Aldrich, St Louis to use Glucose (HK) AssayKit.The nutrient media components and the condition that are adopted in bio-reactor partly elaborate at correlated results.

B) result

Developed collection and screening method, by this, used pollen to lure the collection method multiple protobiont Labyrinthulida (family Labyrinthulida) especially schizochytrium limacinum to be belonged to and separate with genus thraustochytrium with the selectivity bacteria culture medium.20 different collecting locations that are distributed in whole Atlantic Canada are contained in this research, and have obtained through 68 of microscopical identification pure bacterial strains.According to Lewiset al., the method of J Microbiol Meth 43:107-116 (2000), the screening of produce oil bacterial strain (its dry cell weight be more than 20% lipid acid) is based on following result: secondly the GC PUFA condition of production, biomass productive rate, the highest TFA, DHA are EPA concentration (Figure 11).The numerical range of biomass, TFA and DHA and EPA productive rate is respectively 100 to 2300mg L ^-1, 27.1 to 321.14,5.18 to 83.63 and 2.97 to 21.25mg g ^-1(Figure 14).

All strain isolateds of growth (in 68 54) have all produced especially DHA of a large amount of omega-3 polyunsaturated fatty acidses in the liquid medium within, and this DHA accounts for 22 to 80% (Figure 11) of these cells C20 to C22 total content.Result before this has affirmed, promptly the DHA of the thraustochytriale of separating from the cool temperature zone environment with production accounts for 53% the lipid acid condition of production (the Bowles et al. of being up to of existing total fatty acids, J Biotechnol 70:193-202 (1999) and Huang et al., Mar Biotechnol 5:450-457 (2003)).It is worth noting ONC-T18 especially, it can produce and account for its C20 to C22 content and be up to 90% DHA, and this DHA is approximately 35% of total fatty acids in the cell.Shown this DHA content and several bacterial strains that are used to commercially produce for example schizochytrium limacinum belong to identical (the Barclay et al of DHA content of kind of ATCC 20888 (32%) and S.limacinum MYA-1381/SR21 (34%), J Appl Phycol 6:123-129 (1994) and Yokochi et al., Appl Microbiol Biotechnol 49:72-76, (2003)).In addition, all synthetic 2% to 20%w/w the timnodonic acid (EPA) that accounts for total PUFA that identifies of all strain isolateds (Figure 11).Except ω-3 oil that is produced, about 80% can synthesize ω-6 PUFA, arachidonic acid (AA) or clupanodonic acid (DPA) in whole strain isolateds, and its concentration is respectively between 1 to 18% and 3 to 7%w/w (Figure 11).

People such as Huang (Mar Biotechnol 5:450-457 (2003)) think, for for the isolating thraustochytriale in tropical coastal waters of Japan and Fiji, describable have five kinds of unsaturated fatty acids conditions of production, be DHA/DPA (n-6), DHA/DPA/EPA, DHA/EPA, DHA/DPA/EPA/AA and DHA/DPA/EPA/AA/ docosatetratenoic acid (Huang et al., Mar Biotechnol5:450-457 (2003)).With regard to this thraustochytriale (obtaining) gleanings from the separation of the temperate waters of Atlantic Canada, can define four kinds of PUFA conditions of production, wherein three kinds identical with above-mentioned those, promptly account for gleanings 7.4% DHA/DPA/EPA, account for the DHA/EPA of gleanings 13% and 74% DHA/DPA/EPA/AA, the 4th kind of mixture that comprises 5.6% DHA/EPA/AA.

By 18S rDNA gene is directly checked order, ONC-T18 is accredited as a member (GenBank numbering: DQ374149) of thraustochytriale (Thraustochytrid) for certain.Phylogenetic analysis shows that ONC-T18 and line shape thraustochytriale T91-6 have formed independent one group (identity is 97.5%) (Figure 12) (Leander and Porter, Mycologia 93:459-464 (2001)).And collect and be found to be the important producer (the Carmona etal. of DHA from Japanese coastal temperate waters, Biosci Biotechnol Biochem 67:884-888 (2003) and Huang et al., Mar Biotechnol 5:450-457 (2003)) the bacterial classification MBIC of thraustochytriale section 11093, N1-27 and genus thraustochytrium kind CHN-1 show respectively has 96,95.5 and 94.5% similarity with ONC-T18.Genetic diversity is quite low between whole members of thraustochytriale shown in Figure 12 section, and the similarity between all bacterial classifications is between 97.5-91.0%.Yet these bacterial classifications spread all over the world, and have 2/3rds to separate from the tropical coastal waters of Japan, China and Israel, and remaining separation is from the U.S., Europe and Canadian temperate waters.

Similar to the lipid acid condition of production of genus thraustochytrium kind KH105 or S.limacinum SR21, the lipid acid condition of production of ONC-T18 comprises high-load C22 PUFA, extremely low-level C18 and C20 FA, and the saturated fatty acid (15:0 and 17:0) of odd number carbochain occurs.In addition, the carbon to bacterial strain ONC-T18, SR21 and KH105 utilizes the analysis of situation to provide similar absorption mode with nitrogen.The content of n-6DPA is considering that n-6DPA has in limited time among the bacterial strain ONC-T18 in biosphere between 6-10%, and it is very high that this content seems.Yet, be reported in people (Comp Biochem Physiol 29:805-81 (1969)) such as schizochytrium limacinum belongs among kind of the SR21 (6-10%), Ellenbogen according to people such as Nakahara (J AmOil Chem Soc 73:1421-1426 (1996)) and be reported in the n-6DPA that similar level is arranged in golden yellow thraustochytriale (9.5%) and the pink thraustochytriale (6.6%).

To the analysis revealed of ONC-T18 in the three kinds of different culture apparatuses ((1) agar plate, (2) erlenmeyer flask and (3) bio-reactor) and the lipid acid condition of production (Figure 13) of on same medium, growing, from the agar plate to the bio-reactor, the diversity of existing PUFA reduces to some extent, and TFA raises generally.Particularly, agar plate shows PUFA array (array), and the culture of growing in flask and bio-reactor is mainly one or both intermediate products (Figure 13).Golden yellow thraustochytriale is grown in culture flask than in stirred fermentor, and more vigorous (by comparison, ONC-T18 grows better in bio-reactor for Ilda et al., JFerment Bioeng 81:76-78 (1996).People (Nakahara et al. such as this result and Nakahara, J Am Oil ChemSoc 73:1421-1426 (1996)) unanimity as a result, they find that schizochytrium limacinum belongs to kind of a SR21 mechanical stirring is shown high resistance, thereby growth is vigorous under the bio-reactor condition.

In addition, find between flat board, flask and the bio-reactor yeast phase of genus thraustochytrium kind ONC-T18, to produce carotenoid pigment, cause taking off and be light orange.The production of these antioxidants is the most active in the bio-reactor fermentation, and accompanies with lipid acid production.In addition, by using the HPLC mass spectroscopy, determine that these anti-oxidant compounds are accredited as astaxanthin, zeaxanthin, keratin, myoxanthin and the β-Hu Luobusu of puting together with various PUFA (Figure 14).Among each member of the thraustochytid group who it is reported protobiont similar result is arranged.Particularly, shown that collecting living schizochytrium limacinum can produce myoxanthin and keratin (Valadon, Trans Br Mycol Soc 67:1-15 (1976)), and people (Mar Biotechnol 5:450-457 (2003)) such as people such as Carmona (Biosci Biotechnol Biochem 67:884-888 (2003)) and Huang confirm, astaxanthin, myoxanthin, keratin, phoenicoxanthin (being not the zeaxanthin among the ONC-T18) and β-Hu Luobusu are produced (Figure 12) by genus thraustochytrium kind CHI-1 (close relative's bacterial classification of ONC-T18).In this research, the order of magnitude of concentration of finding these carotenoid is than its low in CHN-1, and main compound is β-Hu Luobusu but not astaxanthin.Therefore, in some was planted at thraustochytriale, PUFA and carotenoid production can be joined together, thereby make the depot fat of being produced can avoid oxidized.

Definite before, by changing the growth conditions of culture, the relative quantity of main fatty acid component (myristic acid, palmitinic acid and oleic acid) can get change (Ilda et al., J FermentBioeng 81:76-78 (1996)) to a certain degree.In this way, final fatty acid component can be handled, the physical properties (Sijtsma et al., Recent Res Devel Microbiol 2:219-232 (1998)) of required PUFA can be during fermentation handled thus with controllable manner.In the trial of the factor that inhibition biomass and ω-3PUFA produces in determining ONC-T18, the carbon in the nutritional medium, nitrogen and sea salt component (table 9) and incubation time (Figure 15) are controlled.

Table 9: the average biomass yield of genus thraustochytrium kind ONC-T18 (SD＜15%), total fatty acids (TFA) and DHA content

In this research, because nitrogen concentration descends, total fatty acid content raises, and the total fatty acid content the highest (about 80%) that obtains when yeast extract or msg powder type concentration are 1% (w/v).Yet the culture of low nitrogen concentration has limited the cell growth equally, and has limited the generation of total fatty acids thus.In this experiment, use 8g L ^-1Msg powder type and 2g L ^-1Yeast extract can obtain optimum yield: produce 26.1g L ^-1Biomass and 4.5g L ^-1DHA (table 9).In addition, carbon is elevated to and is up to 100g L ^-1Improved DHA output effectively, result (Yokochi et al., Appl Microbiol Biotechnol 49:72-76 that this and genus thraustochytrium kind SR21 obtain, (2003)) unanimity, and opposite with the given result of golden yellow thraustochytriale, in golden yellow thraustochytriale, be higher than 10g L ^-1Glucose concn be inhibition (Ilda et al., J Ferment Bioeng81:76-78 (1996)).In dextrose culture-medium, obtained to be higher than 4.0g L ^-1The highest DHA output, and the golden yellow thraustochytriale of rate ratio (Bajpai et al., J Am Oil Chem Soc68:509-514 (1991)) and pink thraustochytriale (Li and Ward, J Ind Microbiol13:238-241 (1994)) exceed five times, and with schizochytrium limacinum belong to kind of SR21 and KH105 (Aki etal., J Am Oil Chem Soc 80:789-794 (2003)) quite.At last, ONC-T18 shows typical wide salt resistance ability, and tolerant salinity range is 2.0 to 50.0g L ^-1, the biomass productive rate of generation changes (table 9) between 25-30%.In same experiment, find DHA g L ^-1Value is at optimum value (4.6g L ^-1) and minimum value (2.5g L ^-1) between variation be up to 45% (table 9).

Be shown in Figure 15 by ONC-T18 at biomass, TFA and the DHA that generation obtains in the 5L bio-reactor during the 168h.The growth curve of being drawn is several typical curves that obtain under the same conditions.Obtained maximum biomass output after the 120h, exhaust time point near carbon source (with glucose) this moment.This also is that total fatty acid content in the biomass reaches maximum value (account for biomass 70%) time point.Meaningfully, after only cultivating 24h, DHA content reaches peak value, accounts for 30% of total fatty acids, after this maintains 20-25% all the time.These results produce thraustochytriale bacterial strain consistent of lipid acid with other, yet occur aspect the speed different in these reactions.

C) discuss

Before this, most of bacterial strain that the research of Labyrinturomycota is recognized all can not be to be higher than the amount depot fat acid of 20% biomass.For example, isolating before the schizochytrium limacinum that can accumulate the highest fat that accounts for biomass 50% belongs to kind of SR21, golden yellow thraustochytriale is best accumulation person's (20%) (Bajpai et al., J Am Oil Chem Soc 68:509-514 (1991)).Yet ONC-T18 can accumulate the highest lipid that accounts for its biomass 80%.

For accumulating for example ONC-T18 of oily oleaginous microorganism, should in substratum, grow usually with a certain amount of nitrogen (exhausting to 36h 24 usually) and capacity carbon source.In case nitrogen is depleted, oleaginous microorganism can continue to absorb carbon source, but no longer can carry out cell fission owing to lack nitrogen (having stoped the synthetic of albumen and nucleic acid thus).The result is that these carbon sources (being carbohydrate such as glucose) are converted to and are oil in reserve.In this respect, think that ONC-T18 is than other thraustochytriale bacterial strain more slow (Bowles et al. of G13 growth for example, J Biotechnol 70:193-202 (1999) and Huang etal., Mar Biotechnol 5:450-457 (2003)), yet it produces DHA with rapid rate more, and shows the synthetic more unique ability of a large amount total fatty acids.At last, it should be noted that ONC-T18 can grow and has the ability of high biomass and total fatty acids productive rate under extremely low salt concn.This makes it can reduce the corrodibility of salt solution to industrial fermentation equipment when scale operation.

Sequence table

＜110〉Ocean Nutrition Canada Ltd.

A.M. uncle is outstanding

H. Rui Diyaningtasi

C.J. Barrow

A.J. Wim Wenders spy

＜120〉eukaryotic microorganisms of generation lipid and antioxidant

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<160>3

<170>FastSEQ for Windows Version 4.0

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<211>1724

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＜213〉artificial sequence

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＜223〉to the description of artificial sequence; Annotate=

Synthetic construct

<400>1

<210>2

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉to the description of artificial sequence; Annotate=

Synthetic construct

<400>2

<210>3

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉to the description of artificial sequence; Annotate=

Synthetic construct

<400>3

Claims (100)

1. eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.

2. the microorganism of claim 1, the unsaturated fatty acids of wherein said microorganisms has feature shown in Figure 2.

3. the microorganism of claim 1, wherein said microorganism is from net slime mould door (phylumlabyrinthulomycota).

4. the microorganism of claim 1, wherein said microorganism is from net slime mould guiding principle (classlabyrinthulomycetes).

5. the microorganism of claim 1, wherein said microorganism is from thraustochytriale subclass (subclassthraustochytridae).

6. the microorganism of claim 1, wherein said microorganism is from thraustochytriale (orderthraustochytriales).

7. the microorganism of claim 1, wherein said microorganism is from thraustochytriale section (familythraustochytriaceae).

8. the microorganism of claim 1, wherein said microorganism is from genus thraustochytrium (genusthraustochytrium).

9. the microorganism of claim 1, wherein said microorganism is genus thraustochytrium kind (thraustochytrium sp.).

10. the microorganism of claim 1, wherein said microorganism is golden yellow thraustochytriale (Thraustochytrium aureum).

11. the microorganism of claim 1, wherein said microorganism are pink thraustochytriale (Thraustochytrium roseum).

12. the microorganism of claim 1, wherein said microorganism are line shape thraustochytriale (Thraustochytrium striatum).

13. the microorganism of claim 1, wherein said microorganism belongs to (genusSchizochytrium) from schizochytrium limacinum.

14. being schizochytrium limacinum, the microorganism of claim 1, wherein said microorganism belong to kind of (a Schizochytrium sp.).

15. the microorganism of claim 1, wherein said microorganism contain ω-3 or ω-6 lipid acid.

16. the microorganism of claim 1, wherein said microorganism contains DHA or DPA.

17. the microorganism of claim 1, the lipid or the fatty acid component of wherein said microorganisms are at least about 4wt% to 6wt%.

18. the microorganism of claim 17, wherein said lipid comprises DHA.

19. the microorganism of claim 17, wherein said lipid composition contain account for about 25wt% fatty acid component to the n-3DHA of about 40wt% fatty acid component, account for about 6wt% fatty acid component extremely about 10wt% fatty acid component n-6DPA and account for the extremely n-3EPA of about 3wt% fatty acid component of about 0wt% fatty acid component.

20. the microorganism of claim 1, wherein said microorganism is from thraustochytriale section and have ATCC numbering 20888,20889,20890,20891 or 20892.

21. a composition that comprises the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 94% identity.

22. the composition of claim 21, wherein said composition further includes substratum.

23. the composition of claim 21, wherein said composition further includes nutritive substance.

24. the composition of claim 21, wherein said composition are biomass.

25. the composition of claim 21, wherein said eukaryotic microorganisms is from net slime mould door (phylumlabyrinthulomycota).

26. the composition of claim 21, wherein said eukaryotic microorganisms is from net slime mould guiding principle (classlabyrinthulomycetes).

27. the composition of claim 21, wherein said eukaryotic microorganisms is from thraustochytriale subclass (subclass thraustochytridae).

28. the composition of claim 21, wherein said eukaryotic microorganisms is from thraustochytriale (orderthraustochytriales).

29. the composition of claim 21, wherein said eukaryotic microorganisms is from thraustochytriale section (family thraustochytriaceae).

30. the composition of claim 21, wherein said eukaryotic microorganisms is from genus thraustochytrium (genusthraustochytrium).

31. the composition of claim 21, wherein said eukaryotic microorganisms are genus thraustochytrium kind (thraustochytrium sp.).

32. the composition of claim 21, wherein said eukaryotic microorganisms are golden yellow thraustochytriale (Thraustochytrium aureum).

33. the composition of claim 21, wherein said eukaryotic microorganisms are pink thraustochytriale (Thraustochytrium roseum).

34. the composition of claim 21, wherein said eukaryotic microorganisms are line shape thraustochytriale (Thraustochytrium striatum).

35. the composition of claim 21, wherein said eukaryotic microorganisms belongs to (genusSchizochytrium) from schizochytrium limacinum.

36. being schizochytrium limacinum, the composition of claim 21, wherein said eukaryotic microorganisms belong to kind of (a Schizochytrium sp.).

37. the composition of claim 21, the lipid acid that wherein said eukaryotic microorganisms produces has feature shown in Figure 2.

38. the composition of claim 37, wherein said unsaturated fatty acids comprise ω-3 or ω-6 lipid acid.

39. the composition of claim 37, wherein said unsaturated fatty acids comprises DHA or DPA.

40. the composition of claim 21, producible lipid of wherein said eukaryotic microorganisms or fatty acid component are at least about 4wt% to 6wt%.

41. the composition of claim 40, wherein said lipid comprises DHA.

42. the composition of claim 40, wherein said lipid composition contain account for about 25wt% fatty acid component to the n-3DHA of about 40wt% fatty acid component, account for about 6wt% fatty acid component extremely about 10wt% fatty acid component n-6DPA and account for the extremely n-3EPA of about 3wt% fatty acid component of about 0wt% fatty acid component.

43. the composition of claim 21, wherein said eukaryotic microorganisms is from thraustochytriale section and have ATCC numbering 20888,20889,20890,20891 or 20892.

44. a composition that comprises the eukaryotic microorganisms with 18S sequence, the sequence that wherein said 18S sequence and SEQ ID NO:1 list has at least 80% identity.

45. a composition, contain account for about 25wt% fatty acid component to the n-3DHA of about 40wt% fatty acid component, account for about 6wt% fatty acid component extremely about 10wt% fatty acid component n-6DPA and account for the extremely n-3EPA of about 3wt% fatty acid component of about 0wt% fatty acid component.

46. a method for preparing lipid composition, described method comprises: cultivate each eukaryotic microorganisms of claim 1-20 in different oxygen substratum, and separate described lipid composition.

47. the lipid composition for preparing according to the method for claim 46.

48. a delivery device includes claim 21-45 or 47 each compositions.

49. the delivery device of claim 48, wherein said delivery device comprise micro-capsule, microballoon, nanometer ball or nano particle, liposome, noisome, nanometer rhodoplast, solid-liquid nano particle, Leuprolide, gel, gel capsule, tablet, lotion, paste, sprays, emulsion or pulvis.

50. micro-capsule, the aggregation and the load material that comprise basic micro-capsule, each basic micro-capsule individuality all has main casing, and wherein said load material contains claim 21-45 or 47 each compositions, and sealed by main casing, and wherein said aggregation is by shell enclosure.

51. the micro-capsule of claim 50, wherein said main casing and/or shell comprise tensio-active agent, gelatin, poly phosphate, polysaccharide or their mixture.

52. the micro-capsule of claim 50, wherein said main casing and/or shell comprise the Type B gelatin, poly phosphate, gum arabic, alginate, chitin, carrageenin, pectin, starch, modified starch, alpha-lactalbumin, beta-lactoglobulin, ovalbumin, polysorbate, maltodextrin, cyclodextrin, Mierocrystalline cellulose, methylcellulose gum, ethyl cellulose, HPMC, carboxymethyl cellulose, milk protein, whey-protein, soybean protein, rapeseed protein, albumin, the kosher gelatin, the non-kosher gelatin, Halal gelatin or non-Halal gelatin, or their mixture.

53. the micro-capsule of claim 50, wherein said main casing and/or shell comprise complex coacervate.

54. the micro-capsule of claim 50, wherein said main casing and/or shell comprise A type gelatin.

55. the micro-capsule of claim 50, wherein said main casing and/or shell comprise isinglass.

56. comprising the Bloom value, the micro-capsule of claim 50, wherein said main casing and/or shell be about 0 to about 300 gelatin.

57. comprising the Bloom value, the micro-capsule of claim 50, wherein said main casing and/or shell be about 0 to about 50 gelatin.

58. comprising the Bloom value, the micro-capsule of claim 50, wherein said main casing and/or shell be about 51 to about 300 gelatin.

59. comprising the Bloom value, the micro-capsule of claim 50, wherein said main casing and/or shell be about 0, about gelatin of 210, about 220 or about 240.

60. the micro-capsule of claim 50, wherein said main casing and/or shell comprise the coacervate of gelatin and poly phosphate.

61. the micro-capsule of claim 50, wherein said load material comprises the oil from thraustochytriale, schizochytrium limacinum or their mixture.

62. the micro-capsule of claim 50, the mean diameter of wherein said shell are that about 1 μ m is to about 2,000 μ m.

63. the micro-capsule of claim 50, the mean diameter of wherein said shell are that about 20 μ m are to about 1,000 μ m.

64. the micro-capsule of claim 50, the mean diameter of wherein said shell are that about 30 μ m are to about 80 μ m.

65. the micro-capsule of claim 50, the mean diameter of wherein said shell are that about 40 μ m are to about 10 μ m.

66. the micro-capsule of claim 50, the mean diameter of wherein said shell are that about 0.1 μ m is to about 5 μ m.

67. the micro-capsule of claim 50, wherein said load material are about 20% to about 90% of micro-capsule by weight.

68. the micro-capsule of claim 50, wherein said load material are about 50% to about 70% of micro-capsule by weight.

69. a nutritional additive comprises claim 21-45 or 47 each each delivery device or each micro-capsules of claim 50-68 of composition, claim 48-49.

70. the nutritional additive of claim 69, the form of wherein said additive are tablet, gel capsule, capsule, liquid or syrup form.

71. a food comprises claim 21-45 or 47 each each each micro-capsule or each nutritional additives of claim 69-70 of delivery device, claim 50-68 of composition, claim 48-49.

72. the food of claim 71, wherein said food are bakery, wheaten food, meat product, frozen dairy product, milk-product, cheese product, egg-products, seasonings, powder, dessert, nut goods, plant protein preparation, hard candy, soft sweets, poultry prod, fruit juice, granulated sugar, soy sauce, gravy, syrup, nourishing beverage, beverage, drink dried powder, jam or jelly, infant formulas or infant food through processing.

73. the food of claim 71, wherein said food are fish product, pet companion food, cattle food or aquaculture feed.

74. the food of claim 71, wherein said food are bread, corn-dodger, cereal food, sausage, chicken, ice-creams, sour milk, milk, salad food flavouring, rice bran, fruit juice, drink dried powder, swiss roll, biscuit, crispbread, fast food, fruit pies or cake.

75. one kind is sent the method for passing the experimenter with composition, comprises giving described experimenter's claim 48-49 each each each nutritional additive or each food of claim 71-74 of micro-capsule, claim 69-70 of delivery device, claim 50-68.

76. the method for claim 75, wherein said experimenter is a Mammals.

77. the method for claim 75, wherein said experimenter behaves.

78. each micro-capsule of claim 50-68 is used to prepare the purposes of the load material being sent the medicine of passing the experimenter.

79. a method that reduces subject inner cholesterol level, triglyceride levels or their combination comprises giving described experimenter's significant quantity claim 21-45 or 47 each each each each nutritional additive or each steps of food of claim 71-74 of micro-capsule, claim 69-70 of delivery device, claim 50-68 of composition, claim 48-49.

80. the method for the interior essential trace element of additional subject, described method comprises and gives described experimenter's significant quantity claim 21-45 or 47 each each each each nutritional additive or each steps of food of claim 71-74 of micro-capsule, claim 69-70 of delivery device, claim 50-68 of composition, claim 48-49 that wherein said composition, delivery device, micro-capsule, additive and food contain essential trace element.

81. a method that improves insulin sensitivity in the subject comprises giving described experimenter's significant quantity claim 21-45 or 47 each each each each nutritional additive or each steps of food of claim 71-74 of micro-capsule, claim 69-70 of delivery device, claim 50-68 of composition, claim 48-49.

82. a method that alleviates experimenter's hyperglycemia comprises giving described experimenter's significant quantity claim 21-45 or 47 each each each each nutritional additive or each steps of food of claim 71-74 of micro-capsule, claim 69-70 of delivery device, claim 50-68 of composition, claim 48-49.

83. a method that alleviates experimenter's hypercholesterolemia comprises giving described experimenter's significant quantity claim 21-45 or 47 each each each each nutritional additive or each steps of food of claim 71-74 of micro-capsule, claim 69-70 of delivery device, claim 50-68 of composition, claim 48-49.

84. a method that reduces subject fat comprises giving described experimenter's significant quantity claim 21-45 or 47 each each each each nutritional additive or each steps of food of claim 71-74 of micro-capsule, claim 69-70 of delivery device, claim 50-68 of composition, claim 48-49.

85. one kind promotes experimenter's ways of preventing obesity, comprises giving described experimenter's significant quantity claim 21-45 or 47 each each each each nutritional additive or each steps of food of claim 71-74 of micro-capsule, claim 69-70 of delivery device, claim 50-68 of composition, claim 48-49.

86. the method for treatment or prevention experimenter diabetes comprises giving described experimenter's significant quantity claim 21-45 or 47 each each each each nutritional additive or each steps of food of claim 71-74 of micro-capsule, claim 69-70 of delivery device, claim 50-68 of composition, claim 48-49.

87. a pharmaceutical preparation comprises claim 21-45 or 47 each each each micro-capsule and pharmaceutical carriers of delivery device, claim 50-68 of composition, claim 48-49.

88. a food comprises the composition of claim 21.

89. the food of claim 88, wherein said food baking food, wheaten food, meat product, frozen dairy product, milk-product, cheese product, egg-products, seasonings, powder, dessert, nut goods, plant protein preparation, hard candy, soft sweets, poultry prod, fruit juice, granulated sugar, soy sauce, gravy, syrup, nourishing beverage, beverage, drink dried powder, jam or jelly, infant formulas or infant food through processing.

90. the food of claim 88, wherein said food are fish product, pet companion food, cattle food or aquaculture feed.

91. the food of claim 89, wherein said food are bread, corn-dodger, cereal food, sausage, chicken, ice-creams, sour milk, milk, salad food flavouring, rice bran, fruit juice, drink dried powder, swiss roll, biscuit, crispbread, fast food, fruit pies or cake.

92. a method that reduces subject inner cholesterol level, triglyceride levels or their combination, the delivery device of the composition that comprise the composition, the nutritional additive that contains the composition of claim 21 that give significant quantity claim 21, contains claim 21 or contain the step of food of the composition of claim 21.

93. the method for essential trace element in the additional subject, the delivery device of the composition that comprise the composition, the nutritional additive that contains the composition of claim 21 that give significant quantity claim 21, contains claim 21 or contain the step of food of the composition of claim 21.

94. a method that improves insulin sensitivity in the subject, the delivery device of the composition that comprise the composition, the nutritional additive that contains the composition of claim 21 that give significant quantity claim 21, contains claim 21 or contain the step of food of the composition of claim 21.

95. a method that alleviates experimenter's hyperglycemia, the delivery device of the composition that comprise the composition, the nutritional additive that contains the composition of claim 21 that give significant quantity claim 21, contains claim 21 or contain the step of food of the composition of claim 21.

96. a method that alleviates experimenter's hypercholesterolemia, the delivery device of the composition that comprise the composition, the nutritional additive that contains the composition of claim 21 that give significant quantity claim 21, contains claim 21 or contain the step of food of the composition of claim 21.

97. a method that reduces subject fat, the delivery device of the composition that comprise the composition, the nutritional additive that contains the composition of claim 21 that give significant quantity claim 21, contains claim 21 or contain the step of food of the composition of claim 21.

98. one kind promotes experimenter's ways of preventing obesity, the delivery device of the composition that comprise the composition, the nutritional additive that contains the composition of claim 21 that give significant quantity claim 21, contains claim 21 or contain the step of food of the composition of claim 21.

99. the method for treatment or prevention experimenter diabetes, the delivery device of the composition that comprise the composition, the nutritional additive that contains the composition of claim 21 that give significant quantity claim 21, contains claim 21 or contain the step of food of the composition of claim 21.

100. a pharmaceutical preparation comprises the composition of claim 21.

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