Eukaryotic microorganisms for producing lipids and antioxidants

[0001] 1. The present application claims the use of priority from US provisional application No. 60 / list 207, filed on June 7, 2005, and from US provisional application No. 60 / 751,401, filed December 16, 2005, both of which are incorporated herein by reference in their entirety reference.

I. BACKGROUND OF THE INVENTION [0002] 2. There is clear scientific evidence that highly unsaturated fatty acids (n-3), such as docosahexaenoic acid (DHA), have a positive effect on cardiovascular diseases, chronic inflammations and brain disorders. On the other hand, fatty acids (n-6) have been perceived as intermediate metabolites of eicosanoid steroids such as prostaglandin, leukotrienes or the like.

[0003] 3. At present, the main source of these highly unsaturated fatty acids are fish, where DHA and eicosapentaenoic acid (EPA) are found in various seagoing fish (such as sardines and tuna) respectively in amounts of about 20% and 10%.

However, if someone intends to use fish oil as the sole source of these lipids, there are several drawbacks, such as problems with doubtful smell, unmanageable availability fluctuations, natural variability of fish oil content, as well as the ability to collect harmful pollutants. In addition, if one of these sources intends to obtain highly purified oil (n-3) or (n-6), it is very difficult to selectively separate and purify it.

II. SUMMARY [0004] 4. Compositions and methods are disclosed relating to Thraustochytriales eucarionts and the Thraustochytriaceae family, which in the culture produce amounts of unsaturated fatty acids, such as omega 3 (n-3) and / or omega 6 (n-6) oils, such as DHA, EPA and DPA, suitable for purification and used as all such compositions are used, and on a larger scale, due to their production methods.

III. BRIEF DESCRIPTION OF THE DRAWINGS [0005] The attached drawings, which are included in this specification and form a part thereof, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

6. Figure 1 shows a graph showing the results obtained from methylating fatty acids from lipids derived from the ONC-T18 strain.

Figure 2 graphically depicts a comparison of fatty acid methyl ester profiles between the primary isolate of the ONC-T18 strain collected in Advocate Harbor and the isolate of the ONC-T18 strain Thraustochytrium sp. Deposited at the ATCC under the accession number PTA-6245. All peaks were identified by gas chromatography and mass spectrometry.

8. Figure 3 shows a scatterplot of the results from the biomass optimization experiments carried out for the ONC-T18 strain. These experiments used a technique known as the Taguchi method to determine optimal growth conditions for the ONC-T18 strain under various media conditions.

9. Figure 4 shows a bar graph of the fatty acid profile of the ONC-T18 strain grown under optimal conditions (example 4) for a period of nine days.

10. Figure 5 shows a table of oil producing organisms isolated as described elsewhere herein.

11. Figure 6 shows a branched phylogenetic tree of the relationship between the 18S rRNA gene of the ONC-T18 strain and other Thraustochytriales.

12. Figure 7 shows the production of lipids and DHA by the ONC-T18 strain under various conditions.

13. Figure 8 shows a modified diagram with information on growth conditions for the eukaryotes disclosed herein. (Modification from Ratledge, C. (2004), Lipid Technol.

16: 34-39).

14. Figure 9 shows the proposed metabolic pathway for the production of PUFAs for the disclosed eukaryotes.

15. Figure 10 shows a comparison of fatty acid production maxima and compositions at various alternative low-cost carbon sources.

16. Figure 11 shows the grouping of collected isolates based on their C20 and C22 PUFA profiles. The results were compared with two reference strains: ATCC 20891 and MYA-1381.

17. Figure 12 shows a tree obtained by neighboring-joining tree for 18S rRNA of ONC-T18 strain. The dash means the genetic distance, while the square brackets illustrate the phylogenetic sequences used in this tree

GenBank.

Figure 13 shows the fatty acid profile of the ONC-T18 strain grown in a medium containing 2 gL^-1 yeast extract, 8 g L^-1 L-glutamate, 6 g L^-1 sea ​​salt and 60 g L^-1 glucose in 3 different types of fermentation: on agar plates (1.5% agar, 25 ° C, 27 days), in the flasks (50 ml in the flask 250 ml, 120 rpm, 25 ° C, 3 days) and in the bioreactor 5 L (4 liters of air per minute, pO2 90%, 25 ° C, 3 days).

19. Figure 14 shows the HPLC chromatogram of carotenoid compounds isolated from the ONC-T18 strain Thraustochytrium sp. For example, from the ONC-T18 strain Thraustochytrium sp., Astaxanthin, zeaxanthin, canthaxanthin, echinenone, and β-carotene were isolated.

20. Figure 15 shows typical results for the production of biomass, total fatty acids (TFA), DHA and the use of glucose for the ONC-T18 strain maintained in the bioreactor 5.

L for 168 h with medium consisting of 60 g L^-1 glucose, 2 g L^-1 yeast extract, 8 g L^-1glutamic acid and 6 g L^-1 salt (4 liters of air per minute, pO2 90%, 25 ° C, pH 7-9).

21. Figure 16 shows the postulated pathways of metabolism involved in the formation of astaxanthin in the ONC-T18 strain of Thraustochytrium sp.

IV. DETAILED DESCRIPTION [0006] 22. Before disclosing and describing the present compounds, compositions, articles, devices, and / or methods, it should be understood that they are not limited to specific synthetic methods or specific biotechnological recombination methods, unless otherwise specified, or individual reagents, unless otherwise specified, as these can, of course, be changed. It is also to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting.

[0007] 23. Those skilled in the art will recognize, or, without using anything other than routine experimentation, will be able to determine many equivalents of specific embodiments of the method and compositions described herein. Such equivalents are intended to be covered by the following claims.

A. Definitions [0008] 24. {noun} crowd. - the first sentence refers to the English text} As used in the description of the invention and the appended claims, the generics of the singular "a", "an" and "the"

include the plural unless the context clearly dictates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, etc.

[0009] 25. Value ranges can be expressed herein as "about" one particular value, and / or to "about" another particular value. When such a range is expressed, a different embodiment covers from one particular value and / or another particular value. Similarly, when values ​​are expressed as approximations, by using the preceding term "about", it will be understood that a particular value creates a different embodiment. It will be further understood that the ends of each range are relevant to both the other end and independently of the other ends. It is also understood that there are a number of values ​​disclosed herein and that each value is also disclosed herein as & quot; about & quot; this particular value, in addition to the same value. For example, if the value "10" is disclosed, "about 10" is also disclosed. It is also understood that when a value is disclosed, the "value less than or equal to" this value, "greater than or equal to" this value and the possible ranges between the values ​​are also disclosed, as will be well understood by the skilled person. For example, if the value "10" is disclosed, the value "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout the description data is provided in a number of different formats, and that these data represent endpoints and starting points and ranges for any combination of data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is understood that values ​​greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as between 10 and 15 are considered to be disclosed. It is also understood that each unit between two is disclosed. individual units. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0010] 26. By "reducing" or other forms of reducing is meant lowering the appearance or characteristic. It is understood that this is typically the case for some pattern or expected value, in other words it is relative, but it is not always necessary to refer to a pattern or a related value. For example, "reduces phosphorylation" means a reduction in the degree of phosphorylation that occurs with reference to a standard or a comparison. It is understood that unless otherwise specifically indicated, the amount of compound or composition or intensity of the state may be reduced with respect to another compound or composition or condition.

[0011] 27. By "braking" or other forms of braking is meant inhibiting or limiting a particular feature. It is understood that this is typically the case for some pattern or expected value, in other words it is relative, but it is not always necessary to refer to a pattern or a related value. For example, "inhibits phosphorylation"

- 5 means a reduction in the degree of phosphorylation that occurs with reference to a reference or a comparison. It is understood that unless otherwise specifically indicated, the amount of compound or composition or intensity of the state may be reduced with respect to another compound or composition or condition.

[0012] 28. By "prevention" or other forms of prevention is meant retention of a particular trait or condition. Prevention does not need to be compared with the reference, as it is typically more absolute than, for example, reduction or inhibition. According to the present, something can be reduced, but not inhibited or prevented, but something that is reduced can also be inhibited or prevented. It is understood that where words are used to reduce, inhibit or prevent, unless otherwise specifically indicated, the use of the other two words is clearly disclosed. Thus, if the inhibition of phosphorylation is revealed, then the reduction and prevention of phosphorylation is also disclosed.

[0013] 29. The term "therapeutically effective" means that the amount of composition used is sufficient to ameliorate one or more of the causes or symptoms of the disease or disorder.

Such improvement requires only reducing or changing, and not necessarily eliminating.

[0014] 30. The term "carrier" means a compound, composition, substance, or structure that, when used in combination with a compound or composition, aids in the manufacture or manufacture, storage, administration, delivery, selectivity, or any other characteristic of the compound or composition. composition for its intended use or purpose. For example, the carrier can be selected to minimize any degradation of the active ingredient and to minimize any side effects in the subject.

[0015] 31. Within the description and claims of this application, the word "include" and variations of the word, "including" and "includes", means "including, but are not limited to," and it is not excluded, for example, , other additions, ingredients, integers or steps.

[0016] 32. The term "cell" as used herein also refers to separate microbial cells, or cultures derived from such cells. "Breeding" refers to a composition comprising isolated cells of the same or different types.

[0017] 33. The term "metabolite" refers to active derivatives produced after the compound has been incorporated into a biological environment, such as a patient.

[0018] 34. In the field, the term "stable" as used in reference to pharmaceutical and nutraceutical compositions is generally understood to mean loss of

- 6 specific storage conditions for a certain period of time of less than a certain amount, usually 10%. The time it takes to consider a composition to be stable refers to the use of each product and is dictated by practical considerations of product manufacture, storage for quality control and inspection, shipment to a wholesaler or directly to the buyer, where it is stored again before its final use. Taking into account the safety factor of a few months, the lowest shelf life of the product is usually one year, and preferably more than 18 months. The term "stable" as used herein refers to these commercial circumstances and the ability to store and transport the product under ambient conditions that are easy to obtain, such as refrigeration conditions, 2 ° C to 8 ° C.

[0019] 35. References in the description and the final claims to parts by weight, individual element or component in the composition or manufacture, indicate the weight relationship between the element or component and any other elements or ingredients in the composition or article for which parts by weight are expressed. Thus, in a blend containing 2 parts by weight of component X and 5 parts by weight of component Y, components X and Y are present in a weight ratio of 2: 5, and are present in such a proportion irrespective of whether additional components are included in the blend.

[0020] 36. The weight percentage of ingredient, unless otherwise specifically stated, refers to the total weight of the formulation or composition in which the ingredient is contained.

[0021] 37. "Separation" and any form such as "separate" refers to a situation where something is in a form in which it can be manipulated or further purified. Separated and its forms indicate that something is in its current state, which is different from the previous state. For example, a ribosomal RNA molecule can be "secreted" if it is, for example, removed from the body, synthesized, or produced recombinantly.

Often, the "separation" of one thing is related to something else. For example, a eukaryote, as discussed herein, can be isolated as discussed herein, for example, by culturing a eukaryote, such that eukaryote survives in the absence of significant (detectable) amounts of other organisms. It is understood that unless otherwise specifically indicated, any of the disclosed compositions may be isolated as disclosed herein.

[0022] 38. "Purify" and any form such as "purification" refers to a state in which the substance or compound or composition is in a more uniform state than it was before. It is understood that as disclosed herein, something may have purity, unless otherwise indicated, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. , 16, 17, 18, 19, 20,

- 7 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, if the composition of component A had a purity of 90%, it meant that 90% of the composition was component A, and that 10% of the composition was one or more things, such as molecules, compounds, or other substances. For example, if the eukaryotic microorganism disclosed, for example, produces 35% DHA, this component can be further "purified" so that the final lipid composition has more than 90% DHA. Unless otherwise indicated, this purity will be determined by the relative "weights" ingredients in the composition. It is understood that unless specifically indicated otherwise, any of the disclosed compositions can be purified as disclosed herein.

[0023] 39. "Possible" or "optionally" means that the next described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs, and instances where it does not occur.

[0024] 40. "Primers" means a subset of probes that may support some type of enzymatic manipulation and that may hybridize to a target nucleic acid such that such enzymatic manipulation may occur. The primer can be made from any combination of nucleotides or derivatives or analogs of nucleotides available in the art that do not interfere with enzymatic manipulation.

[0025] 41. Reference is made to various publications throughout the present application. The disclosure of these publications in their entirety constitutes a reference for the present application, in order to more fully describe the state of the art which this application relates to. Disclosed references are also separately and specifically incorporated into this reference by reference to the material contained therein, which is discussed in the sentence that is based on it.

[0026] 42. The components for use in making the disclosed compositions as well as the compositions themselves for use in the methods disclosed herein are disclosed. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, whereas a specific reference to each of the various individual and collective combinations and permutations of these compounds may not be explicit disclosed, each of them is specifically contemplated and described herein. For example, if a particular species of the Thraustochytriaceae family is disclosed and discussed, and a variety of modifications that can be made to a range of organisms including species from the Thraustochytriaceae family are discussed, all combinations and permutations of these species are specifically considered.

- 8 species from the Thraustochytriaceae family and modifications that are possible unless specifically indicated to the contrary. Thus, if the class of molecules A, B, and C is disclosed, as well as the class of molecules D, E, and F, and the example of the molecule in combination AD is disclosed, even if each of them is not separately calculated, each of them it is separately and collectively considered, indicating the combinations, and AE, AF, BD, BE, BF, CD, CE, and CF are considered to be disclosed. Similarly, any subset or combination thereof is also disclosed. Thus, for example, subgroup AE, BF, and CE would be considered disclosed. This approach applies to all aspects of the present application, including, but not limited to, steps in the methods of making and using the disclosed compositions. So if there is a variety of additional stages,

B. Compositions [0027] 43. Thiruses of the Thraustochytriales order, preferably of Thraustochytrium or Schizochytrium species, having the ability to produce lipids, such as fatty acids, such as unsaturated fatty acids such as omega-3 fatty acids such as omega-6 fatty acids, are disclosed. , and omega-9 fatty acids, such as a series of (n-3) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), a series of (n-6) docosapentaenoic acid (DPA) and a series of (n-9) palmitic and stearic acids . The disclosed eukaryotic microorganisms may also produce antioxidants, such as, but not limited to, a carotenoid compound, carotene (e.g. β-carotene) and xanthophyll compounds, astaxanthin, zeaxanthin, canthaxan, and echinenone.

[0028] 44. Also disclosed are the conditions for the secretion and growth of eukaryotic microorganisms. For example, including heterotrophic growth conditions for the production of disclosed lipids and antioxidants, for example, both individually and in combination. Accordingly, by the use of this unique eukaryote, it is possible to efficiently produce a series of (n-3) DHA and / or a series of (n-6) DPA and / or a series of antioxidant carotenoid and / or a series of xanthophyl antioxidants that are useful as or in nutraceuticals, additives for food, pharmaceuticals or industry.

[0029] 45. Compositions comprising a eukaryotic microorganism comprising or consisting of Thraustochytrium species are disclosed, wherein the example, as disclosed herein, is an ONC-T18 strain that is deposited with the ATCC under the accession number PTA-6245.

[0030] 46. It is understood that the eukaryotic microorganism and any clones, modified organisms or genes secreted from said organism as disclosed in ONC-T18 are disclosed. The disclosed organisms have the ability to produce unsaturated fatty acids, such as lipids containing a range of omega-3 DHA and EPA, and a series of omega-6 DPAs and a variety of antioxidants, such as carotenoids, xanthophylls and phenolic compounds.

[0031] 47. Also disclosed are methods for producing a biomass containing said compounds. Further disclosed are methods for the preparation of omega-3, omega-6 and carotenoids using a eukaryotic microorganism. Also disclosed are methods for the production of oils derived from microorganisms (or individual cells).

48. Furthermore, fatty acids and carotenoids produced by the disclosed eukaryotic microorganism and any progeny (genetically modified or otherwise), various food items, nutraceuticals, pharmaceuticals and foods supplemented with lipids and antioxidants, as well as the method of using these compounds as disclosed, are disclosed. addition to various food and food items.

[0033] 49. US Patent No. 5,130,242 to Barclay discloses a collection and screening method for the isolation of microorganism strains with the following characteristics for producing omega-3 fatty acids: 1) capable of heterotrophic growth; 2) produce a high content of omega-3 fatty acids; 3) single-celled; 4) produce low content of normal and omega-6 fatty acids; 5) cells without pigment, white or colorless; 6) temperature-tolerant (e.g., capable of growing above 30 ° C); and 7) euryhalic (e.g., capable of growing in a wide range of environmental salinity, but preferably at low salinity).

50. The disclosure in the '242 patent also describes a method for the heterotrophic production of whole cell or extracted microbial products with a high concentration of omega-3 fatty acids that can later be used in animal or human food products. This method uses the microorganisms identified by the method of collection and screening disclosed herein. These microorganisms, which belong to the order Thaustochytriales, are grown in ground cereal. To increase the production of omega-3 fatty acids, low-temperature stress and high dissolved oxygen content are used, as well as the addition of antioxidants, growth factors, vitamins, and phosphorus. Extracted products contain high concentrations of omega-3 fatty acids (e.g., C20: 5w3, C22: 5w3; and C22: 6w3) and low concentrations of acids

- 10 fatty omega-6 (e.g., C20: 4w6 and C22: 5w6). Specifically, the ratio of C20: 5w3 to C22: 6w3 fatty acids is from 1: 1 to 1:30. The proportions of C22 fatty acids: 5w3 to C22: 6w3 are from 1:12 to only trace amounts of C22: 5w3. In addition, the microorganisms produce 0.6 to 0.72% DHA, 0 to 5% DPA, and 0 to 18.9% EPA, based on the weight of fatty acids together.

[0035] 51. U.S. Patent No. 6,451,567 to Barclay discloses a method for breeding Thraustochytrium and

Schizochytrium in a chloride-free medium (<3 g / L) containing sodium salts (e.g., sodium sulfate). The chloride free medium gives cell cluster sizes of less than 150 μm. The disclosed method produces microorganisms and extracts that are useful in nutritional products for aquaculture. Further components of food products include flaxseed, rapeseed, soy, and avocado flour. Microorganisms can produce 1.08 g / L nutrient per day of omega-3 fatty acids. Further disclosure in the '567 patent describes a variety of culture media that contains seawater, glucose (1, 3, 5, or 10 g / L), yeast extract (0.01, 0.2, 0.4 and 5 g / L). ), additional sources of nitrogen, such as protein hydrolyzate (1 g / L), liver extract (1 g / L), glutamate (5 g / L), MSG (monosodium glutamate, monosodium glutamate, 3 g / L) .

[0036] 52. US Patent No. 6,582,941 to Yokochi et al. discloses the Schizochytrium species, strain SR21 and another Schizochytrium strain belonging to the same species that have the ability to produce fatty acid fractions having a high concentration of DHA omega-3 and / or DPA omega-6 and low concentration of EPA. Also, methods are disclosed for the cultivation of such microorganisms and the secretion of such fatty acids. The applied medium contains sea salt, yeast extract (0.2, 1.0, or 10 g / L), maize soak extract (0.5, 1.0, or 10 g / L), glucose (10-120 g / L), plus additional salts (e.g., NH 4 OAc, phosphates). The fatty acid compositions contain about 15 to 20% DHA based on the weight of the biomass (about 28% with respect to the total weight of fatty acids). The compositions may be used in food products (e.g. infant milk).

[0037] 53. U.S. Patent No. 6,607,900 to Bailey et al. discloses a method for cultivating eukaryotic microorganisms (e.g., Schizochytrium sp. ATCC 20888) that are capable of producing at least 20% of their biomass as polyunsaturated lipids (particularly omega-3 and -6 fatty acids). The process involves cultivating microorganisms in a medium containing a carbon and nitrogen source. It also reveals the use of low levels of dissolved oxygen (less than 3%) and low levels of ions

- chloride (less than 3 g / L) for increased production. The microorganisms have a lipid production rate of at least 0.5 g / L / h. The lipid fraction is from 15 to 20% DHA based on the weight of biomass (about 35% based on the weight of fatty acid methyl esters together).

[0038] 54. Publication of US Application No. 2004/0161831 to Komazawa et al. discloses a Thraustochytrium strain (LEF1; ATCC No. FERM BP-08568) that has the ability to produce DHA. By culturing in conventional media, the microorganism can produce an oil having at least 50% by weight of DHA. The oil can be treated with lipase before the release of DHA. The oil can be used in food or beverages or DHA can be hydrolyzed to produce behenic acid.

1. Fatty acids 55. The fatty acids are carboxyl terminated hydrocarbon chains, which are called unsaturated ones, if they contain at least one carbon-carbon double bond and polyunsaturated when they contain a plurality of such bonds. Long-chain polyunsaturated fatty acids (PUFAs) or highly-unsaturated fatty acids (HUFAs) can be divided into ranks (n-3) and (n-6) due to the location of these double bonds. There is clear scientific evidence that (n-3) highly unsaturated fatty acids, such as DHA, have a positive effect on cardiovascular diseases, chronic inflammation and brain disorders. On the other hand, fatty acids (n-6) were observed as intermediate metabolites of eicosanoid steroids,

[0040] 56. The polyunsaturated fatty acids may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 double and / or triple carbon-carbon bonds. For example, polyunsaturated fatty acids may contain 3-8, 4-7, or 5-6 double bonds and / or triple carbon-carbon bonds.

[0041] 57. Currently, the main source of these highly unsaturated fatty acids is fish, with various seawater fish (such as sardines and tuna) accounting for DHA and EPA in approximately 20% and 10%, respectively. However, if a fish oil intends to be used as the only source of these lipids, there are several drawbacks, such as problems with doubtful fragrance, unmanageable availability fluctuations, natural variability of fish oil content, as well as the ability to collect harmful pollutants. In addition, if someone intends to obtain highly purified oil (n-3) or (n-6) from these sources, it is very difficult to selectively

- separate and clean. In particular, a large market share for highly concentrated DHA is available in the market for neonatal dietary supplements. If the source of these products were to be fish oil, DHA would have to be selectively separated from EPA in large quantities. An alternative source adapted to the production of highly purified and highly unsaturated fatty acids is clearly needed.

[0042] 58. In addition to fish oils, various microorganisms (mainly marine) are able to produce and / or accumulate a series of (n-3) docosahexaenoic acid. Of particular importance is the fact that the production by micro-organisms is not affected by fluctuations caused by external parameters, such as seasonality, weather, and food supply. For example, the ability to produce DHA is known to have the following microorganisms: the subcutaneous bacterium Vibrio marinus (ATCC 15381), Vibrio sp. T3615, Photobacterium profundum SS9, Mortierella marina MP-1 and Psychromonas kaikoae (ATCC BAA-363T); microalgae species, such as Crypthecodinium cohmi, Cyklotella cryptica, and Mortieralla alpina (IS-4); and protista Thraustochytrium sp. (ATCC 20892), Thraustochytrium aureum (ATCC 34304) and

Thraustochytrium roseum. However, according to the method using these purified organisms, the amount of docosahexanenoic acid produced per gram of biomass per liter is low, and is in the range of 10 to 500 mg. Some examples and representative oil producing microorganisms are shown in Figure 5.

[0043] 59. Omega 3 compounds have been shown to have beneficial effects, and the oils and compositions disclosed herein can be used for anti-inflammatory effects on cystic fibrosis (Cochrane Database Syst Rev. 3), rheumatoid arthritis (Drugs 63: 845-53), asthma (Ann Allergy Asthma Immunol 90: 371-7) and thrombotic stroke (Prev Cardiol 6: 38-1), a protective effect for the heart, as well as direct action on arteriosclerosis and arrhythmia (Prostaglandins Leukot Essent Fatty Acids 63: 351-62 ), inhibition of proliferation of breast cancer cell lines and prostate cancer, and reduction in animal experiments (Am J Clin Nutr 77: 532-43), antipsychotic effect on schizophrenia (J Neural Transm Suppl 64: 105-17) and other psychiatric diseases (Can J Psychiatry 48: 195-203),as a nutritional supplement strengthening the immunity used in normal neonatal development and in the treatment of neonatal infections (Eur J Pediatr 162: 122-8), and for the treatment of pathological pain by directly weakening the neuronal and ganglionic processes that underlie neuropathic and inflammatory pain (Prostaglandins Leukot Essent Fatty Acids 68: 219-24).

2. Thraustochytriaceae

- 13 a) ONC-T18 [0044] 60. The marine organism ONC-T18, as disclosed herein, was collected as part of a research expedition to isolate PUFA-producing microorganisms, where over 60 pure cultures were isolated (for details see Table 7). Further, ONC-T18 was isolated from grass leaves from salt marshes in Advocate Harbor, Fundy Bay, Nova Scotia, Canada. On the basis of microscopic examination and techniques of subsequent cultivation, the strain was considered to be a single microorganism belonging to the genus Thraustochytrium. All strains and two comparative cultures of ATCC (ATCC 20891 & MYA-1381) were cultured in 0.5% glucose medium, 0.2% peptone, 0.2% yeast extract in seawater (SW), and analyzed by GC (methyl acid methyl ester). fatty acids methyl esters, FAME).

[0045] 61. Figure 6 shows the proposed phylogenetic tree of the relationship between ONCT18 and other closely related organisms.

[0046] 62. ONC-T18 was originally isolated as a single microorganism, using classical bait techniques from pine pollen and then growing on selective medium.

Specifically a nutrient medium containing 5 g L was prepared^-1 glucose, 2 g L^-1 peptone, 2 g

L^-1 yeast extract in 1 L of sea water filtered through a 0.2 μm filter. The fatty acid profile was then determined for ONC-T18 using the Bligh and Dyer extraction and fractionation method as well as PUFA gas chromatography techniques.

The chromatographic results showed the ability of this strain to produce increased amounts of TFA, DHA as well as significant amounts of EPA and DPA.

[0047] 63. The eukaryotic microorganism disclosed may be used in a method for producing a lipid or fat containing DHA, EPA and DPA, but is not limited to the above-mentioned ONC-T18 strain or PTA-6245, to any derivative of said strain, whether obtained by genetic modification, chemical mutagenesis, fermentation adaptation, or any other means of producing mutant strains, whereby the product of these modifications has genetic or morphological and functional features such as a eukaryotic microorganism as disclosed herein.

[0048] 64. A eukaryotic microorganism which is capable of producing a lipid composition having lipid-class-specific properties is disclosed, and a solution to maintaining a stable, reliable and economic source of such a lipid having high functionality and thus added value is disclosed. Thus, the wild-type strains are disclosed herein

- producing a series of (n-3) DHA and a number of (n-6) DPAs to a greater extent, as well as variant and recombinant strains for producing these polyunsaturated fatty acids to a greater extent. Such variant or recombinant microorganisms include those for obtaining the content of said lipids higher than those produced by the original wild-type strain, when grown using the same conditions and media. In addition, microorganisms for producing a lipid containing a number of (n-3) DHA, EPA and a series of (n-6) DPAs similar to those of the corresponding wild-type strains can be selected efficiently using substrates, with improved cost efficiency, and also they are covered by the disclosure.

[0049] 65. Compositions comprising a eukaryotic microorganism of the order Thraustochytriales are disclosed, wherein the eukaryotic microorganism produces unsaturated fatty acids. The polyunsaturated fatty acids can be, for example, omega 3 or omega 6 fatty acids, such as DHA and DPA.

[0050] 66. Compositions comprising eukaryote are disclosed wherein the composition produces a lipid.

[0051] 67. Also disclosed are compositions where the lipid comprises a lipid as disclosed herein.

[0052] 68. Also disclosed are compositions wherein the eukaryote includes an organism of the order Thraustochytriales.

[0053] 69. Also disclosed are compositions wherein the eukaryote has a 18S ribosomal RNA gene sequence having at least 80% identity to SEQ ID NO. ID NO: 1.

[0054] 70. It is understood that any form of characterization described herein, such as by genetics or by lipid signatures or by classification, for eukaryotic microorganisms can be used to characterize microorganisms as disclosed herein. The eukaryotic microorganism may comprise one or more microorganisms of the Thraustochytriaceae family, and examples are access numbers

ATCC 20888, 20889, 20890, 20891, and 20892. There are various characteristics that can be applied to organisms and the unsaturated fatty acids that they produce. It is understood that they can be used in any combination or permutation to define a kit or kits, for example, organisms or oils or antioxidants. One of the characteristics is, for example, the classification of the organisms themselves, the genetic identification of organisms, the profiles of lipids and antioxidants of organisms, and the conditions for the growth of organisms.

b) Classification

[0055] 71. The eukaryotic microorganism may belong to the Labyrinthulomycota type. The eukaryotic microorganism may belong to the Labyrinthulomycetes group. The eukaryotic microorganism may belong to the Thraustochytridae subgroup. The eukaryotic microorganism may belong to the order Thraustochytriales. The eukaryotic microorganism may belong to the Thraustochytriaceae family. The eukaryotic microorganism may belong to the genus Thraustochytrium. The eukaryotic microorganism can be a species

Thraustochytrium. The eukaryotic microorganism may be Thraustochytrium aureum. The eukaryotic microorganism may be Thraustochytrium roseum. The eukaryotic microorganism may be Thraustochytrium striatum. The eukaryotic microorganism may belong to the genus Schizochytrium. The eukaryotic microorganism may be a species of Schizochytrium. The eukaryotic microorganism may be a modified version of any of the listed eukaryotic microorganisms. The eukaryotic microorganism may also include any currently unknown or isolated members of said group, subgroup, order, family or type of prokaryotes. The combination of eukaryotic microorganisms may be any combination of any organisms disclosed herein, including one or more of Thraustochytrium sp., Schizochytrium sp.

[0056] 72. The eukaryotic microorganisms of the Thraustochytriaceae family may be any of those disclosed above. The eukaryotic microorganism may include the body having the ATCC access number PTA-6245.

c) Genetics [0057] 73. The eukaryotic microorganism may have the 18S rRNA sequence SEQ. SEQ ID NO: 1. A eukaryotic microorganism may have the 18S rRNA sequence which, for example, is about

90% homology, or any other identity disclosed herein, from SEQ. The eukaryotic microorganism may have the 18S rRNA sequence which hybridizes under the acute or any other conditions as disclosed herein, in SEQ ID NO: 1. ID NO: 1, or with part of SEQ. ID NO: 1.

[0058] 74. Sequence similarity / identity and hybridization of nucleic acids for nucleic acids of organisms may be as described herein. Specifically, a comparison of SEQ. ID NO: 1 with nucleic acid sequences found in the genomic database, GenBank (National Center for Biotechnology Information, National Institute of Health, Bethesda, MD, USA) using the BLAST algorithm (Basic local alignment search

- 16 tool) identified the SEQ. ID NO .: 1 as a related (91% similarity) to several eukaryotic species of the Thraustochytridae subgroup, closely related to Thraustochytrium sp. CHN-1 [AB126669] (94.5% similarity) and Thraustochytriidae sp. N1-27 [AB073308] (95, 5% similarity), and the closest related to Thraustochytrium striatum [AF265338] (97.5% similarity).

3. (1) Sequence similarity [0059] 75. It is understood that the terms homology and identity discussed herein are the same as the similarity. Thus, for example, if the word homology is used between two non-natural sequences, it is understood that this does not necessarily indicate the evolutionary relationship between the two sequences, but rather shows the similarity or link between their nucleic acid sequences. Many methods for determining homology between two evolutionarily dependent molecules are routinely applied to two or more nucleic acids or proteins to measure sequence similarity, whether they are evolutionarily related or not.

[0060] 76. In general, it is understood that one method of defining any known or those that may arise, variants and derivatives of the genes and proteins disclosed herein, is to define variants and derivatives in the homology category to specific known sequences. This identity of the individual sequences disclosed herein is also discussed elsewhere herein. In general, the variants of the nucleic acids and proteins disclosed herein typically have 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 percent homology to the sequence given or the native sequence. Those skilled in the art will readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, homology can be calculated after the arrangement of two sequences so that homology is at its highest level.

[0061] 77. Another method of calculating homology can be performed by published algorithms. The optimal alignment of sequences for comparison can be carried out using the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482, 1981, the homology algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443, 1970, by seeking similarity by the method

Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 57 = 5 Science Dr., Madison, WI), or by review.

[0062] 78. The same types of homology can be obtained for nucleic acids, for example, the algorithms disclosed in: 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, which is herein incorporated by reference to at least material associated with the arrangement of nucleic acids. It is understood that any of the methods may be typically used and that in some cases the results of these various methods may vary, but a skilled person understands that if the identity is found in at least one of these methods, it finds that the sequences have established identity, and will be disclosed herein.

[0063] 79. For example, as used herein, a sequence referred to having a particular percentage of homology to another sequence refers to sequences that have the homology as quoted as calculated by one or more of the calculation methods described above. For example, the first sequence has 80 percent homology, as defined herein, for the second sequence if it is calculated that the first sequence has 80 percent homology to the second sequence using the Zaver calculation method, even if the first sequence does not have 80 percent homology to the other sequence as calculated from any of the other calculation methods. For another example, the first sequence has 80 percent homology, as defined herein, for the second sequence, if calculated, that the first sequence has 80 percent homology to the second sequence using both the Zaver calculation method and the calculation method of Pearson and Lipman, even if the first sequence does not have 80 percent homology to the second sequence as calculated by Smith and Waterman calculation method for the calculation of Needleman and Wunsch, methods of calculating Jaeger, or any of the other methods of calculation. For yet another example, the first sequence has 80 percent homology as defined herein to the second sequence if it is calculated that the first sequence has 80 percent homology to the second sequence using each of the calculation methods (although, in practice, different calculation methods will often be give different calculated percentages of homology). even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the method of calculating Needleman and Wunsch, methods for calculating Jaeger, or any of the other calculation methods. For yet another example, the first sequence has 80 percent homology as defined herein to the second sequence if it is calculated that the first sequence has 80 percent homology to the second sequence using each of the calculation methods (although, in practice, different calculation methods will often be give different calculated percentages of homology). even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the method of calculating Needleman and Wunsch, methods for calculating Jaeger, or any of the other calculation methods. For yet another example, the first sequence has 80 percent homology as defined herein to the second sequence if it is calculated that the first sequence has 80 percent homology to the second sequence using each of the calculation methods (although, in practice, different calculation methods will often be give different calculated percentages of homology). or any of the other methods of calculation. For yet another example, the first sequence has 80 percent homology as defined herein to the second sequence if it is calculated that the first sequence has 80 percent homology to the second sequence using each of the calculation methods (although, in practice, different calculation methods will often be give different calculated percentages of homology). or any of the other methods of calculation. For yet another example, the first sequence has 80 percent homology as defined herein to the second sequence if it is calculated that the first sequence has 80 percent homology to the second sequence using each of the calculation methods (although, in practice, different calculation methods will often be give different calculated percentages of homology).

(2) Hybridization / selective hybridization [0064] 80. Hybridization typically means a sequence-guided interaction between at least two nucleic acid molecules, such as a primer or probe, and a gene. A sequence-mediated interaction is an interaction that occurs between two nucleotides or nucleotide analogues or nucleotide derivatives in a nucleotide specific manner. For example, sequence-driven interactions are G

18 interacting with C or A interacting with T. Typically, sequence-directed interactions occur on the side of Watson-Crick or on the Hoogsteen side of the nucleotide. The hybridization of two nucleic acids is affected by a number of conditions and parameters known to those skilled in the art. For example, both salt concentrations, pH and reaction temperature affect whether two nucleic acid molecules will hybridize.

[0065] 81. Parameters of selective hybridization between two nucleic acid molecules are known to those skilled in the art. For example, in some embodiments, the selective hybridization conditions can be defined as acute hybridization conditions. For example, the stringency of hybridization is regulated by both the temperature and salt concentration of any or both of the hybridization and washing steps. For example, the hybridization conditions for achieving selective hybridization may include hybridization in a high ionic strength solution (6x SSC or 6x SSPE) at a temperature that is about 1225 ° C below Tm (melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing with a combination of temperature and salt concentration selected so that the washing temperature is about 5-20 ° C below Tm. The temperature and salt conditions are readily determined empirically in pre-experiments where the reference DNA samples immobilized on the filters hybridize to the labeled nucleic acid of interest, and then washed under conditions of various stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridization. Conditions as described above may be used to achieve stringency, or as is known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al., Methods Enzymol. 154: 367, 1987, which is incorporated herein by reference. the reference principle to a material at least associated with nucleic acid hybridization). A preferred condition for stringent hybridization for DNA: DNA hybridization may be a temperature of about 68 ° C (in aqueous solution) in 6x SSC or 6x SSPE, followed by washing at 68 ° C. The stringency of hybridization and washing, if desired, may be reduced as the desired degree of complementarity decreases, and further depending on the abundance of GC or AT in any area in which variation is sought. Similarly, the stringency of hybridization and washing, if desired, can be increased as desired homology increases, and further, depending on the abundance of GC or AT in any area where high homology is desired, as is known in the art. The stringency of hybridization and washing, if desired, may be reduced as the desired degree of complementarity decreases, and further depending on the abundance of GC or AT in any area in which variation is sought. Similarly, the stringency of hybridization and washing, if desired, can be increased as desired homology increases, and further, depending on the abundance of GC or AT in any area where high homology is desired, as is known in the art. The stringency of hybridization and washing, if desired, may be reduced as the desired degree of complementarity decreases, and further depending on the abundance of GC or AT in any area in which variation is sought. Similarly, the stringency of hybridization and washing, if desired, can be increased as desired homology increases, and further, depending on the abundance of GC or AT in any area where high homology is desired, as is known in the art.

[0066] 82. Another way to define selective hybridization is by looking at the amount (percent) of one of the nucleic acids bound to another nucleic acid. For example, in some embodiments, selective hybridization conditions exist when 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, 97, 98, 99, 100 percent restriction nucleic acid is bound to a non-limiting nucleic acid. Typically, the non-limiting starter is in excess, for example, 10 or 100 or 1000 fold. This type of assay can be performed under conditions where both the limiting and non-limiting primers are in quantities, for example, 10, 100 or 1000 times below their kd, or where only one of the nucleic acid molecules is 10, 100 or 1000 times or where one or both nucleic acid molecules are in amounts above their kD.

[0067] 83. Another way to define selective hybridization is to look at the percentage of the primer that undergoes enzymatic manipulation under conditions where hybridization is required to induce the desired enzymatic manipulation. For example, in some embodiments, the selective hybridization conditions exist when 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 percent of the primer undergoes enzymatic manipulation under conditions that trigger enzymatic manipulation, for example if enzymatic manipulation involves extension of the DNA, then selective hybridization conditions exist when 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 percent of the primer molecules. Preferred conditions also include those suggested by the manufacturer or indicated in the art as being suitable for performing enzymatic manipulation.

[0068] 84. As with homology, it is understood that there are various methods disclosed herein for determining the level of hybridization between two nucleic acid molecules.

It is understood that these methods and conditions may give different percentages of hybridization between two nucleic acid molecules, but, unless otherwise indicated, it is sufficient to satisfy the parameters of either of these methods. For example, if 80% of the hybridization would be required and if hybridization would occur within the required parameters in any of these methods, it is considered to be disclosed herein.

[0069] 85. It is understood that those skilled in the art understand that if a composition or method meets any of these criteria for determining hybridization either collectively or individually, then that composition or method is disclosed herein.

D) Composition of the produced molecules [0070] 86. It is understood that the eukaryotes disclosed herein are capable of producing a series of compounds and compositions. The compounds and compositions can be used as a signature, a way of identifying the organism. For example, one of the ways to characterize an organism is the profile of lipids that the body produces. As disclosed herein, these various lipid profiles can be used to characterize an organism as well as for purification, manipulation, and collection for a variety of reasons.

(1) Lipids [0071] 87. It is understood that each organism can produce certain profiles of unsaturated fatty acids as disclosed herein. These profiles are the characteristics of organisms. The following are some examples of unsaturated and other lipid profiles for organisms.

[0072] 88. The eukaryotic microorganism can produce, for example, a lipid or fatty acid fraction of at least about 4 wt%. up to 6 wt% (e.g., about 5 wt.%), which contains from about 0 wt.%. up to about 2 wt.% myristic acid (e.g., about 1 wt%), from about 16 wt% up to about 20 wt.% (e.g., about 18 wt.%) of palmitic acid, from about 0 wt.%. up to about 2 wt.% (e.g., about 1 wt.%) of palmitoleic acid, from about 4 wt.%. up to about 8 wt.% (e.g., about 6 wt.%) of stearic acid, from about 30 wt.%. up to about 34 wt% (e.g., about 32 wt%) oleic acid, from about 40 wt% up to about 44% by weight (e.g., about 42 wt.%) of linoleic acid, and from about 0 wt.%. up to about 3 wt.% (e.g., about 2 wt.%) of n-3 EPA on dry cellular biomass.

[0073] 89. A eukaryotic microorganism may also, for example, produce a lipid or fatty acid fraction of at least about 1 wt.%. up to 3% by weight (e.g., about 1.25 wt.%), which contains from about 2 wt.%. up to about 4 wt.% (e.g., about 3 wt%) myristic acid, from about 50 wt% up to about 60 wt.% (e.g., about 55 wt.%) of palmitic acid, from about 2 wt.%. up to about 4 wt.% (e.g., about 3 wt.%) of palmitoleic acid, from about 16 wt.%. up to about 20 wt.% (e.g., about 18 wt.%) stearic acid, from about 9 wt.%. up to about 13 wt.% (e.g., about 11 wt.%) of oleic acid, from about 1 wt.%. up to about 3 wt.% (e.g., about 2 wt.%) of eicosadienoic acid, and from about

6 wt% up to about 10 wt.% (e.g., about 8 wt.%) of n-3 EPA on dry cellular biomass.

[0074] 90. A eukaryotic microorganism, e.g., such as ONC-T18, can produce at least about 30 wt.%, 40 wt.%, 50 wt.%, 60 wt.%, 70 wt.% Wt. or 80% by weight (Eg.,

- about 80% by weight) of the lipid composition to dry cellular biomass. For example, a eukaryotic microorganism can produce a lipid composition comprising from about 25% to about 40% of an omega-3 fatty acid, such as DHA n-3, (e.g., at least 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55% or 60% by weight), and from about 0% to about

3% omega-3 fatty acid, EPA, (e.g., at least 1% or 2% by weight) and from about 4% to about 12% omega-6 fatty acid, such as DPA n-6, (e.g. at least 4%, 5%, 6%, 7%, 8%, 9%, or 10% by weight).

[0075] 91. It is understood that the composition of the lipids produced by the eukaryotic microorganism can be manipulated based on the culture conditions in which the eukaryotic microorganism is located. By varying the various parameters as disclosed herein, compositions can be manipulated to produce, for example, better DHA or DPA yields. For example, manipulation can not actually produce more grams, but manipulation can give a better ratio of DHA or DPA to EPA and other desired PUFAs, which may be desirable from the viewpoint of purification. Changing conditions are discussed herein.

[0076] 92. Figure 10 shows the possible metabolic pathway for the various PUFAs produced by the disclosed eukaryotic microorganism following the tracing of metabolites of fatty acid methyl esters. In the biological transformation pathways, as disclosed herein, proteins such as a polyketide synthase can be identified, using, for example, a degenerate primer test (Metz et al (2001) Science 293: 290-3 and

Kaulmann & Hertweck (2002) Angew. Chem. Int. Ed. 41: 1866-9). Elongases and desaturases can also be identified using, for example, a hybridization probe. Fatty acid synthases can also be identified by, for example, testing with a hybridization probe or degenerate primer.

4. Growth and breeding [0077] 93. A phenotypic study was carried out on microplates, including carbon; nitrogen (peptide nitrogen); phosphorus and sulfur; osmolyte, and pH.

[0078] 94. The Taguchi method with an orthogonal experimental design was used to determine optimal media configurations and changes in nitrogen, carbon and salt concentrations (Joseph J & Piganatiells

JR (1998) IIE Trans, 20: 247-254).

[0079] 95. If you increase mixing or dO2, you increase the production of biomass and TFA, but you reduce DHA. If you reduce the mixing or dO2, you reduce cellular biomass (g) and reduce TFA, but you increase DHA, but also reduce C16: 0, C16: 1 & C18: 1.

[0080] 96. If you increase the temperature, you increase the production of biomass and TFA, but you reduce DHA. If you reduce the temperature, you reduce cellular biomass (g) and reduce TFA, but increase DHA, but reduce C16: 0, C16: 1 & C18: 1.

[0081] 97. Cell biomass derived from the disclosed eukaryotic microorganism can be obtained by inoculating a suitable medium with natural or artificial sea water containing from about 2% to about 100% seawater. This eukaryotic microorganism has the ability to utilize various nutrients in this medium. Examples of the carbon source used in the medium are carbohydrates such as glucose, fructose, dextrose, lactulose, galactose, maltotriose, maltose, lactose, glycogen, gelatin, starch (corn or wheat), as well as sugar derivatives such as acetate, m-inositol (originating from maize corn extract), galacturonic acid (derived from pectin), L-fucose (derived from galactose), gentenobiosis, glucosamine, alpha-D-glucose-1-phosphate (derived from glucose), cellobiose (derived from cellulose), dextrin (derived from maize) and α-cyclodextrin (derived from starch) and polyols such as maltitol, erythritol, adonitol and oleic acid, such as glycerol and Tween 80, and aminosugars, such as N-acetyl-Digalactosamine, N-acetyl D-glucosamine and N-acetyl-eD-mannosamine. In contrast, examples of the nitrogen source are natural sources of nitrogen, such as peptone, yeast extract, malt extract and fish meal, or organic nitrogen sources, such as monosodium glutamate, but are not limited thereto. In addition, if necessary, phosphate, such as potassium phosphate, and sodium phosphate, inorganic salts such as ammonium sulfate, sodium bicarbonate, sodium orthovanadate, potassium chromate, sodium molybdate, selenum acid, nickel sulphate, copper sulfate, can be used as trace nutrients. , zinc sulphate, cobalt chloride, iron chloride, manganese chloride and calcium chloride, together with a chelating agent, ethylenediaminetetraacetic acid, alone or in combination with vitamins such as pyridoxine hydrochloride, thiamine hydrochloride, calcium pantothenate, p-aminobenzoic acid, riboflavin, nicotinic acid, biotin, folic acid and vitamin B12. After the preparation of the medium, the pH is adjusted to between 3.0 and 10.0 using an acid or base to adjust, where appropriate, e.g., between pH 4.0 and 6.5, and the medium is sterilized, for example, in an autoclave. The culture can be carried out for 1 to 30 days, 1 to 21 days, 1 to 15 days, nicotinic acid, biotin, folic acid and vitamin B12. After the preparation of the medium, the pH is adjusted to between 3.0 and 10.0 using an acid or base to adjust, where appropriate, e.g., between pH 4.0 and 6.5, and the medium is sterilized, for example, in an autoclave. The culture can be carried out for 1 to 30 days, 1 to 21 days, 1 to 15 days, nicotinic acid, biotin, folic acid and vitamin B12. After the preparation of the medium, the pH is adjusted to between 3.0 and 10.0 using an acid or base to adjust, where appropriate, e.g., between pH 4.0 and 6.5, and the medium is sterilized, for example, in an autoclave. The culture can be carried out for 1 to 30 days, 1 to 21 days, 1 to 15 days,

1 to 12 days, 1 to 9 days, or preferably 3 to 5 days, at temperatures between 4 to 30 ° C, preferably 18 to 28 ° C, by culture with aeration and shaking, culture with shaking, stationary culture, batch culture, cultivation continuous, rearing with batch change, or wave cultivation, or the like.

[0082] 98. The following conditions are examples of conditions that allow the formation of a lipid set with efficiencies that allow them to be used as a commodity. Examination of culture conditions for ONC-T18 revealed that the eukaryotic microorganism disclosed herein grows well in natural or artificial seawater or in a medium containing not less than 5% concentration of natural or artificial seawater. The carbon and nitrogen sources added to the medium may be those conventionally used as described above. One thing is whether the nitrogen source is natural or organic, while in the nutrient solution a constant total nitrogen concentration is maintained. These sources are added to the medium at normal concentrations. If these conditions are met, a small effect on the lipid content, shares or amount of accumulated DHA, DPA and EPA is obtained,

[0083] 99. Several methods of increasing both cellular biomass and lipid production rates can be used for the ONC-T18 high-concentration fermentation. These include increasing the concentration of both carbon and nitrogen in the medium (in a ratio between 6: 1 and 15: 1, preferably between 6: 1 and 13: 1 and temperatures between 4 to 30 ° C, preferably 18 to 28 ° C) respectively range 5 g L^-1 up to 60 g L^-1 to the range of 100 g L^-1 and 160 g L^-1 and from the range of 4 g

L^-1 up to 10 g L^-1 to the range of 40 g L^-1 up to 60 g L^-1. By using this method, the share of biomass and produced lipids in comparable proportions is also increased. In addition, lipid production can be increased by using carbon sources increased from the 5 g L range^-1 to g L^-1 to the range of 100 g L^-1 up to 160 g L^-1while the nitrogen source remains constant. In addition, the production of biomass can be increased, while maintaining the lipid content, by using increased amounts of nitrogen sources from the 10 g L range.^-1 up to 60 g L^-1while the carbon source remains constant. In addition, experiments have shown that the production of biomass and lipids increases enormously with increased mixing from the range of 100 and 1000 rpm, preferably between 350 and 600 rpm and optimally between 350 and 450 rpm, with only a marginal decrease in lipid content and without dropping in fatty acid profiles, mixing being particularly suitable for the early stages of heterotrophic fermentation. Experiments have also shown that optimal lipid production is achieved when the dissolved oxygen content in the culture medium is between 1 and 10%, optimally at 5%. Finally, the addition of acetate, trace elements, metals and vitamins to the production medium (as mentioned above) increases the production of DHA, EPA and DPA with respect to other fatty acids,

100. By heterotrophic fermentation as described above, a cellular biomass that produces a lipid containing a series of (n-3) DHA in culture can be cohesive.

- 24 with a high concentration of not less than 5 g, and more preferably not less than 20 g / L medium. In addition, experiments have shown that most of these lipids accumulate during later exponential / transitional culture steps after reaching maximum biomass levels. During the fermentation process, the lipid content typically does not fall below 25% biomass together, typically reaching a maximum of about 80%. The culture under the above conditions can be carried out using a conventional mixer-fermenter. It is also possible to use a fermentor with a bubble column (batch or continuous cultures) or a wave fermenter.

[0085] 101. Collection of cellular biomass prior to processing to isolate lipids can be performed using a variety of conventional methods such as centrifugation (such as solid-waste centrifuges) or filtration (such as cross-flow filtration), and can also include use a precipitating agent for the accelerated collection of cellular biomass (such as sodium phosphate, calcium chloride or polyacrylamide).

5. Lipid secretion [0086] 102. Figure 7 shows lipid and DHA profiles as a function of various parameters. All of these data can be used to obtain specific characteristics for the ONC-T18 eukaryotic microorganism. Figure 13 shows the general profile of fatty acids for the disclosed eukaryote.

[0087] 103. A fat containing a series of (n-3) DHA and a series (n-6) DPA can be obtained by breaking or tearing harvested cellular biomass, for example, by milling, ultrasound treatment, followed by extraction with a solvent such as chloroform. , hexane, methanol, ethanol or supercritical fluid extraction means. The content of fat obtained containing a series of (n-3) DHA and a series (n-6) of DPA per gram of dry cellular biomass is preferably more than 0.25 g, and more preferably more than 0.6 g.

[0088] 104. The ability to produce the thus obtained lipids has disclosed eukaryotic microorganisms, such as ONC-T18, any of its variants and any assemblies of members of the same species of eukaryotic microorganisms, so that the lipid profile is as follows. The percentage of neutral lipids can be at least 95% by weight of the total lipids. The fatty acid composition typical of a eukaryotic microorganism, such as ONC-T18, in neutral lipids is as follows: 15% myristic acid, 8% pentadecanoic acid, 35% palmitic acid, 7% palmitolic acid, 1% stearic acid, 2% oleic acid .

- 1% eicosapentaenoic acid, 6% docosapentaenoic acid and 25% docosahexaenoic acid (gas chromatograms shown in Figure 1).

[0089] 105. The ability to produce the thus obtained lipids has disclosed eukaryotic microorganisms, such as ONC-T18, any of its variants and any assemblies of members of the same species of eukaryotic microorganisms, so that the lipid profile is as follows. The percentage of mono-, di- and tri-glycerides in the neutral lipid fraction with ONC-T18 is 0% to about 2%, 0 to about 2% and 96 to about 100%, respectively. In contrast, the polar lipid fraction, which is between 5% and about 10% of the lipid fraction, includes phosphatidylcholine, phosphatidylserine and phosphatidic acid, both bound and unrelated to neutral lipids.

[0090] 106. It is understood that these lipids can be found in the body in any combination or permutation. It is also understood that the concentrations of these lipids can be manipulated by altering growth conditions and media conditions as discussed herein.

(1) Lipid as concentration [0091] 107. The eukaryotic microorganism can produce a lipid fraction containing n-3

DHA, EPA and n-6 ​​DPA in amounts greater than or equal to about 4.0 g L^-1 medium. The eukaryotic microorganism can produce a lipid composition containing n-3 DHA,

EPA and n-6 ​​DPA in amounts greater than or equal to about 20.0 g L^-1 medium. The eukaryotic microorganism can produce a lipid composition containing n-3 DHA,

EPA and n-6 ​​DPA in amounts greater than or equal to about 14.0 gL^-1 medium. The eukaryotic microorganism can produce from about 1.5 g L^-1 to about 5.0 g L^-1 (e.g., about 4.6 g L^-1) n-3 DHA, from about 0.5 g L^-1 up to about 1.5 g L^-1 (e.g., about 0.22 g L^-1) n-3

EPA, and from about 0.5 g L^-1 up to about 1.5 g L^-1 n-6 DPA. In addition, the eukaryotic microorganism can produce a lipid fraction containing myristic, myristyl, pentadecanoic, palmitic, palmitolic, stearic oleic, linoleic, eicosadienoic, arachidonic, eicosapentaenoic, docosahexanoic and docosapentaenoic acids between 301.2 and 360.3 mg g.^-1 or up to 790 mg g^-1 cellular biomass. The eukaryotic microorganism can also produce a fraction containing between 44.3 and 57 mg g^-1 myristic acid (corresponding to 1134.5 to 1448.1 mg L)^-1), 0.5 to 0.65 myristic acid (corresponding to 13.3 to 16.63 mg L)^-1), 33.5 to 34.6 mg g^-1 pentadecanoic acid (corresponding to 856.9 to 885.1 mg L)^-1), 121.9 and 165.1 mg g^-1 palmitic acid (corresponding to 3118.2 to 4223.3 mg L)^-1)

- 26 7.9 to 28.5 mg g^-1 palmitolic acid (corresponding to 202.1 to 729 mg L)^-1), 4.38 to 5.9 mg g^-1 stearic acid (corresponding to 112 to 151 mg L)^-1), 6.94 to 9.9 mg g^-1 oleic acid (corresponding to 177.5 to 253.2 mg L)^-1), 0.4 to 1.3 mg g^-1 linoleic acid (corresponding to 11.26 to 33.3 mg L)^-1), 0.5 to 1.0 mg g^-1 eicosadienoic acid (corresponding to

12.8 to 25.6 mg L^-1), 0.4 to 0.5 mg g^-1 arachidonic acid (corresponding to 10.2 to 13 mg L)^-1), 75 to 100 mg g^-1 docosahexaenoic acid (corresponding to 1918 to 2560 mg L)^-1), 1.9 to 6 mg g^-1 eicosapentaenoic acid (corresponding to 48.6 to 153.5 mg L)^-1) and 17.1 to 33.7 mg g^-1 docosapentaenoic acid (corresponding to 437.4 to 866.1 mg L)^-1), having a total fatty acid content in the biomass of between 301 to 790 mg g (corresponding to 7,700 to 20209 mg L^-1).

(2) Other molecules [0092] 108. The eukaryotic microorganism can further produce carotenoids and xanthophylls. Examples of such carotenoids and xanthophylls include beta-carotene, lycopene, astaxanthin, canthaxanthin, phycikoxanthin, zeaxanthin, echinenone, beta-cryptoxanthin, capsanthin, lutein, annatto, beta-apo-8-carotenal and beta-apo-8-carotenal ester.

[0093] 109. The xanthophylls produced by the disclosed eukaryotic microorganisms can be conjugated to a variety of PUFAs also produced by the disclosed eukaryotic microorganisms.

(a) Antioxidants [0094] 110. In general, antioxidants are compounds that react with oxygen and are typically consumed by oxygen. Since antioxidants typically react with oxygen, antioxidants also typically react with free radicals and free radicals. (Publication "The Antioxidants - The Nutrients that Guard Your Body", author Richard A. Passwater, Ph.D., 1985, Keats Publishing Inc., which is incorporated herein by reference at least as regards the material referring to antioxidants) . The compositions may contain any antioxidants, and a non-limiting list could include, but are not limited to, non-flavonoid antioxidants and nutrients that can directly sweep free radicals, including multi-carotenes, beta-carotenes, alpha-carotenes,

- B3, nicotinic acid and nicotinamide), vitamin A, 13-cis-retinoic acid, N-acetyl-L-cysteine ​​(NAC), sodium ascorbate, pyrrolidine dithiocarbamate, and coenzyme Q10; enzymes that catalyze the destruction of free radicals, including peroxidases, such as glutationoperoxidase (GSHPX), which acts on H2O2 and such as organic peroxides, including catalase (CAT), which acts on H2O2, superoxide dismutase (SOD), which disproportionates O2H2O2, glutathione reductase (GSHTx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD), and their mimetics, analogs and polymers (analogs and polymers of antioxidant enzymes such as SOD, are described in, for example, US Pat. No. 5,171,680, which is incorporated herein by reference at least in the scope of material related to antioxidants and antioxidant enzymes); glutathione; ceruloplasmin; cysteine, and cysteamine (beta-mercaptoethylamine) and flavonoids and flavonoid-like molecules such as folic acid and folate. A review of the enzymes of antioxidants and their mimetics and of antioxidant nutrients can be found in: Kumar et al., Pharmac. Ther. 39: 301, 1988 and Machlin LJ and Bendich, FASEB Journal 1: 441-445, 1987, which are incorporated herein by reference in the field of material relating to antioxidants.

[0095] 111. F Lawoniids, also known as "phenylchromons", are naturally occurring, water-soluble compounds that have the characteristics of antioxidants. Flavonoids are widely distributed in vascular plants and are found in many vegetables, fruits and beverages such as tea and wine (especially red wine). Flavonoids are conjugated aromatic compounds. The most widely occurring flavonoids are flavones and flavonols (for example, myricetin (3,5,7,3 ', 4', 5'-hexahydroxyflavone), quercetin (3,5,7,3 ', 4'-pentahydroxyflavone), kemferol ( 3,5,7,4'-tetrahydroxyflavone), and apigenin flavonoids (5,7,4'-trihydroxyflavone) and luteolin (5,7,3 ', 4'-tetrahydroxyflavone) and their glycosides and quercetin).

112. Carotenoids are important natural pigments produced by many microorganisms and plants, usually red, orange or yellow. Traditionally, carotenoids have been used in the food, food and nutraceutical industries. They are known to be essential for plant growth and photosynthesis, and in the human diet they are the main source of vitamin A. Antioxidants in the diet, such as carotenoids (beta-carotene, lycopene, astaxanthin, canthaxanthin, zeaxanthin, capsanthin, lutein, annatto, beta-apo -8-carotenal and beta-apo-8-carotenal ester), show significant anti-cancer activities and play an important role in the prevention of chronic diseases. Carotenoids are strong antioxidants

- biologicals that the singlet oxygen excitation energy can absorb on the carotenoid chain, leading to the decomposition of the carotenoid molecule, but preventing damage to other molecules or tissues.

[0097] 113. Oxygen is needed for metabolic functions, but also a problem for cells. The human body has a wide range of metabolic enzymes and antioxidants to remove molecules from oxygen from cells. It is assumed that this oxidative stress is a causative factor in conditions such as rheumatoid arthritis, ischemic heart disease and stroke, Alzheimer's dementia, cancer and aging.

Thus, antioxidants have the potential to protect against a wide range of diseases. Several antioxidant compounds have been isolated from the sources of marine microorganisms; they include astaxanthin, beta-carotene and other carotenoids.

[0098] 114. Carotenoids are a widespread group of pigments found in nature, including more than 700 natural lipid-soluble pigments, predominantly produced by species of microalgae, macroalgae, bacteria and fungi, with astaxanthin and its derivatives being of particular economic importance. Astaxanthin is an extremely effective protective antioxidant. Moreover, unlike beta-carotene, astaxanthin easily overcomes the blood-brain / retina barrier, and therefore also has the potential to protect against diseases of the brain and eyes. Preclinical studies suggest various beneficial effects of astaxanthin consumption, such as: (i) inhibiting the formation of cancer and growth in the bladder, colon, liver, breast and oral cavity; (ii) protection of the retina from oxidative damage, therefore, action against macular degeneration associated with age; (iii) promoting increased activity of the immune system, (iv) providing protection against damage caused by ultraviolet light, as well as (v) providing increased muscle strength.

b) Isolation of microorganisms [0099] 115. The microorganisms of the Thraustochytriaceae family are obtained by a method involving the application of a bait from pollen grains to a vegetative sample in salt water (natural marine or artificial) and incubation; separation and transfer of the seeds to heterotrophic medium and incubation; identification of the isolate that produces fatty acids, isolation from the identified isolate of the microorganism from the Thraustochytriaceae family. Additional forms of isolation include media with the addition of appropriate antibiotics and identification by either microscopic means as mentioned above or by the use of

- 29 primers or probes of the 18S rRNA gene. The heterotrophic medium may be as described below.

6. Lipids and other molecules produced by the eukaryotic microorganism [0100] 1. Lipid compositions comprising from about 25 wt.% Are disclosed. up to about 40% 5 wt. DHA n-3, from about 6 wt.% up to about 10 wt.% DPA n-6, and from about 0 wt% to around

3 wt% EPA n-3.

[0101] 117. Lipid compositions may further contain from about 11 wt.%. up to about 15 wt.% (e.g., about 13 wt.%) myristic acid, from about 7 wt.%. up to about 11 wt.% (e.g., about 9 wt.%) of pentadecanoic acid, from about 37 wt.%. up to about 41 wt.% (e.g., about 39 wt.%) of palmitic acid, from about 3 wt.%. up to about 7 wt% (e.g., about 5 wt.%) of palmitoleic acid, from about 0 to about 3 wt.%. (e.g., about 1 wt.%) stearic acid, or from about 1 wt.%. up to about 4 wt.% (e.g., about 2 wt.%) of oleic acid.

[0102] 118. The lipid composition may contain DHA n-3 in concentrations of over 400 mg of biomass, DPA n-6 in concentrations of over 100 mg of biomass.

[0103] 119. The lipid compositions may further comprise carotenoids. Examples of such carotenoids include beta-carotene, lycopene, astaxanthin, zeaxanthin, canthaxanthin, echinenone, phycikoxanthin, capsanthin, lutein, annatto, beta-apo-8-carotenal, and beta-apo-8-carotenal ester.

[0104] 120. In one aspect, the composition may contain at least about 24 wt.

DHA n-3, about 1 wt.% DPA, about 6 wt.% DPA n-6, and about 1 wt.% EPA n-3.

7. Compositions comprising molecules produced by a eukaryotic microorganism [0105] 121. A foodstuff, supplement, pharmaceutical composition for both humans and animals (including marine) can contain a composition (lipids, lipids with antioxidants and antioxidants alone).

[0106] 122. An infant formula containing the composition (lipids, lipids with antioxidants and anti-oxidants themselves) is also disclosed.

C. Ways

1. Methods of Producing Lipids [0107] 123. Methods of making a lipid composition are disclosed, the method comprising:

culturing a eukaryotic microorganism comprising one or more microorganisms of the Thraustochytriaceae family, and secreting lipid compositions.

[0108] 124. A variety of procedures may be employed to recover the resulting cell biomass from fermentation in a variety of culture media, such as by filtration or centrifugation. The cells can then be washed, frozen, lyophilized, or spray dried, and stored in a non-oxidizing atmosphere to eliminate the presence of oxygen before being incorporated into processed foods or a food product.

[0109] 125. Cell-containing lipids containing PUFA (n-3) DHA, EPA and (n-6) DPA can also be extracted from cell biomass by methods such as extraction with supercritical fluid, or by extraction with solvents such as chloroform, hexane, methylene chloride, or methanol, and the obtained extract was evaporated under reduced pressure to obtain a sample of concentrated lipid material. The omega-3 and omega-6 PUFAs can be further concentrated by hydrolysing the lipids and concentrating the highly unsaturated fraction using traditional methods such as urea or fractional distillation, column chromatography, or supercritical fluid fractionation. Cells can also be torn or lysed, and lipids extracted into vegetable or animal oils (e.g. fish oil).

The extracted oils can be refined by known methods routinely used for refining vegetable oils (e.g., by chemical or physical refining). These refining methods remove impurities from extracted oils before they are used or sold as edible oils. After refining, the oils can be used directly as a feed or food additive for the production of omega-3 and / or omega-6 enriched products. Alternatively, the oil can be further processed and purified as outlined below, and then used in the above uses as well as in pharmaceutical applications.

[0110] 126. In another method of producing enriched (concentrated) omega-3 or omega-6 oils, harvested cellular biomass (fresh or dried) can be disrupted or transmissive to cell walls by known techniques such as ultrasound treatment, shear disruption in liquid , ball milling, high pressure crushing, freezing and thawing, or enzymatic digestion of the cell wall. The lipids from the disrupted cells are extracted by the use of a solvent or a mixture of solvents such as hexane, chloroform, ether, or methanol.

The solvent is removed and the lipids are hydrolyzed by using any known methods for converting the triglycerides into free fatty acids or fatty acid esters including basic, acidic or enzymatic hydrolysis. After hydrolysis, the non-saponifiable compounds are extracted into a solvent such as ether,

31 hexane or chloroform, and removed. The remaining solution is then acidified by the addition of acid, and the free fatty acid is extracted into a solvent such as hexane, ether or chloroform. The solvent solution containing free fatty acids can then be cooled to a temperature sufficiently low for the crystallization of compounds other than PUFA, which can then be removed by filtration, centrifugation or standing. This results in the concentration of other PUFAs used as food supplements for humans, as a food additive, or in pharmaceutical applications.

[0111] 127. Also disclosed is a lipid composition made by the method disclosed above.

[0112] 128. Microorganisms of the Thraustochytriaceae family may be any of the microorganisms disclosed above.

a) Medium [0113] 129. The heterotrophic medium may contain a sea salt (artificial or natural), one or more carbon sources, and one or more nitrogen sources. The sea salt may be present in an amount of from about 2.0 to about 40.0 g L^-1. The concentration of the carbon and nitrogen source used in normal farming conditions (not for high-concentration but rather economic fermentation) is in the range of 5 g L respectively^-1 up to 0 g L^-1 and 4 g L^-1 up to 10 g L^-1. For high-concentration fermentation, the concentration of the carbon and nitrogen source in normal culture conditions is in the range of 100 g L, respectively^-1 and 160 g L^-1 and 40 g L^-1 up to 60 g

L^-1. The tendency is that in order to accumulate the oil the eukaryotic microorganism is grown in a culture medium (as described above) in which the nitrogen supply after 24 to 48 hours is limited, and the carbon supply remains in abundance. This eukaryotic microorganism still assimilates the carbon (in the form of simple sugars), but can no longer undergo cell division due to the lack of nitrogen for the production of appropriate proteins and nucleic acids. As a result, these sugars are converted into reserve oils, quite as described by C. Ratledge (Lipid Tech. 16: 34-39, 2004), while Figure 9 presents this phenomenon specific to this organism.

[0114] 130. The nitrogen source may be one or more of peptone, yeast extract, malt extract, and monosodium glutamate. The source of nitrogen may also be maize extract or cottonseed extract. The nitrogen source may include yeast extract and / or peptone or monosodium glutamate. For example, the nitrogen source may include, but is not limited to, EMD ™ YE-MSG, EMD ™ YE, EMD ™ Peptone-MSG, Sigma ™ YEMSG, Sigma ™ YE, Fermtech ™ YE-MSG, Fermtech ™ YE, or meal fish (62% protein).

Yeast extract (YE) may be present in an amount of about 2 g L^-1. The monosodium glutamate may be present in an amount of about 8 gL^-1.

In one aspect, the carbon source may be D, L-malic acid, D-fructose, D-xylose, fumaric acid, D-cellobiose, 5-keto-D-gluconic acid, pyruvic acid, alpha-D-lactose, corn dextrin, gelatin. , corn starch or wheat starch. The carbon source may be present in an amount of about 1 gL^-1 to around g L^-1 and up to about 200 g L^-1.

[0116] 132. In one example, the medium may contain about 5 g of D-glucose, about 2 g of peptone, and about 2 g of yeast extract per liter of salt water (natural or artificial). In another medium, it may contain about 60 g of D-glucose, about 10 g of yeast extract per liter of salt water (natural or artificial). In another, the medium may contain about 8 g of yeast extract, 32 g of MSG, 24 g of sea salt (natural and artificial) and 300 g of D-glucose per liter.

[0117] 133. The medium may further contain phosphates (e.g., potassium phosphate and sodium phosphates). The medium may further contain inorganic salts (e.g., ammonium sulfate, sodium bicarbonate, sodium orthovanadate, potassium chromate, sodium molybdate, selenum acid, nickel sulfate, copper sulfate, zinc sulphate, cobalt chloride, iron chloride, manganese chloride). The medium may further contain a chelating compound (e.g., EDTA). The medium may further contain vitamins (e.g., pyridoxine hydrochloride, thiamine hydrochloride, calcium pantothenate, paminobenzoic acid, riboflavin, nicotinic acid, biotin, folate, and vitamin B12). The medium may have a pH of from about 4.0 to about 6.5.

[0118] 134. Incubation may take place from about 1 to about 9 days (e.g., from about 3 to about 5 days). Incubation may take place at a temperature of about 18 to about 30 ° C (e.g., from about 1825 ° C). Incubation may further include shaking or aerating.

[0119] 135. Lipid isolation may involve contacting microorganisms with an extraction solvent. The solvent may include one or more solvents selected from chloroform, hexane, methanol, or ethanol, or supercritical CO 2.

[0120] 136. The method may produce any of the compositions as disclosed above.

[0121] 137. The eukaryotic microorganism can produce a lipid composition containing n3 DHA in amounts greater than or equal to about 20 gL^-1 medium. The eukaryotic microorganism can produce a lipid composition containing n-3 DHA in amounts greater than or equal to about 40 gL^-1 medium. The eukaryotic microorganism can produce a lipid composition containing n-3 DHA in amounts greater than or equal to about 80 gL^-1 medium.

2. Methods of screening and identification [0122] 138. The eukaryotic microorganism as disclosed herein can produce a lipid comprising a series of (n-3) docosahexaenoic acid and eicosapentaenoic acid and a series (n156) of DPA. These eukaryotic microorganisms can be selected, for example, using the following screening method. The vegetative samples may be placed (and placed) in 20 mL vials containing 10 mL of sterile filtered 0.2 μm natural seawater containing penicillin and streptomycin at 300 and 500 mg L, respectively.^-1. Subsequently, the bait was placed from sterile pollen grains and incubated for 48 hours at 18 to 25 ° C. The pollen grains were then transferred to agar plates containing antibiotics (as above) and incubated under the same conditions. Single, irregular, glassy colonies composed of spherical or cochlear cells and atypical for yeast or bacterial colonies were harvested and then grown on the same medium and in the same conditions as above. These isolates were then screened for growth and fatty acids using a liquid nutrient medium prepared using a 0.2 μm filtered natural seawater containing 5 g L.^-1 glucose, 2 g L^-1 peptone and 2 g L^-1 yeast extract, the resulting cellular biomass was collected by centrifugation or sedimentation in a liquid medium. The fatty acids were transesterified directly using conventional methods, whereby the composition of fatty acid methyl esters was analyzed by gas chromatography, selecting for further work the strains which produced the appropriate amounts of the n-3 series of DHA and the n-6 DPA series.

[0123] 139. Methods for identifying a eukaryotic microorganism are disclosed, including: treating a vegetative sample in salt water (natural marine or artificial) with pollen grains and incubating; transferring the grains to heterotrophic medium and incubating; and identifying the isolates that produce fatty acids.

[0124] 140. Lipid compositions produced by eukaryotic microorganisms identified above are also disclosed.

[0125] 141. Lipid compositions produced by methods using the disclosed eukaryotic microorganisms and methods disclosed herein, are also disclosed.

[0126] 142. Also disclosed are eukaryotic microorganisms (ONC-T18) having accession number PTA-6245 in the American Type Culture Collection.

[0127] 143. Also disclosed are eukaryotic microorganisms belonging to the order

Thraustochytriales (ONC-T18) having 18S rRNA, such as SEQ ID NO: 1. ID NO. 1, and identified as Thraustochytrium sp.

[0128] 144. Also disclosed is a eukaryotic microorganism, Thraustochytrium sp. Capable of producing DHA and DPA at concentrations over 400 mg L^-1 and 100 mg L^-1, respectively.

[0129] 145. Also disclosed is a eukaryotic microorganism, Thraustochytrium sp. Capable of producing carotenoids by heterotrophic fermentation as mentioned above in the range of 50 to 1250 mg kg.^-1 and astaxanthin, zeaxanthin, canthaxanthin, echinenin and beta-carotene in the range of 1 to 20 mg kg respectively^-10.25 to 10 mg kg^-1, 1 to 20 mg kg^-1 up to 20 mg kg^-1 and

1 to 200 mg kg^-1.

[0130] 146. Also disclosed are methods for culturing a eukaryotic microorganism comprising culturing a eukaryotic microorganism under conditions wherein the conditions include a medium containing sodium chloride in the form of artificial sea salt (tropic marine) between 2.0 and 15.0 g L^-1; source of nitrogen in the form of yeast extract and monosodium glutamate in the amount of 2.0 and 8.0 g L^-1 respectively; and carbon in the form of glucose up to 130 g L^-1.

[0131] 147. The disclosed methods may, for example, cultivate ONC-T18, whereby at least 24% by weight is DHA, at least 6% by weight is DPA and at least 1% is EPA with respect to fatty acids together.

[0132] 148. The culture methods disclosed can also, for example, cultivate ONC-T18 such that at least 1% by weight is carotenoid material, 1 to 2% and at least 1.2% of which is astaxanthin, from 0 , 25 and 1% and at least 0.45% is zeaxanthin, from 5 to 16% and co

- at least 9% is canthaxanthin, from 1 to 2% and at least 1.2% of this is echinenone, and from 12 to 16% and at least 14% by weight is beta-carotene.

3. Nucleic Acids [0133] 149. There are various molecules disclosed herein that are based on nucleic acids, including, for example, nucleic acids that encode, for example, rRNA, as well as any of the other proteins disclosed herein, as well as various functional nucleic acids. The disclosed nucleic acids consist, for example, of nucleotides, nucleotide analogs, or nucleotide substitutes. Here, non-limiting examples of these and other molecules are discussed. It is understood that, for example, when a vector is expressed in a cell, the expressed mRNA will typically consist of A, C, G, and U / T. Similarly, it is understood that if, for example, the antisense molecule is introduced into a cell or cell environment by, for example, exogenous delivery,

a) Nucleotides and related molecules [0134] 150. A nucleotide is a molecule that contains a basic moiety, a sugar moiety, and a phosphate moiety. Nucleotides may be joined together by their phosphate moieties and sugar moieties to form an internucleotide linkage. The basic nucleotide moiety may be adenine-9-yl (A), cytosin-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), and thymine-1-yl (T). ). The sugar moiety of the nucleotide is ribose or deoxyribose. The phosphate moiety of the nucleotide is pentavalent phosphate. A non-limiting example of a nucleotide would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).

[0135] 151. A nucleotide analogue is a nucleotide that includes some type of modification of either basic or sugar or phosphate moieties. Nucleotide modifications are known in the art and may include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, and 2-amino adenine, as well as modifications on sugar or phosphate moieties.

[0136] 152. Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in the interaction of Watson-Crick or Hoogsteen, but which are

36 taken together by a moiety other than the phosphate moiety. Nucleotide substitutes are able to accept the conformation of a double helix structure when interacting with an appropriate target nucleic acid.

[0137] 153. Other types of molecules (conjugates) can also be linked to nucleotides or analogs of nucleotides to increase, for example, cellular uptake. The conjugates may be chemically linked to nucleotides or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 86: 6553-6556, 1989).

[0138] 154. Watson-Crick interaction means at least one interaction with the party

Watson-Crick of a nucleotide, a nucleotide analogue, or a nucleotide substitute. The Watson site of a nucleotide, a nucleotide analogue or a nucleotide substitute includes C2, N1, and C6 positions of a nucleotide, a purine nucleotide or nucleotide analogue substitute and positions C2, N3, C4 of a nucleotide, a nucleotide analogue, or a pyrimidine-based nucleotide substitute.

[0139] 155. Hoogsteen interaction means an interaction that occurs on the Hoogsteen side of a nucleotide or nucleotide analog that is exposed in the groove of a large DNA duplex. The Hoogsteen site includes the N7 position and the reactive groups (NH2 or O) at the C6 position of the purine nucleotides.

b) Sequences [0140] 156. There are various sequences associated with, for example, SEQ. And ID No.1: as well as any other nucleic acids and proteins disclosed herein that may be disclosed in Genbank, and these sequences and others are hereby incorporated by reference in their entirety as well as into the separate sequences therein.

[0141] 157. A variety of sequences are provided herein, and these and others can be found in

Genbank, on the site <a href="http://www.pubmed.gov">www.pubmed.gov</a>. Those skilled in the art understand how to resolve discrepancies and sequence differences and match compositions and methods relating to a particular sequence to other related sequences. The primers and / or probes can be designed for any sequence, having the information disclosed herein and known in the art.

c) Primers and Probes [0142] 158. Compositions comprising primers and probes that are capable of interacting with the genes disclosed herein are disclosed. In certain embodiments, the primers

37 is used to enable DNA amplification reactions. Typically the primers will be able to be extended in a sequence-specific manner. Prolonger extension in a sequence specific manner includes any method in which the sequence and / or composition of the nucleic acid molecule to which the primer hybridizes or otherwise binds, directs or affects the composition or sequence of the product produced by the primer extension. Primer extension in a sequence-specific manner thus includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred. In some aspects, the primers can be used as species or type specific probes for the Thraustochytrium or Bacillus mentioned herein. In this case, primers should be constructed so that they are specific for the eukaryotic microorganism and, in turn, perform PCR reactions. The presence of target species could then be determined by successfully creating a PCR product. In some aspects, the primers can also be used for DNA amplification reactions, such as PCR or direct sequencing. It is understood that in some aspects the primers can also be extended using non-enzymatic techniques, where, for example, the nucleotides or oligonucleotides used to prolong the primer are modified so that they will react chemically with the primer in a sequence-specific manner.

d) Delivery of nucleic acids [0143] 159. In the methods described above which comprise the administration and uptake of exogenous DNA into host cells (i.e., transduction or gene transfection), the disclosed nucleic acids may be in the form of pure DNA or RNA, or the nucleic acids may be found in the nucleic acid delivery vector into cells, thereby the DNA fragment encoding the antibody is regulated transcriptionally by the promoter, as should be understood by one of ordinary skill in the art. The vector may be a commercially available preparation such as an adenoviral vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada).

Delivery of the nucleic acid or vector to the cells may take place via a variety of mechanisms. As one example, the delivery may be via the liposome, using commercially available liposome formulations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT

- 38 (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to normal procedures in this field. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology of which is available from Genetronics, Inc. (San Diego, CA) as well as using the SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

[0144] 160. As one example, vector delivery can be mediated by a viral system, such as a retroviral vector system that can package a recombinant retroviral genome (see, e.g., Pastan et al., Proc. Natl. Acad. Sci. USA 85: 4486, 1988, Miller et al., Mol. Cell Biol. 6: 2895, 1986). The recombinant retrovirus can then be used to infect and thereby deliver to the infected cells the nucleic acid coding for the broadly neutralizing antibody (or active fragment thereof). The exact method of introducing altered nucleic acid into mammalian cells, of course, is not limited to the use of retroviral vectors. Other techniques are widely available for this procedure, including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther. 5: 941-948, 1994), Adenovirus-associated viral vectors (AAV) (Goodman et al., Blood 84: 1492-1500, 1994), lentiviral vectors (Naidini et al., Science 272: 263-267, 1996), pseudotypic retroviral vectors (Agrawal et al. , Exper. Hematol. 24: 738-747, 1996). Physical transduction techniques can also be used, such as delivery in liposomes and via receptors and other mechanisms of endocytosis (see, e.g., Schwartzenberger et al., Blood 87: 472-478, 1996). These disclosed compositions and methods may be used in conjunction with any of these or other commonly used gene transfer methods. 1996). Physical transduction techniques can also be used, such as delivery in liposomes and via receptors and other mechanisms of endocytosis (see, e.g., Schwartzenberger et al., Blood 87: 472-478, 1996). These disclosed compositions and methods may be used in conjunction with any of these or other commonly used gene transfer methods. 1996). Physical transduction techniques can also be used, such as delivery in liposomes and via receptors and other mechanisms of endocytosis (see, e.g., Schwartzenberger et al., Blood 87: 472-478, 1996). These disclosed compositions and methods may be used in conjunction with any of these or other commonly used gene transfer methods.

4. Expression Systems [0145] 161. The nucleic acids that are delivered to the cells typically contain expression control systems. For example, genes inserted in viral and retroviral systems typically contain promoters and / or enhancer sequences to help regulate the expression of the desired gene product. A promoter generally means a sequence or DNA sequences that act when they are located at a relatively fixed site relative to the start of transcription. The promoter contains the core elements required for the primary interaction of RNA polymerase and transcription factors, and may include elements upstream and response elements.

[0146] 162. It is understood that there are various transcription control systems that can be used in the organisms disclosed herein, in addition to the general systems discussed

- 39 below. It is understood that the organisms disclosed herein can be transfected and transformed with a variety of genes, such as marker genes as discussed herein, or genes that have other desirable traits, such as increased or unique growth characteristics.

a) Viral promoters and enhancer sequences [0147] 163. Preferred promoters for regulating transcription from vectors in mammalian host cells can be obtained from a variety of sources, e.g., viral genomes, such as: polyoma virus, simian polyomavirus (SV40), adenovirus , retroviruses, hepatitis B virus, and most preferably cytomegalovirus, or heterologous mammalian promoters, e.g. the actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as the SV40 restriction fragment which also contains the SV40 virus origin of replication (Tiers et al. Nature, 273: 113, 1978). The immediate early human cytomegalovirus promoter is conveniently obtained as the HindIII E restriction fragment (Greenway, PJ et al., Gene 18: 355-360, 1982). Of course,

[0148] 164. The enhancer sequence generally refers to a DNA sequence that acts not at a fixed distance from the start of transcription and may be either 5 '(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 , 1981) or 3 '(Lusky, ML, et al., Mol. Cell Bio. 3: 1108, 1983) relative to the transcription unit. In addition, enhancer sequences may reside within the intron (Banerji, JL et al., Cell 33: 729, 1983) as well as within the coding sequence itself (Osborne, TF, et al., Mol. Cell Bio. 4: 1293, 1984). . They have a length usually between 10 and 300, and they work in yew. Enhancer sequences work by increasing transcription from nearby promoters. Enhancer sequences often also contain elements of the response that mediate transcription regulation. Promoters can also contain elements of the answer, which mediate regulation of transcription. Enhancer sequences often determine the regulation of gene expression. Although many enhancer sequences are currently known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein and insulin), typically a specialist will use a boosting sequence from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the origin of replication (100-270 bp), the early enhancer sequence of the cytomegalovirus promoter, the enhancer substance of the polyoma virus on the late side of the origin of replication, and adenovirus enhancer sequences. alpha-fetoprotein and insulin), typically a specialist will use a boosting sequence from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the origin of replication (100-270 bp), the early enhancer sequence of the cytomegalovirus promoter, the enhancer substance of the polyoma virus on the late side of the origin of replication, and adenovirus enhancer sequences. alpha-fetoprotein and insulin), typically a specialist will use a boosting sequence from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the origin of replication (100-270 bp), the early enhancer sequence of the cytomegalovirus promoter, the enhancer substance of the polyoma virus on the late side of the origin of replication, and adenovirus enhancer sequences.

[0149] 165. The promoter and / or enhancer sequence can be specifically activated either by light or by specific chemical events that trigger their effect. layouts

- they can be regulated by reagents such as tetracycline and dexamethasone. There are also methods for enhancing the expression of genes of a viral vector by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapeutic drugs.

[0150] 166. In certain embodiments, the promoter and / or enhancer region may act as a constitutive promoter and / or enhancer to maximize the expression of the region of the transcription unit to be transcribed. In certain constructs, the promoter region and / or enhancer sequence will be active in all types of eukaryotic cells, even if it is expressed only in a particular cell type at a particular time. A preferred promoter of this type is the CMV promoter (650 bp). Other preferred promoters are the SV40 promoters, the cytomegalovirus (full-length promoter), and the retroviral LTF vector.

[0151] 167. It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types, such as melanoma cells. The promoter of glial fibrillary acetic protein (GFAP) has been used for the selective expression of genes in glial cells.

[0152] 168. Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, human or nucleated cells) may also contain sequences necessary to terminate transcription that may affect the expression of mRNA. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding the tissue factor protein. Untranslated 3 'areas also include the end of transcription. It is preferred that the transcription unit also contains a polyadenylated region. One of the benefits of this area is that it increases the probability that the transcribed unit will be processed and transported like an mRNA. The identification and use of polyadenylation signals in expression constructs is known. It is preferred to use homologous polyadenylation signals in transgenic constructs. In certain transcription units, the polyadenylated area is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other normal sequences alone or in combination with the above sequences to improve the expression or stability of the construct.

b) Markers

[0153] 169. Viral vectors can include a nucleic acid sequence coding for a marker product. This marker product is used to determine whether the gene has been delivered to the cell and whether it is already being expressed. Preferred marker genes are the E. coli lacZ gene that encodes β-galactosidase and the green fluorescent protein.

[0154] 170. In some embodiments, the marker may be a selectable marker. Examples of useful selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin G418 analogue, hydromycin, and puromycin. When such selectable markers are successfully transferred to the mammalian host cell, the transformed host cell of the mammal can survive if placed under selective pressure. There are two widely used different categories of selective regimes. The first category is based on the metabolism of the cell and the use of a mutant cell line that is incapable of growing independently of the supplemented medium. Two examples are: DHFR CHO cells and mouse cells

LTK. These cells do not have the ability to grow without the addition of nutrients such as thymidine or hypoxanthine. Because these cells do not have certain genes necessary for the full pathway of nucleotide synthesis, they can not survive unless the missing nucleotides are provided in the supplemented media. An alternative to supplementing nutrients is to introduce an intact DHFR or TK gene into cells that do not have relevant genes, thus changing their growth requirements. Individual cells that have not been transformed with the DHFR or TK gene will not be able to survive in non-supplemented media.

[0155] 171. The second category is dominant selection, which refers to the selection scheme used in any cell type and does not require the use of mutant cell lines. These schemes typically use the drug to arrest the growth of the host cell. Those cells that have a new gene should express the drug-resistant protein and should survive the selection. Examples of such dominant selection are neomycin drugs (Southern P. and Berg, P., J Molec. Appl. Genet. 1: 327, 1982), mycophenolic acid, (Mulligan, RC and Berg, P. Science 209: 1422, 1980 ) or hygromycin (Sugden, B. et al., Mol. Cell Biol. 5: 410-413, 1985).

Three examples use bacterial genes under eukaryotic control to confer resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the G418 neomycin analog and puramycin.

5. Peptides

- 42 a) Variants of proteins [0156] 172. As discussed herein, there are numerous variants of the disclosed body proteins that are known and contemplated herein. In addition to the known active Thraustochytriales strain, there are derivatives of Thraustochytriales proteins that are also active in the disclosed methods and compositions. Variants and derivatives of proteins are understood by those skilled in the art and may include amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitution, insertion or deletion variants. Insertions include amine and / or carboxyl termination fusions as well as insertions within the sequence of single or multiple amino acid residues. Insertions will usually be smaller insertions than at amine or carboxyl terminus fusions, e.g. of one to four residues. An immunogenic fused derivative protein, such as those described in the examples, is made by fusion of a polypeptide large enough to confer immunogenicity to the target sequence by in vitro crosslinking or by growing recombinant cells transformed with a DNA coding fusion.

Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, anywhere within the protein molecule, no more than about 2 to 6 residues are deleted. These variants are usually produced by site-specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant and then expressing DNA in the culture of recombinant cells. There are known techniques for generating substitution mutations at specific sites in DNA having a known sequence, e.g. M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions typically refer to individual residues, but can occur in a variety of different sites at once; the insertions will usually be on the order of about 1 to 10 amino acid residues; and the deletions will be in the range of about 1 to 30 residues. Deletions or insertions are preferably performed in adjacent pairs, it is as a deletion of 2 residues or an insertion of 2 residues. Substitutions, deletions, insertions or any combination of them may be combined to arrive at the final construct. Mutations can not derive sequences beyond the reading frame, and preferably they will not create complementary regions that could produce a secondary mRNA structure. The substitution variants are those in which at least one of the rest has been removed, and the rest are inserted in its place. Such substitutions are generally prepared according to the following Tables 1 and 2 and are referred to as conservative substitutions. that could produce a secondary mRNA structure. The substitution variants are those in which at least one of the rest has been removed, and the rest are inserted in its place. Such substitutions are generally prepared according to the following Tables 1 and 2 and are referred to as conservative substitutions. that could produce a secondary mRNA structure. The substitution variants are those in which at least one of the rest has been removed, and the rest are inserted in its place. Such substitutions are generally prepared according to the following Tables 1 and 2 and are referred to as conservative substitutions.

[0157] 173. Significant changes in immune function or identity are generated by the selection of substitutions that are less conservative than those in Table 2, i.e. residue selection,

Those which more significantly differ in their effect on the behavior of (a) the polypeptide backbone structure in the substitution region, e.g. as a sheet or helix conformation, (b) alteration of the hydrophobicity of the molecule at the target site, or (c) side chain volume. The substitutions for which the greatest changes in protein properties should generally be expected will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, substitutes (or is substituted by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) the cysteine ​​or proline substitutes (or is substituted by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, substitutes (or is substituted with) an electronegative residue, e.g., glutamyl or aspartyl;

Table 1: Abbreviations of amino acid names

<td>Amino acid</td><td colspan="2">Shortcuts</td>

<td>alanine</td><td>ala</td><td>AND</td>

<td>arginine</td><td>Arg</td><td>R</td>

<td>asparagine</td><td>own</td><td>N</td>

<td>Aspartic acid</td><td>Asp</td><td>D</td>

<td>Cysteine</td><td>Cys</td><td>C</td>

<td>glutamine</td><td>Gln</td><td>Q</td>

<td>Glutamic acid</td><td>Glu</td><td>E</td>

<td>glycine</td><td>Gly</td><td>G</td>

<td>histidine</td><td>His</td><td>H</td>

<td>isoleucine</td><td>He</td><td>AND</td>

<td>leucine</td><td>Leu</td><td>L</td>

<td>lysine</td><td>lys</td><td>K</td>

<td>methionine</td><td>Underworld</td><td>M</td>

<td>phenylalanine</td><td>phe</td><td>F</td>

<td>Amino acid</td><td colspan="2">Shortcuts</td>

<td>proline</td><td>Pro</td><td>P</td>

<td>serine</td><td>Cheese</td><td>S</td>

<td>threonine</td><td>Thr</td><td>T</td>

<td>tryptophan</td><td>Trp</td><td>IN</td>

<td>tyrosine</td><td>Tyr</td><td>Y</td>

<td>valine</td><td>val</td><td>V</td>

Table 2: Amino acid substitutions

<td>The rest is original</td><td>Examples of conservative substitutions *</td>

<td>ala</td><td>Cheese</td>

<td>Arg</td><td>Lys; Gln</td>

<td>own</td><td>Gln; His</td>

<td>Asp</td><td>Glu</td>

<td>Cys</td><td>Cheese</td>

<td>Gln</td><td>Asn; lys</td>

<td>Glu</td><td>Asp</td>

<td>Gly</td><td>Pro</td>

<td>His</td><td>Asn; Gln</td>

<td>How much</td><td>Leu; val</td>

<td>Leu</td><td>How much; val</td>

<td>lys</td><td>Arg; Gln</td>

<td>Underworld</td><td>Leu; How much</td>

<td>phe</td><td>Underworld; Leu; Tyr</td>

<td>Pro</td><td>Gly</td>

<td>Cheese</td><td>Thr</td>

<td>The rest is original</td><td>Examples of conservative substitutions *</td>

<td>Thr</td><td>Cheese</td>

<td>Trp</td><td>Tyr</td>

<td>Tyr</td><td>Tip; phe</td>

<td>val</td><td>How much; Leu</td>

<td>* Known and different in the prior art.</td><td></td>

[0158] 174. For example, replacing one amino acid residue with another that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitute can replace one or the other polar residue with one hydrophobic residue to replace another. Substitutions include combinations such as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Cheese, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variants of each of the overtly disclosed sequences are included in the mosaic polypeptides provided herein.

[0159] 175. Substitution or deletion mutagenesis can be used at N-linked glycosylation sites (Asn-X-Thr / Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine ​​or other labile residues may also be desirable. Deletions or substitutions of potential proteolytic sites, e.g. Arg, are achieved by deleting one of the basic residues or substituting it with glutaminyl or histidyl residues.

[0160] 176. Certain post-translational derivatives are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are often post-translationally deamidated for glutamyl and asparyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or treonyl residues, methylation of o-amino groups of lysine, arginine, and histidine side chains (TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman &amp; Co., San Francisco, pp. 79-86, 1983), acetylation of the N-terminal amine and, in some cases, amidation of the C-terminal carboxyl group.

[0161] 177. It is understood that one of the ways to define variants and derivatives of the proteins disclosed herein is to identify variants and derivatives in terms of homology / identity with specific known sequences. It reveals itself in detail

- variants of the proteins disclosed herein which have at least, 60%, 70% or 75% or 80% or 85% or 90% or 95% homology with the given sequence. Those skilled in the art will easily understand how to determine the homology of two proteins. For example, homology can be calculated after the arrangement of two sequences so that homology is at its highest level.

[0162] 178. Another method of calculating homology can be made by published algorithms. The optimal alignment of sequences for comparison can be carried out using the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482, 1981, the homology algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443, 1970, by looking for similarity by the Pearson and Lipman method, Proc. Natl. Acad. Sci. USA 85: 2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by browsing.

[0163] 179. The same types of homology can be obtained for nucleic acids, for example, the algorithms disclosed in: 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, 198, 188 which are incorporated herein by reference to at least a material relating to the nucleic acid arrangement.

[0164] 180. It is understood that the description of conservative mutations and homology can be combined together in any combination, such as embodiments that have at least 70% homology with a particular sequence, where the variants are conservative mutations.

[0165] 181. Since the present description discusses a variety of protein and protein sequences, it is understood that nucleic acids that can encode these protein sequences are also disclosed. This may include all degenerate sequences referring to a particular protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence, as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequence. Thus, while each individual nucleic acid sequence can not be stored herein, it is understood that each and every sequence is actually disclosed and described herein by the disclosed protein sequence. It is also understood that while no amino acid sequence indicates that

[0166] 182. It is understood that there are numerous amino acid and peptide analogs that can be included in the disclosed compositions. For example, there are numerous D-amino acids or amino acids that have a functional substituent different from the amino acids shown in Table 1 and Table 2. The isomers with stereochemistry opposite to naturally occurring peptides as well as stereoisomers of peptide analogs are disclosed. These amino acids can be easily incorporated into polypeptide chains by introducing the selected amino acid into the tRNA molecules and constructing genetic engineering constructs that use, for example, codons of UAG (amber codons), to insert the amino acid analog into the peptide chain in a site-specific manner. (Thorson et al., Methods in 10 Mol 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), all of which are incorporated herein by reference at least to the material related to amino acid analogs).

[0167] 183. Molecules that resemble peptides may be produced, but which are not joined by a natural peptide bond. For example, the binding of amino acids or amino acid analogs may include CH2NH--, -CH2S--, -CH2-CH2-, -CH = CH-- (cis and trans), -COCH2--, -CH ( OH) CH2--, and - CHH2SO--. (These and others can be found in: Spatola, AF, Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, ed. B. Weinstein, Marcel Dekker, New York pp. 267, 1983; Spatola, AF, Vega Data (March 1983) ), volume 1, issue 3, Peptide Backbone Modifications (general overview); Morley, Trends Pharm. Sci. 463468, 1980; Hudson, D. et al., Int. J. Pept. Res. 14: 177-185 , 1979 (--CH2NH--, CH2CH2 -); Spatola et al., Life Sci. 38: 1243-1249, 1986 (- CH H2 - S); Hann, J. Chem. Soc. Perkin Trans. 1307-314, 1982 (- CH - CH--, cis and trans); Almquist et al., J. Med. Chem. 23: 1392-1398, 1980 (-COCH2--); Jennings-White et al., Tetrahedron Lett., 23: 2533, 1982 (--COCH 2--); Szelke et al., European Application EP45665 CA: 97: 39405, 1982 (- CH (OH) CH2-); Holladay et al., Tetrahedron Lett. 24: 4401-4404, 1983 (- C (OH) CH2--); and Hruby, Life Sci. 31: 189-199, 1982 (-CH2 - S--); each of which is incorporated herein by reference. A particularly preferred non-peptide bond is --CH2NH--. It is understood that peptide analogs may have more than one atom between bond atoms, such as β-alanine, γ-aminobutyric acid, and the like. 189-199, 1982 (-CH2 - S--); each of which is incorporated herein by reference. A particularly preferred non-peptide bond is --CH2NH--. It is understood that peptide analogs may have more than one atom between bond atoms, such as β-alanine, γ-aminobutyric acid, and the like. 189-199, 1982 (-CH2 - S--); each of which is incorporated herein by reference. A particularly preferred non-peptide bond is --CH2NH--. It is understood that peptide analogs may have more than one atom between bond atoms, such as β-alanine, γ-aminobutyric acid, and the like.

[0168] 184. Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties such as more efficient production, greater chemical stability, increased pharmacological properties (half-life, absorption, potency,

- 48 efficacy, etc.), altered specificity (e.g., a broad spectrum of biological activities), reduced antigenicity, and others.

[0169] 185. D-Amino acids can be used to produce more stable peptides because D-amino acids are not recognized by peptidases etc. Systematic substitution of one or more amino acids of a sequence consistent with the D-amino acid of the same type (e.g., D-lysine instead of L-lysine) can be used to produce more stable peptides. Cysteine ​​residues can be used to cyclize or combine two or more peptides together. This may be beneficial for reducing peptides in particular conformations. (Rizo and Gierasch, Ann. Rev. Biochem. 61: 387, 1992, incorporated herein by reference).

6. Supplements [0170] 186. Nutritional supplements are also disclosed herein. A nutritional supplement is any compound or composition that can be administered to a subject or taken by a subject to form, deliver or elevate the level of a nutrient (s) (e.g., vitamin, mineral, essential trace element, amino acid, peptide, nucleic acid, oligonucleotide). , lipid, cholesterol, steroid, carbohydrate, and the like). In one aspect, disclosed herein are nutritional supplements comprising any of the compounds disclosed herein. For example, the nutritional supplement may contain any of the lipids disclosed herein. The fatty acid residues of these lipids may be any fatty acids as disclosed herein (e.g., unsaturated or saturated fatty acid residues).

[0171] 187. The nutrient supplement may contain any amount of the compounds disclosed herein, but will typically contain an amount specified to deliver the desired dose of a benzenediol derivative (e.g., Cab)) and / or fatty acids to the subject. The exact amount of compound needed in the nutritional supplement will vary between those being treated, depending on the species, age, weight and general condition being treated, the severity of the dietary deficiency being treated, the particular mode of administration, and the like. Thus, you can not give the exact amount for each nutritional supplement. However, the correct amount can be determined using only routine experimentation by an ordinary person skilled in the art who has the teachings herein. In one particular example, the nutritional supplement may contain from about 0.05 to about 20%, from about 1 to about 7.5%, or from about 3 to about 5% by weight of the compound. In another example, the nutritional supplement may contain from about 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% by weight of the compound, where any of the given values ​​may form the upper or lower end of the range, as appropriate. In another aspect, as a nutritional supplement, the supplement may be composed of up to 100% of the supplement.

[0172] 188. The nutrient supplement may also contain other nutrients, such as vitamins, trace elements, minerals, and the like. Further, the nutritional supplement may contain other ingredients such as preservatives, antibacterial agents, antioxidants, chelating agents, thickeners, flavors, diluents, emulsifiers, dispersing agents, and / or binders.

[0173] 189. Nutritional supplements are generally taken orally and may be in any form suitable for oral administration. For example, a nutritional supplement may typically be in the form of a tablet, gel capsule, capsule, liquid, bag, or syrup.

7. Delivery devices [0174] 190. Any of the compounds described herein can be incorporated into a delivery device. Examples of delivery devices include, but are not limited to, microcapsules, microspheres, nanospheres or nanoparticles, liposomes, niosomes (from non-ionic surfactant based vesicles), nanoerythrosomes, solid lipid nanoparticles, gels, gel capsules, tablets, lotions, creams, sprays, or emulsions. Other examples of delivery devices that are suitable for non-oral administration include PulmoSpheres. Examples of individual delivery devices useful herein are described below.

[0175] 191. The disclosed compounds can be incorporated into liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid suitable for forming liposomes can be used. The disclosed liposome formulations may contain, in addition to the compound disclosed herein, stabilizers, preservatives, excipients, and the like. Examples of suitable lipids are phospholipids and phosphatidylcholines (lecithins), both natural and synthetic. Methods for forming liposomes are known in the art. See, e.g., Prescott, ed., Methods in Cell Biology, vol. XIV, Academic Press, New York, pp. 33 and next; 1976, which is a reference to this reference reference to the instructions on liposomes and their preparation.

[0176] 192. In other examples, the liposomes can be cationic liposomes (e.g., DOTMA, DOPE, DC cholesterol) or anionic liposomes. Liposomes may further contain proteins to facilitate targeting to a particular cell, if desired. The composition containing the compound and the cationic liposome may be administered to the blood leading to the target organ or may be inhaled into the target cells of the respiratory system. For liposomes, see, e.g., Brigham, et al., Am J Resp Cell Mol Biol 1: 95100, 1989; Feigner, et al., Proc Natl Acad Sci USA 84: 7413-7, 1987; and U.S. Patent No. 4,897,355, which are incorporated herein by reference to their teaching of liposomes. As one example, the delivery may be via the liposome using commercially available liposome formulations, such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI) as well as other liposomes formulated according to normal procedures in this field . Liposomes can also be used where diffusion of the compound or delivery of the compound from the liposome is planned at a particular rate or dosage.

[0177] 193. As described herein, the niosomes are delivery devices that can be used to deliver the compositions disclosed herein. Niosomes are multi-layered or single-layered vesicles containing non-ionic surfactants. The aqueous solution of the dissolved substance is enclosed by a bilayer formed as a result of organizing the macromolecules of the surfactant. Similar to liposomes, niosomes are used in the directed delivery of, for example, anti-cancer drugs, including methotrexate, doxorubicin, and immunoadjuvants. It is believed that they are generally different from transferosomes, vesicles prepared from amphiphilic carbohydrate and polymers containing amino groups, e.g., chitosan.

[0178] 194. As described herein, nanoerythrosomes are delivery devices that can be used to deliver the compositions disclosed herein. Nanoerythrosomes are nanowires generated from red blood cells by dialysis through filters with a defined pore size. These vesicles can be filled with a variety of biologically active molecules, including proteins and compositions disclosed herein. In general, they serve as ideal carriers for anti-cancer agents such as bleomycin, actinomycin D, but can be used for steroids, other lipids, etc.

[0179] 195. Artificial red cells as described herein are further delivery devices that can be used to deliver the compositions disclosed herein. Artificial red blood cells can be produced by emulsion polymerization at the interface. Generally, the "cell" wall is made of polyalphthalyl-L-lysine / polystyrene polymer and the core is made from hemoglobin solution from sheep hemolysate. Hemoglobin-filled microspheres typically have particle sizes of from about 1 to about 10 mm. Their size, flexibility, and oxygen carrying capacity are similar to red blood cells.

[0180] 196. Solid lipid nanoparticles, as described herein, are other delivery devices that can be used to deliver the compositions disclosed herein.

Solid lipid nanoparticles are nanoparticles that are dispersed in an aqueous solution of surfactant. They consist of a hydrophobic core having a monolayer of a phospholipid coating and are usually made by high-pressure homogenisation techniques. Examples of solid lipid nanoparticles are immunomodulatory complexes (ISCOMS). They are similar to the 40 nm cage of supramolecular assemblies composed of phospholipid, cholesterol, and hydrophobic antigens, and are mainly used as immunoadjuvants. For example, ISCOM is used to prolong subcutaneous levels of cyclosporin in the blood plasma.

[0181] 197. Microspheres and microcapsules as described herein are yet other delivery devices that can be used to deliver the compositions disclosed herein. In contrast to liposomal delivery systems, microspheres and microcapsules typically do not have a water core, but a solid matrix or polymeric membrane. These delivery devices are obtained by controlled precipitation of polymers, chemical crosslinking of soluble polymers, and polymerization at the interface of two monomers or by high pressure homogenisation techniques. The encapsulated compound is gradually released from the drug portion by erosion or diffusion from particles. Successful formulations of short-acting peptides such as LHRH agonists such as leuprorelin and triptorelin have been developed. Poly (lactide co-glycolide) microspheres (PLGA) are currently used as monthly and three-monthly dosages in the treatment of advanced prostate cancer, endometriosis, and other hormone-responsive states. Leuprolide, the LHRH superagonist, was incorporated into a variety of PLGA templates using a solvent extraction and evaporation method. As mentioned, all of these delivery devices can be used in the methods disclosed herein.

[0182] 198. PulmoSpheres are yet further examples of delivery devices that can be used herein. PulmoSpheres are empty porous particles with low density (less than about 0.1 m mL^-1). PulmoSpheres typically have an excellent dispersibility and are usually produced by a supercritical fluid condensation technology.

Spray drying together with certain matrices, such as carbohydrates, human serum albumin, etc., can improve the stability of proteins and peptides (e.g., insulin) and other biological molecules for pulmonary delivery. This type of delivery can also be carried out using microemulsions and lipid emulsions, which are ultrafine, thin, transparent oil in water (o / w) emulsions formed spontaneously without significant mechanical energy input. In this technique, the emulsion can be produced at a temperature which must be higher than the phase inversion temperature of this system. At elevated temperature, the emulsion is of the water-in-oil (w / o) type and, as it cools down, at the temperature of the phase inversion, this emulsion inverts, becoming o / w. Due to their very small internal phase, they are extremely stable and used for the sustained release of steroids and vaccines. The lipid emulsions contain an inert lipid core (i.e., triglycerides) stabilized by a lipid monolayer (i.e., a phospholipid) using surfactants such as chicken egg lecithin, triglycerides and miglyol. They are useful for passive and active targeting of medicines.

[0183] 199. There are other test systems for oral delivery that are based on osmotic pressure modulation, pH modulation, swelling modulation, altered density and floating systems on the surface of the liquid, mucosal adhesion, etc. These formulations and formulations are retarded to the delivery of drugs in accordance with the circadian rhythm of the disease that are currently in use or study, may be used to deliver the compositions disclosed herein.

[0184] 200. In one particular aspect disclosed herein, the disclosed compounds, including a nutritional supplement and its pharmaceutical formulations, may be incorporated into microcapsules as described herein.

[0185] 201. In one of the aspects disclosed herein, the disclosed compounds may be incorporated into microcapsules. In one aspect, the microcapsule includes microcapsules of primary and chromium compounds described herein, each individual primary microcapsule having a primary shell, wherein the chromium compound is enclosed by the primary shell, where the cluster is enclosed by the outer shell. These microcapsules are referred to herein as &amp; quot; multi-core microcapsules &amp; quot ;.

[0186] 202. In another aspect, described herein are microcapsules comprising a chromium compound, a primary shell, and a secondary shell, wherein the primary shell encloses a chromium compound and the secondary shell closes the filling substance and the primary coating. These microcapsules are referred to herein as &amp; quot; single-core microcapsules &amp; quot ;.

[0187] 203. Optionally, other filler substances may be closed with the chromium compound. The filling substance can be any substance that is not completely soluble in the aqueous mixture. In one aspect, the filler substance is a solid, a hydrophobic liquid, or a mixture of a solid and a hydrophobic liquid. In another aspect, the filler comprises a fat, oil, lipid, drug (e.g., small molecule), a biologically active substance, a nutritional supplement (e.g., vitamins), a fragrance compound, or a mixture thereof. Examples of oils include, but are not limited to, animal oils (e.g., fish oil, marine mammal oil, etc.), vegetable oils (e.g., low erucic rape or rapeseed oil), mineral oils, derivatives thereof, or mixtures thereof. The filling substance may be a purified or partially purified oily substance, such as a fatty acid, its triglyceride or ester, or a mixture thereof. In another aspect, the filler substance may be a carotenoid (e.g., lycopene), a satiety sensing agent, a fragrance compound, a drug (e.g., a water insoluble drug), particulate matter, agricultural chemicals (e.g., herbicides, insecticides, fertilizers) or a hydroponic component (e.g., feed, pigment).

[0188] 204. In one aspect, the filler substance may be an omega-3 fatty acid. Examples of omega-3 fatty acids include, but are not limited to, α-linolenic acid (18: 3n3), octadatatetraenoic acid (18: 4n3), eicosapentaenoic acid (20: 5n3) (EPA), docosahexaenoic acid (22: 6n3) (DHA), docosapentaenoic acid (22: 5n3) (DPA), eicosatetraenoic acid (20: 4n3), unopinenoic acid (21: 5n3), docosapentaenoic acid (22: 5n3) and their derivatives and mixtures thereof. Numerous types of omega-3 fatty acid derivatives are known in the art. Examples of useful derivatives include, but are not limited to, esters such as phytosterol esters, branched or unbranched C1-C30 alkyl esters, branched or unbranched C2-C30 alkenyl esters, or branched or unbranched C3-C30 cycloalkyl esters, such as phytosterol esters and C 1 -C 6 alkyl esters. Sources of oils may come from aquatic organisms (e.g., anchovy, capelin, Atlantic cod, oceanic herring, Atlantic mackerel, Atlantic menhaden, salmonids, sardines, shark, tuna, etc.) and plants (e.g., flax, vegetables, etc. ) and microorganisms (e.g., fungi and algae).

[0189] 205. In one aspect, the filler may contain an antioxidant. Examples of antioxidants include, but are not limited to, vitamin E, CoQ10, tocopherols, lipid soluble derivatives of more polar antioxidants, such as ascorbic esters of fatty acids (e.g., ascorbyl palmitate), plant extracts (e.g., rosemary, sage and rosmarin oil) marjoram), algal extracts, and synthetic antioxidants (e.g., BHT, TBHQ, ethoxychine, alkyl gallates, hydroquinones, tocotrienols).

[0190] 206. A variety of polymers may be used to produce the shell layers of single and multicore microcapsules. Examples of such polymers include, but are not limited to, a protein, a polyphosphate, a polysaccharide, or a mixture thereof. In another aspect, the envelope material used to make the mono- and multicore microcapsules further includes Type A gelatine, Type B gelatine, polyphosphate, gum arabic, alginate, chitosan, carrageenan, pectin, starch, modified starch, alpha-lactalbumin, beta-lactoglobulin, hen egg albumin, polysorbitone, maltodextrin, cyclodextrin, cellulose, methylcellulose, ethylcellulose, hydropropylmethylcellulose, carboxymethylcellulose, milk protein, whey protein, soy protein, low-rapeseed protein, albumin, chitin, polylactides, poly (lactide-co-glycolide), chitin derivative . chitosan, poly-lysine, various inorganic-organic composites, or any mixture thereof. It is also contemplated that derivatives of these polymers may also be used. In another aspect, the polymer may be kosher gelatin, non-kosher gelatin, halal gelatin, or non-halal gelatin.

[0191] 207. In one aspect, the one or more shell layers in the single- and multicore microcapsules comprise gelatin having a Bloom number of less than 50. This gelatin is referred to herein as "low Bloom" gelatin ". Bloom numbers describe the shear resistance of a gel prepared at 10 ° C with a 6.67% solution gelled for 18 hours. In one aspect, gelatin with a low Bloom value has a Bloom value of less than 40, less than 30, less than 20, or less than 10. In another aspect, gelatin has a Bloom number of 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0, where any two values ​​can be used to obtain the range. In another aspect, gelatin with a low Bloom value is found both in the primary envelope and in the outer shell of the multicore microcapsules. In one aspect, low Bloom gelatin is type A gelatin. In another aspect, low Bloom gelatin is Type A gelatine manufactured by Kenney &amp; Ross Ltd., RR # 3 Shelburne, NS Canada. In another

In an aspect, the gelatin having the Bloom number value equal to zero is found both in the primary shell and in the outer shell of the multicore microcapsules.

[0192] 208. In one aspect, the material used to make shells of single- or multicore microcapsules is a bi-component system made from a mixture of two different types of polymers. In one aspect, the material is a complex coacervate of polymeric components. Complex coacervation is caused by interaction between two polymers with opposite charges. In one aspect, the envelope material used to make the mono and multi-core microcapsules is composed of (1) gelatin with a low Bloom value and (2) type B gelatine, polyphosphate, gum arabic, alginate, chitosan, carrageenan, pectin, carboxymethylcellulose, whey protein, soy protein, low-erosive rape protein, albumin, or mixtures thereof. The molar ratio of different polymers can vary. E.g, the molar ratio of gelatin with a low Bloom value to another polymer component is from 1: 5 to 15: 1. For example, when low in Bloom gel and polyphosphate gelatin is used, the molar ratio of low gelatin Bloom to polyphosphate is about 8: 1 to about 12: 1; when using low Bloom gelatin and type B gelatine, the molar ratio is 2: 1 to 1: 2; and when gelatin with a low Bloom and alginate value is used, the molar ratio is 3: 1 to 8: 1. when using low Bloom gelatin and type B gelatine, the molar ratio is 2: 1 to 1: 2; and when gelatin with a low Bloom and alginate value is used, the molar ratio is 3: 1 to 8: 1. when using low Bloom gelatin and type B gelatine, the molar ratio is 2: 1 to 1: 2; and when gelatin with a low Bloom and alginate value is used, the molar ratio is 3: 1 to 8: 1.

[0193] 209. Processing aids may be included in the shell material (e.g., primary or outer shells). Processing aids can be used for a variety of reasons. For example, they may be used to induce aggregation of primary microcapsules, stabilize the emulsion system, improve the properties of the outer coatings, control the size of the microcapsules, and / or act as an antioxidant. In one aspect, the processing aid may be an emulsifier, a fatty acid, a lipid, a wax, a microbial cell (e.g., yeast cell lines), a clay, or an inorganic compound (e.g., calcium carbonate). Without wishing to be bound by theory, the inventors believe that these processing aids can improve the barrier properties of microcapsules. In one aspect, one or more antioxidants can be added to the shell material. The antioxidant properties are useful both during processing (e.g. during coacervation and / or spray drying) and in microcapsules after their preparation (i.e., for shelf life, etc.). Preferably, a small number of processing aids that fulfill a large number of functions may be used. In one aspect, the antioxidant may be a phenolic compound, a plant extract, or an amino acid

- 56 containing sulfur. In one aspect, ascorbic acid (or a salt thereof, such as sodium or potassium ascorbate) can be used to induce aggregation of primary microcapsules, to regulate the size of microcapsules, and to act as an antioxidant. The antioxidant may be used in an amount of about 100 ppm to about 12000 ppm, or from about

1000 ppm to about 5000 ppm. Other processing aids may also be used, such as, for example, metal chelating compounds. For example, ethylenediaminetetraacetic acid may be used to bind metal ions, which may reduce catalytic oxidation of the filling substance.

[0194] 210. In one aspect, the primary microcapsules (primary shells) have an average diameter of about 40 nm to about 10 μm, 0.1 μm to about 10 μm, 1 μm to about 10 μm, 1 μm to about 8 μm, 1 μm to about 6 μm, 1 μm to about 4 μm, or 1 μm to about 2 μm, or 1 μm. In another aspect, the multi-core microcapsules may have an average diameter of from about 1 μm to about 2000 μm, 20 μm to about 1000 μm, from about 20 μm to about 100 μm, or from about 30 μm to about 80 μm. In another aspect, the single-core microcapsules have an outer diameter of 1 μm to 2,000 μm.

[0195] 211. The microcapsules described herein generally have a combination of high payload and structural strength. For example, the usable charge of the filling substance may be from 20% to 90%, 50% to 70% by weight, or 60% by weight of one- or multi-core microcapsules.

[0196] 212. In one aspect, the methods disclosed in U.S. Patent Application Publication No. 2003/0193102, which are incorporated by reference in its entirety, may be used to encapsulate the chromium compounds described herein. It is also contemplated that one or more additional envelope layers may be provided on the outer shell of the single- or multicore microcapsules. In one aspect, the techniques described in International Application No. WO 2004/041251 A1, which are incorporated in their entirety by reference, may be used to add additional envelope layers to single- or multicore microcapsules.

a) Pharmaceutical and Nutraceutical Compositions [0197] 213. These lipids and antioxidants are intended for use in animal feed, pharmaceuticals, nutraceuticals (especially infant formulas) as well as in industry. This is also intended to include nutraceutical delivery forms, such as gel capsules and the like, ordinary microcapsules, etc.

[0198] 214. As described above, the compositions may also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to the subject, together with the nucleic acid or vector, without causing any adverse biological effects or adversely affecting any of the other. the components of the pharmaceutical composition in which it is contained. Naturally, the carrier should be selected to minimize any degradation of the active ingredient and to minimize any side effects in the subject, as will be known to the person skilled in the art.

[0199] 215. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, intraperitoneal, transdermally, extracorporeally, locally or the like, including topical nasal administration or administration via an inhaler. As used herein, "nasal delivery" means delivery of a composition to the nose and nasal passages through one or both of the nostrils and may include delivery by a mechanism that generates a jet or mechanism that produces droplets, or by forming an aerosol of nucleic acid or vector. The administration of the composition by the inhalation agent can take place through the nose or mouth, by delivery through a mechanism producing a stream or droplets. The delivery can also take place directly to any area of ​​the respiratory system (e.g. lungs) by intubation. The exact amount of composition needed for the subject will vary depending on the species, age, weight and general condition being treated, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, the mode of its administration and the like. Thus, the exact amount for each composition can not be determined. However, the correct amount can be determined using only routine experimentation by an ordinary person skilled in the art who has the teachings herein. you can not specify the exact amount for each composition. However, the correct amount can be determined using only routine experimentation by an ordinary person skilled in the art who has the teachings herein. you can not specify the exact amount for each composition. However, the correct amount can be determined using only routine experimentation by an ordinary person skilled in the art who has the teachings herein.

[0200] 216. Parenteral administration of a composition, if used, is generally characterized by an injection. Injectables can be prepared in conventional forms either as liquid solutions or suspensions, solid forms suitable for dissolving the suspension in liquid prior to injection, or as emulsions. A recently proven approach for parenteral administration includes the use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is herein incorporated by reference.

[0201] 217. Materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells). They can be targeted to a particular type of cell by antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology for targeting specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2: 447-451, 1991; Bagshawe, KD, 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 and McKenzie, Immunologist Reviews, 129: 57-80, 1992, and Roffler, et al., Biochem. Pharmacol., 42: 2062-2065, 1991). Substrates such as "hidden" and other liposomes conjugated to antibodies (including targeting of lipid-mediated drugs to colon cancer), receptor-mediated targeting of cell-specific ligands, tumor targeting by lymphocytes, and highly specific therapeutic targeting of retroviruses to murine glioma cells in vivo. The following references are examples of the use of this technology for targeting specific proteins to tumor tissue (Hughes et al., Cancer Research, 49: 6214-6220, 1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104: 179-187, 1992). In general, receptors are involved in either endocytic or constitutive or ligand-induced pathways. These receptors accumulate in clathrine cavities, enter cells through clathrin-coated vesicles, pass through acidified endosomes, in which the receptors are sorted, and then either recycle to the cell surface, they are stored intracellularly, or they are broken down into lysosomes. Internalisation pathways serve a variety of functions such as uptake of nutrients, removal of activated proteins, purification of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of the ligand, and regulation of receptor levels. Many receptors participate in more than one intracellular pathway, depending on cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. There is an overview of the molecular and cellular mechanisms of receptor-mediated endocytosis (Brown and Greene, DNA and Cell Biology 10: 399-409, 1991). or they are spread out in lysosomes. Internalisation pathways serve a variety of functions such as uptake of nutrients, removal of activated proteins, purification of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of the ligand, and regulation of receptor levels. Many receptors participate in more than one intracellular pathway, depending on cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. There is an overview of the molecular and cellular mechanisms of receptor-mediated endocytosis (Brown and Greene, DNA and Cell Biology 10: 399-409, 1991). or they are spread out in lysosomes. Internalisation pathways serve a variety of functions such as uptake of nutrients, removal of activated proteins, purification of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of the ligand, and regulation of receptor levels. Many receptors participate in more than one intracellular pathway, depending on cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. There is an overview of the molecular and cellular mechanisms of receptor-mediated endocytosis (Brown and Greene, DNA and Cell Biology 10: 399-409, 1991). dissociation and degradation of the ligand, and regulation of receptor levels. Many receptors participate in more than one intracellular pathway, depending on cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. There is an overview of the molecular and cellular mechanisms of receptor-mediated endocytosis (Brown and Greene, DNA and Cell Biology 10: 399-409, 1991). dissociation and degradation of the ligand, and regulation of receptor levels. Many receptors participate in more than one intracellular pathway, depending on cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. There is an overview of the molecular and cellular mechanisms of receptor-mediated endocytosis (Brown and Greene, DNA and Cell Biology 10: 399-409, 1991).

(1) Pharmaceutically acceptable carriers [0202] 218. Compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0203] 219. Useful carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th Edition), edited by AR Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, a suitable amount of a pharmaceutically acceptable salt is used. in the formulation

59 to give an isotonicity formulation. Examples of a pharmaceutically acceptable carrier include, but are not limited to, saline solution, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release formulations, such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be clear to those skilled in the art that certain carriers may be more preferred, depending on, for example, the route of administration and the concentration of the composition administered.

[0204] 220. Pharmaceutical carriers are known to those skilled in the art. Most typically, these will be normal carriers for administering drugs to humans, including solutions such as sterile water, saline solution, and buffered solutions at physiological pH. The compositions may be administered intramuscularly or subcutaneously. Other compounds will be administered according to the normal procedures used by specialists.

[0205] 221. The pharmaceutical compositions may contain carriers, thickeners, diluents, buffers, preservatives, surface-active agents and the like in addition to the molecule of choice. The pharmaceutical compositions may also contain one or more active ingredients such as anti-bacterial agents, anti-inflammatories, anesthetics, and the like.

[0206] 222. The pharmaceutical composition can be administered in a variety of ways, depending on whether local or systemic treatment is desired, and the area being treated. Administration may be local (including ophthalmic, vaginal, rectal, nasal), oral, inhalation, or parenteral, e.g. by intravenous infusion, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracisternally, or transdermally.

[0207] 223. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including salt solutions and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present,

Such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases and the like.

[0208] 224. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oil substrates, thickeners, etc. may be necessary or desired.

[0209] 225. Compositions for oral administration include powders or granules, suspensions or solutions in water or nonaqueous media, capsules, bags, or tablets. Thickeners, flavors, diluents, emulsifiers, dispersing agents or binders may be desirable.

[0210] 226. Some of the compositions can potentially be administered as a pharmaceutically acceptable acid or base addition salt formed by reaction with inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanate acid, sulfuric acid, and acid. phosphoric acid, and organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base, such like sodium hydroxide, ammonium hydroxide, potassium hydroxide, and with organic bases such as mono-, di-, trialkyl- and aryl-amines and substituted ethanolamines.

(2) Therapeutic Uses [0211] 227. Effective dosages and schedules for administering the compositions can be determined empirically, and making such determinations is within the skill of the skilled person. Dosage ranges for the administration of compositions are ranges large enough to produce the desired effect that affects the symptoms of the disorder. The dosage should not be so great as to cause harmful side effects, such as undesirable cross reactions, anaphylactic reactions, and the like. Generally, the dosage will vary depending on the age, condition, sex and degree of the disease in the patient, the route of administration, or whether other drugs are included in the treatment regimen, and can be determined by a person skilled in the art. Dosage can be selected by an individual doctor in case of any contraindications. Dosage may change, and can be administered in one or more doses of a day, for one or several days. The literature provides tips for dosages appropriate for given classes of pharmaceutical products. For example, guidance on the selection of appropriate doses of antibodies can be found in the literature

- 61 therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., Ed., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and pp. 303-357; Smith et al.

Antibodies in Human Diagnosis and Therapy, Haber et al., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone can range from about 1 mg / kg to up to 100 mg / kg of body weight or more per day, depending on the factors mentioned above.

b) Targeted delivery [0212] 228. The disclosed liposomes and microcapsules can be targeted to a particular cell type, such as islet cells, via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology for targeting specific tissue (Senter, et al., Bioconjugate Chem 2: 447-51, 1991; Bagshawe, Br J Cancer 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., Biochem Pharmacol

42: 2062-5, 1991). These techniques can be used for a variety of other specific cell types.

8. Foodstuffs [0213] 229. Disclosed herein are also foodstuffs comprising any of the microcapsules and emulsions disclosed herein. By "foodstuff" is meant any product that can be consumed (e.g., eaten, drunk, or taken) by the person being treated. In one aspect, the microcapsules can be used as nutritional supplements for the foodstuff. For example, microcapsules and emulsions can be filled with vitamins, omega-3 fatty acids, and other compounds that provide health benefits. In one aspect, the foodstuff means baking, pasta, meat product, frozen dairy product, milk product, cheese product, egg product, spice, soup mix, snack, nut product, vegetable protein product, hard candy , soft candy, poultry product, processed fruit juice, granulated sugar (eg, white or brown), sauce, gravy, syrup, nutritional bar, drink, dry powder for making a drink, jam or jelly, fish product, or pet food. In another aspect, the foodstuff means bread, tortillas, grain product, sausage, hen, ice cream, yogurt, milk, salad dressing, rice bran, fruit juice, dry powder for making a drink, rolls, biscuits, crackers, snacks, cakes with fruit, or cake.

9. Integrated circuits and chips [0214] 230. Integrated circuits are disclosed wherein at least one address is the sequence or part of the sequences depicted in any of the nucleic acid sequences disclosed herein. Also disclosed are integrated circuits, wherein at least one address is the sequence or part of the sequences depicted in any of the peptide sequences disclosed herein.

[0215] 231. Also disclosed are integrated circuits, wherein at least one address is a variant sequence or portion of the sequences provided in any of the nucleic acid sequences disclosed herein. Also disclosed are integrated circuits, wherein at least one address is a variant sequence or portion of the sequences provided in any of the peptide sequences disclosed herein.

10. Information media in computer-readable form [0216] 232. It is understood that the disclosed nucleic acids and proteins can be represented as a sequence consisting of nucleotides of amino acids. There are various ways to display these sequences, for example the guanosine nucleotide can be represented as G or g. Similarly, the valine amino acid can be represented as Val or V. Those skilled in the art understand how to show and express any nucleic acid or protein sequence in any of the various existing ways, any of which is considered disclosed herein. It is in detail contemplated to show these sequences on computer-readable information carriers, such as commercially available floppy disks, tapes, integrated circuits, hard disks, compact discs, and video discs, or other information media in a computer-readable form. The representations of the disclosed sequences in the binary code are also disclosed. Those skilled in the art understand which information carriers are computer-readable. Thus, information mediums in a computer-readable form on which nucleic acid or protein sequences are stored, stored, or recorded.

[0217] 233. Computer-readable information mediums are disclosed comprising sequences and sequence information provided herein.

11. Kits [0218] 234. We now disclose kits that include reagents that can be used in the performance of the methods disclosed herein or in the use or maintenance of the compositions disclosed herein. Kits can contain any reagent

- 63 or a combination of reagents discussed herein, or which would be considered useful or beneficial in the practice of the methods disclosed. For example, the kits could contain one or more eukaryotic microarrays disclosed herein together with, for example, maintenance media. The kits may also contain, for example, lipids or antioxidants, together with agents for their use or administration.

12. Compositions with similar functions [0219] 235. It is understood that the compositions disclosed herein have certain functions such as the production of certain ratios of lipids. Certain structural, genetic, and functional requirements for performing the disclosed functions are disclosed herein, and it is understood that there are various structures, genetic backgrounds, and functional backgrounds that can perform the same function that are related to the disclosed structures, and that these structures they will eventually achieve the same result, for example the production of a certain portion of lipids.

D. Methods of Making Compositions [0220] 236. The compositions disclosed herein and the compositions necessary to perform the disclosed methods can be prepared using any method known to those skilled in the art for that particular reagent or compound, unless specifically stated otherwise.

1. Nucleic Acid Synthesis [0221] 237. For example, nucleic acids, such as oligonucleotides for use as primers, may be made using standard methods of chemical synthesis or may be prepared using enzymatic methods or any other known method. Such methods may range from normal enzymatic digestion to subsequent secretion of nucleotide fragments (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) chapters 5, 6) purely synthetic, for example, a cyanoethyl phosphoramidate method using a Milligen or Beckman System 1Plus DNA synthesizer (e.g., a Model 8700 automated synthesizer from Milligen-Biosearch, Burlington, MA or ABI Model 380B). Synthetic methods useful for the production of oligonucleotides are also described by Ikuta et al., Ann. Rev. Biochem. 53: 323-356, 1984, (phosphotriester and phosphine-ester methods), and Narang et al., Methods Enzymol., 65: 610-620, 1980, (phosphotriester method).

Peptide nucleic acid molecules can be produced using known methods such as those described by Nielsen et al., Bioconjug. Chem. 5: 3-7, 1994.

2. Peptide synthesis

[0222] 238. One method of producing the disclosed proteins is to combine two or more peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be synthesized chemically using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) groups. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily recognize that a peptide or polypeptide corresponding to the disclosed proteins, for example, can be synthesized by normal chemical reactions. For example, a peptide or polypeptide may be synthesized on a resin and not cleaved from it, while another peptide or protein fragment may be synthesized and subsequently cleaved from the resin, thereby exposing the final group, which on the previous fragment is blocked functionally. By the condensation reactions of the peptides, the two fragments can be covalently linked via a peptide bond at their carboxyl and amino terminus respectively to give the antibody, or a fragment thereof. (Grant GA (1992) Synthetic Peptides: A User Guide WH Freeman and Co., NY (1992); Bodansky M and Trost

B., ed. (1993) Principles of Peptide Synthesis. Springer-Verlag Inc., NY (which is hereby incorporated by reference to at least the material relating to the synthesis of peptides). Alternatively, the peptide or polypeptide is synthesized independently in vivo as described herein. Once isolated, these independent peptides or polypeptides can be linked to form a peptide or fragment thereof by similar peptide condensation reactions.

[0223] 239. For example, enzymatic ligation of cloned or synthetic peptide segments enables the linkage of relatively short peptide fragments to produce larger fragments of peptides, polypeptides or entire protein domains (Abrahmsen L et al., Biochemistry, 30: 4151, 1991). Alternatively, the native chemical ligation of synthetic peptides can be used to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two-step chemical reaction (Dawson et al. Science, 266: 776-779, 1994). The first step is the chemoselective reaction of an unprotected synthetic peptide thioester with another unprotected peptide segment containing an amino-terminal Cys residue to form a thioester linked intermediate as the initial covalent product.

[0224] 240. Alternatively, unprotected peptide segments are chemically combined where the linkage created between the peptide segments by chemical ligation is an unnatural linkage (non-peptide) (Schnolzer, M. et al., Science, 256: 221, 1992). This technique was used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins having full biological activity (de Lisle Milton RC et al., Techniques in Protein Chemistry IV Academic Press, New York, pp. 257-267, 1992).

3. Methods of making the compositions [0225] 241. The methods for preparing the composition as well as the preparation of intermediates leading to the composition are disclosed. For example, eukaryotic microbes are disclosed that can produce desired lipids and antioxidants as well as methods for secretion and purification of desired lipids and antioxidants. There are various ways that can be used to manufacture these compositions, such as synthetic chemical methods and normal molecular biology methods. It is understood that the methods of preparing these and other disclosed compositions are disclosed in detail.

[0226] 242. The cells generated by the cell-transforming method by any nucleic acid are disclosed. Cells produced by the cell transformation method are disclosed using any of the disclosed nucleic acids not found in nature.

[0227] 243. Any of the lipids produced by the disclosed eukaryotic microorganisms are disclosed. Any peptides produced by the process of expressing a peptide in the disclosed organisms are disclosed. Ways of applying the composition.

4. Methods of using the compositions as research tools [0228] 244. The disclosed compositions can be used in various ways as research tools and for the production of, for example, lipids and antioxidants.

[0229] 245. The disclosed compositions can be used as discussed herein as either reagents in microspheres or as reagents for probing or analyzing existing microturbers. The disclosed compositions can be used in any known manner to isolate or identify polymorphisms for a single nucleotide. The compositions may also be used in any method for determining allelic analysis, e.g. strains of the organisms disclosed herein, particularly allelic analysis relating to the production of lipids and antioxidants. The compositions may also be used in any known screening assay method with respect to the modules / chips. The compositions may also be used in any known manner of using readable ones

Computer-aided embodiments of the disclosed compositions, e.g., for linking or for performing molecular modeling analysis associated with the disclosed compositions.

5. Methods of gene modification and gene disruption [0230] 246. The disclosed compositions and methods can be used for targeted disruption of genes and modifications in any animal that may undergo these events. Gene modification and gene disruption relate to methods, techniques, and compositions that surround the selective removal or alteration of a chromosome gene or extension in an organism, such as the eukaryotes disclosed herein, in a manner that promotes modification by replication of an organism. In general, for example, a cell is transformed with a vector that is constructed to homologously recombine with the area of ​​a particular chromosome or nucleic acid contained in a cell, as for example described herein. This act of homologous recombination can produce a chromosome that has introduced exogenous DNA, for example in the reading frame with the surrounding DNA. This type of procedure allows the introduction of very specific mutations into the genome of the cell, such as point mutations. Disclosed herein are methods for performing this type of homologous recombination.

V. SPECIFIC EMBODIMENTS [0231] 247. A eukaryotic microorganism having the sequence 18S is disclosed herein, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. The eukaryotic microorganism may produce unsaturated fatty acids having the profile shown in Figure 2. The eukaryotic microorganism may belong to the Labyrinthulomycota type, the Labyrinthulomycetes group, the Thraustochytridae subgroup, the Thraustochytriales family, the Thraustochytriaceae family, and / or the Thraustochytrium genus.

The eukaryotic microorganism may be some kind of Thraustochytrium,

Thraustochytrium aureum, Thraustochytrium roseum, or Thraustochytrium striatum. The eukaryotic microorganism may also belong to the Thraustochytriaceae family and may have the ATCC accession number 20888, 20889, 20890, 20891, or 20892.

[0232] 248. Also disclosed herein is a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, where the microorganism belongs to the genus Schizochytrium. The eukaryotic microorganism may be some species of Schizochytrium.

[0233] 249. Also disclosed herein is a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, where the eukaryotic microorganism contains omega-3 or omega-6 fatty acid. The eukaryotic microorganism may also contain DHA or DPA.

[0234] 250. Also disclosed herein is a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, wherein the microorganism produces a lipid or fatty acid fraction of at least about 4 wt%. up to 6 wt% The lipid may contain DHA. The lipid composition may also contain from about 25% by weight. fatty acid fractions up to about 40 wt.% the n-3 DHA fatty acid fractions, from about 6 wt.%. fatty acid fractions to about 10 wt.%. n-6 DPA fatty acid fractions, and from about 0 wt.%. fatty acid fractions to about 3 wt.%. of n-3 EPA fatty acid fractions.

[0235] 251. Also disclosed is a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1. The composition may further comprise nutrient medium and / or nutrients.

[0236] 252. Also disclosed is a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, where the composition is biomass. The eukaryotic microorganism in the composition may belong to the Labyrinthulomycota type, the Labyrinthulomycetes group, the Thraustochytridae subgroup, the order Thraustochytriales, the Thraustochytriaceae family, or the Thraustochytrium genus. The eukaryotic microorganism may be Thraustochytrium sp., Thraustochytrium aureum, Thraustochytrium roseum, or Thraustochytrium striatum. The eukaryotic microorganism can also belong to the family

Thraustochytriaceae and may have an ATCC accession number 20888, 20889, 20890, 20891, or 20892.

[0237] 253. Also disclosed is a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, where the microorganism belongs to the genus Schizochytrium.

The eukaryotic microorganism may be Schizochytrium sp.

[0238] 254. Also disclosed is a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, where the eukaryotic microorganism produces unsaturated

Fatty acids having the profile shown in FIG. 2. The unsaturated fatty acid may include omega 3 or omega 6 fatty acids. The unsaturated fatty acid may also include DHA or DPA.

[0239] 255. Also disclosed is a composition comprising a eukaryotic microorganism having an 18S sequence, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, wherein the eukaryotic microorganism can produce a lipid or fatty acid fraction of at least about 4 wt.%. up to 6% weight ..

The lipid may contain DHA. The lipid may also contain from about 25% by weight. fatty acid fractions up to about 40 wt.% the n-3 DHA fatty acid fractions, from about 6 wt.%.

fatty acid fractions to about 10 wt.%. n-6 DPA fatty acid fractions, and from about 0 wt.%. fatty acid fractions to about 3 wt.%. of n-3 EPA fatty acid fractions.

[0240] 256. Also disclosed is a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 80% identity with the sequence set forth in SEQ. [0241] 257. A composition comprising from about 25 wt.% Is also disclosed. fatty acid fractions up to about 40 wt.% the n-3 DHA fatty acid fractions, from about 6 wt.%. fatty acid fractions to about 10 wt.%. n-6 DPA fatty acid fractions, and from about 0 wt.%. fatty acid fractions to about 3 wt.%. of n-3 EPA fatty acid fractions.

[0242] 258. Also disclosed is a method of producing a lipid composition, which method comprises: culturing a eukaryotic microorganism described herein in a heterotrophic medium, and secreting a lipid composition. Also disclosed is a lipid composition produced in accordance with this method.

[0243] 259. Also disclosed is a delivery device comprising any of the compositions described above. For example, a delivery device is disclosed comprising a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the sequence 18S has at least 94% identity with the sequence set forth in SEQ. The delivery device may include a microcapsule, microsphere, nanocube or nanoparticle, liposome, niosome, nanoerythrosome, solid lipid nanoparticle, leuprolide, gel, gel capsule, tablet, liquid, cream, aerosol, emulsion, or powder.

[0244] 260. Also disclosed is a microcapsule, including the focus of primary microcapsules and a filler, wherein each separate primary microcapsule has a primary shell, where the filler comprises any of the above-described compositions, and is encapsulated by the primary shell, and where the focus it is closed by the outer shell.

The primary shell and / or outer sheath may contain a surfactant, gelatin, polyphosphate, polysaccharide, or a mixture thereof. The primary and / or outer sheath may also contain B-type gelatine, polyphosphate, gum arabic, alginate, chitosan, carrageenan, pectin, starch, modified starch, alpha-lactalbumin, beta-lactoglobulin, ovalbumin, polysorbitone, maltodextrin, cyclodextrin, cellulose, methylcellulose, ethylcellulose, hydropropylmethylcellulose, carboxymethylcellulose, milk protein, whey protein, soy protein, low-rapeseed oil, albumin, kosher gelatine, non-kosher gelatin, halal gelatin, non-halal gelatin, or a mixture thereof. The primary shell and / or outer sheath may also contain a complex coacervate, type A gelatine, fish gelatine,

[0245] 261. The filler substance of the microcapsules disclosed may comprise an oil of Thraustochytrium, Schizochytrium, or a mixture thereof. The filling substance may comprise from about 20% to about 90% or 50% to about 70% by weight of the microcapsule.

[0246] 262. The outer shell of the disclosed microcapsules may have an average diameter of from about 1 μm to about 2,000 μm, about 20 μm to about 1,000 μm, about 30 μm to about 80 μm, about 40 nm to about 10 μm, or about 0, 1 μm to about 5 μm.

[0247] 263. Also disclosed is a nutritional supplement that includes any of the compositions, delivery devices, or microcapsules described above. The disclosed nutritional supplements may be in the form of a tablet, gel capsule, capsule, liquid, or syrup.

[0248] 264. Also disclosed is a foodstuff that includes any of the compositions, delivery devices, or microcapsules described above. The food item can be bake, pasta, meat product, frozen dairy product, milk product, cheese product, egg product, seasoning, soup mix, snack, nut product, vegetable protein product, hard candy, soft candy, poultry product, processed fruit juice, granulated sugar, sauce, sauce, syrup, nutritional bar, drink, dry powder for making a drink, jam or jelly, baby formula, or food for

- 70 children. The food item may also be a fish product, pet food, fodder or hydroponic feed. The food item can also be bread, tortillas, grain product, sausage, chicken, ice cream, yogurt, milk, salad dressing, rice bran, fruit juice, dry powder for making a drink, rolls, biscuits, crackers, snacks, fruit cakes, or dough.

[0249] 265. Also disclosed is a method of delivering a composition to a subject, comprising administering to the subject any of the compositions, delivery devices, microcapsules, or foodstuffs described above. The mammal may be treated. People can also be treated.

[0250] 266. Also disclosed is the use of any of the microcapsules described above and for the manufacture of a medicament for delivering a filling substance to the subject.

[0251] 267. Also disclosed is a method for lowering cholesterol levels, triglyceride levels, or a combination thereof in a subject, comprising the step of administering to the subject an effective amount of any of the compositions, delivery devices, microcapsules, nutritional supplements, or foodstuffs described above.

[0252] 268. Also disclosed is a method of topping up the necessary trace elements in a subject, which method comprises administering to the subject an effective amount of any of the compositions, delivery devices, microcapsules, nutritional supplements, or foodstuffs described above, wherein the composition, delivery device, microcapsule , supplement, and foodstuff contains the essential trace element.

[0253] 269. Also disclosed is a method of improving the insulin sensitivity of a subject, comprising the step of administering to the subject an effective amount of any of the compositions, delivery devices, microcapsules, nutritional supplements, or foodstuffs described above.

[0254] 270. Also disclosed is a method of reducing hyperglycemia in a subject, comprising the step of administering to the subject an effective amount of any of the compositions, delivery devices, microcapsules, nutritional supplements, or foodstuffs described above.

[0255] 271. Also disclosed is a method of reducing hypercholesterolemia in a subject, comprising the step of administering to the subject an effective amount of any of the compositions, delivery devices, microcapsules, nutritional supplements, or foodstuffs described above.

[0256] 272. Also disclosed is a method for reducing body fat in a subject, comprising the step of administering to the subject an effective amount of any of the compositions, delivery devices, microcapsules, nutritional supplements, or foodstuffs described above.

[0257] 273. Also disclosed is a method of allowing weight loss in a subject, comprising the step of administering to the subject an effective amount of any of the compositions, delivery devices, microcapsules, nutritional supplements, or foodstuffs described above.

[0258] 274. Also disclosed is a method of treating or preventing diabetes in a subject, comprising the step of administering to the subject an effective amount of any of the compositions, delivery devices, microcapsules, nutritional supplements, or foodstuffs described above.

[0259] 275. Also disclosed is a pharmaceutical formulation comprising any of the compositions, delivery devices, or microcapsules described above, and a pharmaceutical carrier.

[0260] 276. Also disclosed is a foodstuff containing a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO .: 1. The food item may be a bake, pasta, meat product, frozen dairy product, milk product, cheese product, egg product, seasoning, soup mix, snack, nut product, vegetable protein product, hard candy, soft candy, poultry product, processed fruit juice, granulated sugar, sauce, sauce, syrup, nutritional bar, drink, dry powder for making a drink, jam or jelly, baby formula, or baby food. The food item may also be a fish product, pet food, fodder or hydroponic feed.

[0261] 277. Also disclosed is a method for lowering cholesterol levels, triglyceride levels, or a combination thereof in a subject, comprising the step of administering an effective amount of a composition comprising a eukaryotic microorganism having the 18S sequence wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, a nutritional supplement containing a composition comprising a eukaryotic microorganism having

- an 18S sequence, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, a delivery device comprising a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ ID NO: 1. ID NO: 1, or a foodstuff containing a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the sequence 18S has at least 94% identity with the sequence set forth in SEQ. ID NO: 1.

[0262] 278. Also disclosed is a method of supplementing the necessary trace elements in a subject, which method comprises the step of administering an effective amount of a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, a nutritional supplement containing a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ ID NO: 1. ID NO: 1, a delivery device comprising a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ ID NO: 1. ID NO: 1, or a foodstuff containing a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1.

[0263] 279. Also disclosed is a method of improving insulin sensitivity in a subject, comprising the step of administering an effective amount of a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, a nutritional supplement containing a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ ID NO: 1. ID NO: 1, a delivery device comprising a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ ID NO: 1. ID NO: 1, or a foodstuff containing a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1.

[0264] 280. Also disclosed is a method of reducing hyperglycaemia in a subject, comprising the step of administering an effective amount of a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, a nutritional supplement containing the composition

- 73 comprising a eukaryotic microorganism having the sequence 18S, wherein the sequence 18S has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, a delivery device comprising a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ ID NO: 1. ID NO: 1, or a foodstuff containing a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the sequence 18S has at least 94% identity with the sequence set forth in SEQ. ID NO: 1.

[0265] 281. Also disclosed is a method of reducing hypercholesterolemia in a subject, comprising the step of administering an effective amount of a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, a nutritional supplement containing a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ ID NO: 1. ID NO: 1, a delivery device comprising a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ ID NO: 1. ID NO: 1, or a foodstuff containing a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1.

[0266] 282. Also disclosed is a method for reducing body fat in a subject, comprising the step of administering an effective amount of a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the sequence 18S has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, a nutritional supplement containing a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ ID NO: 1. ID NO: 1, a delivery device comprising a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ ID NO: 1. ID NO: 1, or a foodstuff containing a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1.

[0267] 283. Also disclosed is a method of allowing weight loss in a subject, comprising the step of administering an effective amount of a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, a nutritional supplement containing the composition

- 74 comprising a eukaryotic microorganism having the sequence 18S, wherein the sequence 18S has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, a delivery device comprising a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ ID NO: 1. ID NO: 1, or a foodstuff containing a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the sequence 18S has at least 94% identity with the sequence set forth in SEQ. ID NO: 1.

[0268] 284. Also disclosed is a method of treating or preventing diabetes in a subject, comprising the step of administering an effective amount of a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1, a nutritional supplement containing a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ ID NO: 1. ID NO: 1, a delivery device comprising a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ ID NO: 1. ID NO: 1, or food itemcontaining a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1.

[0269] 285. Also disclosed is a pharmaceutical formulation comprising a composition comprising a eukaryotic microorganism having the sequence 18S, wherein the 18S sequence has at least 94% identity with the sequence set forth in SEQ. ID NO: 1.

VI. EXAMPLES [0270] 286. The following examples are provided to provide those skilled in the art with complete disclosure and description of how to formulate and evaluate the compounds, compositions, articles, devices and / or methods claimed herein, and are intended to be exemplary only and are not intended to be limiting. disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., quantity, temperature, etc.), but it should be corrected for some errors and deviations. Unless otherwise indicated, parts are parts by weight, temperature is expressed in ° C or is ambient temperature, and the pressure is atmospheric or near atmospheric.

1. Example 1 Isolation of ONC-T18 strain Thraustochytrium sp.

[0271] 287. Classical bacterial techniques for strain purification were used to isolate ONC-T18 from mangrove leaves harvested in Advocate Harbor, Nova Scotia. ONC-T18 was grown serially at 25 ° C on an agar nutrient medium containing 5 g L^-1 glucose, 2 g L^-1 peptone, 2 g L^-1 yeast extract and 15.0 g L^-1 agar in 1 L of sea water filtered through a 0.2 μm filter to ensure cleanliness. In turn, a liquid medium containing 15% artificial seawater (trophic seawater) was prepared, supplemented with a nitrogen and carbon source, which constituted 60 g L respectively^-1 glucose and 10 g L^-1 yeast extract. This medium (50 ml of medium in 250 ml flasks) was inoculated with ONC-T18, then incubated at 25 ° C and aerated by shaking at room temperature.

120 rpm

[0272] 288. ONC-T18 was separated from the medium by centrifugation, after which the cell biomass was washed, re-centrifuged and lyophilized to completion. The cellular biomass was then weighed to determine the yield of the culture using the recorded biomass values ​​per liter of medium. Extraction of lipid fractions from biomass, and then separation of fatty acid methyl esters was carried out by the Bligh and Dyer method. Transesterification was performed by transferring the lyophilized cell material to a 10 ml tube with a screw cap and adding 10% methanolic HCl and dichloromethane to the tube, the mixture being allowed to react for 2 hours at 90 ° C. Then the fatty acid methyl esters were extracted by adding hexane: chloroform, and methyl esters of the components were measured by gas chromatography (FID) to determine the fatty acid profile for each of the microorganisms and the symbiotic assembly (ONC-T18). The concentrations of each of the fatty acid methyl esters (C14: 0 to C22: 6) were determined by comparing the GC peak areas of the two internal standards (C19: 0 and C23: 0) added in specific amounts both at the beginning (C23: 0) and at the end (C19: 0) transesterification process. The total amount of fatty acids per gram of dried cellular biomass and the percentage of each of the fatty acids calculated using this method are shown in Figure 2. 6) were determined by comparing the GC peak areas of the two internal standards (C19: 0 and C23: 0) added in specified amounts both at the beginning (C23: 0) and at the end (C19: 0) of the transesterification process. The total amount of fatty acids per gram of dried cellular biomass and the percentage of each of the fatty acids calculated using this method are shown in Figure 2. 6) were determined by comparing the GC peak areas of the two internal standards (C19: 0 and C23: 0) added in specified amounts both at the beginning (C23: 0) and at the end (C19: 0) of the transesterification process. The total amount of fatty acids per gram of dried cellular biomass and the percentage of each of the fatty acids calculated using this method are shown in Figure 2.

[0273] 289. From the analysis of these results, in combination with those shown in Figure 1, it can be seen that ONC-T18 has the ability to produce increased amounts of DHA as well as significant amounts of EPA and DPA. In this unoptimised fermentation broth, ONC-T18 produces approximately 25% DHA, 8.0% (n-6) DPA and 1.0% EPA. Then ONCT18 was selected on the basis of the combination of economically desirable features: (1) have the ability to maximize heterotrophic growth (as compared to control strains); (2)

- 76 contain a high percentage of highly unsaturated omega 3 fatty acids; (3) have the ability to grow on cheap nutrients; (4) show thermotolerance, and are (5) euryhalic.

[0274] 290. Furthermore, many different strains of 5 oil-producing microorganisms were compared to the ONC-T18 strain. It is believed that each of these microorganisms is

Thraustochytridae, and produces oil in the amounts shown in Table 3.

Table 3. [] = mg / g

<td></td><td>[J. OM Α</td><td>[1 ΕΡΑ</td><td>lipids together</td><td>Weight &lt;9&gt;</td>

<td>IV! ΥΧΧ-1 38-1</td><td>1 27.96</td><td>5.52</td><td>21 Ο 37</td><td>1 80</td>

<td>ATCC-2O891</td><td>37 97</td><td>7.14</td><td>07 34</td><td>1 30</td>

<td>ONC-T01</td><td>5.18</td><td>7 7 (5th</td><td>50.82</td><td>0 40</td>

<td>ONC-T02</td><td>3 1 84</td><td>4.20</td><td>52 88</td><td>0.50</td>

<td>ONC ΤΟ3</td><td>24 87</td><td>(5 97</td><td>75.00</td><td>Ο 60</td>

<td>ONC-T04</td><td>1 4.39</td><td>4 4 9</td><td>4 1 74</td><td>0.90</td>

<td>ONC-T05</td><td>1 1.37</td><td>3.97</td><td>34 89</td><td>Ο 1 Ο</td>

<td>ONC-TOG</td><td>27.80</td><td>0.7 1</td><td>03.87</td><td>Ο. ΙΟ</td>

<td>ONC-T07</td><td>33.02</td><td>5.49</td><td>(51 81</td><td>0.50</td>

<td>ONC-T08</td><td>24.48</td><td>4.83</td><td>53.35</td><td>0.80</td>

<td>ONC-T09</td><td>63.82</td><td>4.25</td><td>109.12</td><td>0.80</td>

<td>ONC-T10</td><td>22.22</td><td>4.93</td><td>40.99</td><td>0.10</td>

<td>0NC-T11</td><td>18.37</td><td>21.25</td><td>214.98</td><td>0.80</td>

<td>0NC-T12</td><td>57.96</td><td>9.03</td><td>96.26</td><td>0.60</td>

<td>ONC-T13</td><td>12.90</td><td>4.57</td><td>39.52</td><td>1.30</td>

<td>0NC-T14</td><td>15.99</td><td>5.16</td><td>36.46</td><td>0.50</td>

<td>0NC-T15</td><td>15.53</td><td>5.11</td><td>37.72</td><td>0.50</td>

<td>0NC-T16</td><td>18.02</td><td>5.55</td><td>42.12</td><td>0.50</td>

<td>0NC-T17</td><td>36.43</td><td>4.34</td><td>94.26</td><td>0.30</td>

<td>0NC-T18</td><td>83.63</td><td>2.76</td><td>321.14</td><td>2.30</td>

<td>0NC-T19</td><td>34.71</td><td>8.07</td><td>66.14</td><td>0.60</td>

<td>ONC-T20</td><td>19.28</td><td>6.94</td><td>66.74</td><td>0.10</td>

<td>0NC-T21</td><td></td><td></td><td></td><td></td>

<td>ONC-T22</td><td>22.72</td><td>3.26</td><td>47.58</td><td>0.60</td>

<td>ONC-T23</td><td></td><td></td><td></td><td></td>

<td>ONC-T24</td><td>11.73</td><td>3.56</td><td>33.56</td><td>0.70</td>

<td>ONC-T25</td><td>26.99</td><td>6.11</td><td>45.67</td><td>0.60</td>

<td>ONC-T26</td><td>14.50</td><td>6.43</td><td>39.22</td><td>0.60</td>

<td>ONC-T27</td><td>26.83</td><td>7.75</td><td>61.87</td><td>0.70</td>

<td>ONC-T28</td><td>16.62</td><td>6.02</td><td>38.28</td><td>0.90</td>

<td>ONC-T29</td><td>14.67</td><td>4.91</td><td>34.48</td><td>0.80</td>

<td>ONC-T30</td><td>16.56</td><td>5.42</td><td>81.88</td><td>0.80</td>

<td>0NC-T31</td><td>13.36</td><td>5.74</td><td>44.86</td><td>0.30</td>

<td>ONC-T32</td><td>19.12</td><td>6.56</td><td>53 29</td><td>0.20</td>

<td>ONC-T33</td><td colspan="2"></td><td></td><td></td>

<td>ONC-T34</td><td>18.92</td><td>5.98</td><td>53 36</td><td>0.60</td>

<td>ONC-T35</td><td></td><td></td><td></td><td></td>

<td>ONC-T36</td><td colspan="2"></td><td></td><td></td>

<td>ONC-T37</td><td>35.69</td><td>11.06</td><td>82.73</td><td>0.10</td>

<td>ONC-T38</td><td>22.73</td><td>10.94</td><td>51.56</td><td>0.10</td>

<td>ONC-T39</td><td colspan="2"></td><td></td><td></td>

<td>ONC-T40</td><td>26.87</td><td>8.83</td><td>67 87</td><td>0.80</td>

<td>0NC-T41</td><td>22.85</td><td>6.65</td><td>52.63</td><td>0.50</td>

<td>ONC-T42</td><td>33.65</td><td>9.22</td><td>83.93</td><td>0.80</td>

<td>ONC-T43</td><td>12.49</td><td>3.25</td><td>37.93</td><td>0.80</td>

<td>ONC-T44</td><td>11.71</td><td>2.93</td><td>55.05</td><td>1.10</td>

<td>ONC-T45</td><td>26.08</td><td>7.95</td><td>70.45</td><td>0.70</td>

<td>ONC-T46</td><td>33.34</td><td>6.27</td><td>63.76</td><td>0.30</td>

<td>ONC-T47</td><td>10.01</td><td>4.77</td><td>68.02</td><td>0.70</td>

<td>ONC-T48</td><td>26.23</td><td>3.95</td><td>69.06</td><td>0.60</td>

<td>ONC-T49</td><td>16.64</td><td>4.89</td><td>39.76</td><td>0.30</td>

<td>ONC-T50</td><td>13-64</td><td>4.56</td><td>40.30</td><td>1.00</td>

<td colspan="5">0NC-T51</td>

<td>ONC-T52</td><td>26.57</td><td>4.55</td><td>41.36</td><td>0.60</td>

<td>ONC-T53</td><td>11.40</td><td>3.56</td><td>29.20</td><td>0.70</td>

<td>ONC-T54</td><td>10.34</td><td>3.18</td><td>29. 31</td><td>0.70</td>

<td>ONC-T55</td><td></td><td></td><td></td><td></td>

<td>ONC-T56</td><td></td><td></td><td></td><td></td>

<td>ONC-T57</td><td></td><td></td><td></td><td></td>

<td>ONC-T58</td><td>10.30</td><td>3.13</td><td>27. ΙΟ</td><td>0.70</td>

<td>ONC-T59</td><td></td><td></td><td></td><td></td>

<td>ONC-T60</td><td>27.71</td><td>7.01</td><td>66.84</td><td>0.30</td>

<td>0NC-T61</td><td>15.72</td><td>5.62</td><td>52.56</td><td>0.40</td>

<td colspan="5">ONC-T62</td>

<td>ONC-T63</td><td>20.17</td><td>8.25</td><td>62.58</td><td>0.60</td>

<td>ONC-T64</td><td>12.16</td><td>2.97</td><td>44.73</td><td>1.10</td>

<td>ONC-T65</td><td></td><td></td><td></td><td></td>

<td>ONC-T66</td><td></td><td></td><td></td><td></td>

<td>And ONC-T67</td><td>23.71</td><td>5.63</td><td>I 43</td><td>.24 |</td><td>0.50 |</td>

<td>| ONC-T68</td><td>22.72</td><td>6.10</td><td>| 41</td><td>.37 |</td><td>0.50</td>

[0275] 291. It is understood that as for ONC-T18, as described herein, a set of microorganisms represented by oil producing capabilities is disclosed, such as, for example, by a percentage of DHA for total oil production, or by complete production of DHA. .

2. Example 2 Identification of the ONC-T18 strain of the eucaryotic Thraustochytrium genus using genetic techniques [0276] 292. Using polymerase chain reaction (PCR) techniques and primers targeting the 18S gene of ribosomal RNA, universal for all eukaryotic species, it was possible to produce PCR products structural genes of the eukaryotic microorganism isolated from ONC-T18 (as for Example 1). The PCR products were then sequenced and designated SEQ. ID NO: 1 for eukaryotic species (see Figure 2).

[0277] 293. Comparison of SEQ. ID NO: 1 with nucleic acid sequences found in the genomic database, GenBank (National Center for Biotechnology Information, National Institute of Health, Bethesda, MD, USA) using the BLAST algorithm (Basic Local Alignment Search Tool) identified SEQ. ID NO .: 1 as the most related to Thraustochytrium striatum [AF265338] (97.5% similarity).

[0278] 294. The BLAST results for the ONC-T18 strain from Thraustochytrium sp. Are shown below.

<td>Sequences that give significant matches:</td><td>Punctation (Bits)</td><td>Value E</td>

<td>g 114279326 | gb | AF265338.1 | Thraustochytrium striatum small subun ...</td><td>2126</td><td>0.0</td>

<td>gi | 50508012 | dbj | AB183657.1 | Thraustochytriidae sp. MBIC11072 gen ...</td><td>2121</td><td>0.0</td>

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

<td>gi | 50508019 | dbj | AB183664.1 | Thraustochytriidae sp. MBIC11093 gen ...</td><td>1828</td><td>0.0</td>

<td>gi 38524571 | dbj | AB126669.1 | Thraustochytrium sp. CHN-1 gene for ...</td><td>1748</td><td>0.0</td>

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

<td>gi | 50508018 | dbj | AB183663.1 | Thraustochytriidae sp. MBIC11092 gen ...</td><td>1257</td><td>0.0</td>

<td>gi | 50508017 | dbj | AB183662.1 | Thraustochytriidae sp. MBIC11091 gen ...</td><td>1257</td><td>0.0</td>

<td>gi | 50508015 | dbj | AB183660.1 | Thraustochytriidae sp. MBIC11084 gen ...</td><td>1255</td><td>0.0</td>

<td>gi | 50508011 | dbj | AB183656.1 | Thraustochytriidae sp. MBIC11070 gen ...</td><td>1255</td><td>0.0</td>

<td>gi | 50508016 | dbj | AB183661.1 | Thraustochytriidae sp. MBIC11086 gen ...</td><td>1249</td><td>0.0</td>

<td>g 115823623 | dbj | AB052555.1 | Schizochytrium sp. KH105 gene for 18 ...</td><td>1245</td><td>0.0</td>

<td>gi | 50508013 | dbj | AB183658.1 | Thraustochytriidae sp. MBIC11075 gen ...</td><td>1227</td><td>0.0</td>

<td>gi | 50508010 | dbj | AB183655.1 | Thraustochytriidae sp. MBIC11067 gen ...</td><td>1213</td><td>0.0</td>

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

<td>g 114279326 | gb | AF265338.1 | AF265338 Thraustochytrium striatum sma ...</td><td>1106</td><td>0.0</td>

<td>gi | 6492308 | gb | AF155209.1 | AF155209 Labyrinthulid quahog parasite ...</td><td>765</td><td>0.0</td>

<td>g 116209570 | gb | AY052644.1 | Labyrinthulid quahog parasite QPX fried ...</td><td>757</td><td>0.0</td>

<td>gi | 9755031 | gb | AF261664.1 | AF261664 | Labyrinthulid quahog parasite ...</td><td>757</td><td>0.0</td>

<td>gi 58176547 | gb | AY870336.1 | Thraustochytriidae sp. Fngl 18S ribos ...</td><td>735</td><td>0.0</td>

<td>gi | 67624914 | dbj | AB191425.1 | Uncultured eukaryote gene for small ...</td><td>724</td><td>0.0</td>

<td>gi | 5509891 | dbj | AB022112.1 | Thraustochytrium striatum gene for 18 ...</td><td>724</td><td>0.0</td>

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

<td>gi | 50508014 | dbj | AB183659.1 | Thraustochytriidae sp. MBIC11077 gen ...</td><td>686</td><td>0.0</td>

<td>gi | 50508008 | dbj | AB183653.1 | Thraustochytriidae sp. MBIC11060 gen ...</td><td>686</td><td>0.0</td>

<td>gi | 50508009 | dbj | AB183654.1 | Thraustochytriidae sp. MBIC11063 gen ...</td><td>658</td><td>0.0</td>

<td colspan="3">Sequences that give significant matches:</td><td>Punctation (Bits)</td><td>Value E</td>

<td>g</td><td colspan="2">41391986 | emb | AJ535188.1 | Pleurosira cf. laevis 18S rRNA gene, ...</td><td>634</td><td>E-178</td>

<td>g</td><td colspan="2">28316562 | gb | AF525670.1 | Pleurosira laevis small subunit ribos ...</td><td>634</td><td>E-178</td>

<td>g</td><td colspan="2">5509889 | dbj | AB022110.1 | Thraustochytrium aureum gene for 18S ...</td><td>634</td><td>E-178</td>

<td>g</td><td colspan="2">561883 | gb | L34668.1 | TUKRRE Thraustochytrium kinnei 18S ribosom ...</td><td>628</td><td>E-176</td>

<td>g</td><td colspan="2">5509894 | dbj | AB022115.1 | Ulkenia radiata gene for 18S rRNA</td><td>624</td><td>E-175</td>

<td>g</td><td colspan="2">5509893 | dbj | AB022114.1 | Ulkenia profunda gene for 18S rRNA</td><td>624</td><td>E-175</td>

<td>g</td><td colspan="2">5509895 | dbj | AB022116.1 | Ulkenia vizurgensis gene for 18S rRNA</td><td>603</td><td>E-169</td>

<td>g</td><td colspan="2">9027563 | gb | AF257315,2 | Thraustochytriidae sp. BS2 18S ribosom ...</td><td>589</td><td>E-164</td>

<td>g</td><td colspan="2">5509886 | dbj | AB022107.1 | Schizochytrium limacinum gene for 18S ...</td><td>581</td><td>E-162</td>

<td>g</td><td>48727879</td><td>gb | AY620254.1 | Metromonas simplex clone TC-S small s ...</td><td>571</td><td>E-159</td>

<td>g</td><td>33309650</td><td>gb | AF411282.1 | Unidentified cercozoan 18S ribosomal ...</td><td>569</td><td>E-158</td>

<td>g^and</td><td>28076844</td><td>gb | AF530543.1 | Uncultured eukaryote clone AT4-68 18S ...</td><td>531</td><td>E-147</td>

<td>g^and</td><td>30144485</td><td>gb | AY256273.1 | Uncultured eukaryote isolate E170 fried ...</td><td>517</td><td>E-143</td>

<td>g^and</td><td>30144529</td><td>gb | AY256317.1 | Uncultured eukaryote isolate D107 fried ...</td><td>507</td><td>E-140</td>

<td>g^and</td><td>14579477</td><td>gb | AF363207.1 | Eukaryote marine clone ME1-24 18S rib ...</td><td>505</td><td>E-139</td>

<td>g^and</td><td>39578677</td><td>gb | AY426906.1 | Uncultured marine eukaryote clone BL0 ...</td><td>504</td><td>E-139</td>

<td>g^and</td><td>39981869</td><td>gb | AY381216.1 | Uncultured eukaryote clone BL010625,3 ...</td><td>504</td><td>E-139</td>

<td>g^and</td><td>73533408</td><td>gb | DQ103811.1 | Uncultured marine eukaryote clone M4 _...</td><td>504</td><td>E-139</td>

<td>g^and</td><td>73533402</td><td>gb | DQ103805.1 | Uncultured marine eukaryote clone M3 ...</td><td>504</td><td>E-139</td>

<td>g^and</td><td>73533389</td><td>gb | DQ103792.1 | Uncultured marine eukaryote clone M2 _...</td><td>504</td><td>E-139</td>

<td>g^and</td><td>73533382</td><td>gb | DQ103785.1 | Uncultured marine eukaryote clone M1 _...</td><td>504</td><td>E-139</td>

<td>g^and</td><td>30144534</td><td>gb | AY256322.1 | Uncultured eukaryote isolate D179 fried ...</td><td>504</td><td>E-139</td>

<td>g^and</td><td>24817738</td><td>dbj | AB073305,2 | Thraustochytriidae sp. H1-14 gene fo ...</td><td>504</td><td>E-139</td>

<td>g^and</td><td>30268157</td><td>emb | AJ519935.1 | AST519935 Aplanochytrium stocchinoi p ...</td><td>504</td><td>E-139</td>

<td>g^and</td><td>58531881</td><td>gb | AY882527.1 | Uncultured marine eukaryote clone T41 ...</td><td>504</td><td>E-139</td>

<td>g^and</td><td colspan="2">463127 | gb | L27634.1 | LADDLRRNA Labyrinthuloides minute 16S-like ...</td><td>504</td><td>E-139</td>

<td>Sequences that give significant matches:</td><td>Punctation (Bits)</td><td>Value E</td>

<td>gi 39981839 | gb | AY381186.1 | Uncultured eukaryote clone OR000415,1 ...</td><td>502</td><td>E-138</td>

<td>gi 39981824 | gb | AY381171.1 | Uncultured eukaryote clone HE001005,1 ...</td><td>502</td><td>E-138</td>

<td>g 118026024 | gb | AY046848.1 | Uncultured eukaryote isolate C3 E019 ...</td><td>502</td><td>E-138</td>

<td>g 118026022 | gb | AY046846.1 | Uncultured eukaryote isolate C3 E017 ...</td><td>502</td><td>E-138</td>

<td>g 118026014 | gb | AY046838.1 | Uncultured eukaryote isolate C3 E008 ...</td><td>502</td><td>E-138</td>

<td>g 118026008 | gb | AY046832.1 | Uncultured eukaryote isolate C3 E002 ...</td><td>502</td><td>E-138</td>

<td>g 118025980 | gb | AY046804.1 | Uncultured eukaryote isolate C2 E014 ...</td><td>502</td><td>E-138</td>

<td>g 118025969 | gb | AY046793.1 | Uncultured eukaryote isolate C2 E002 ...</td><td>502</td><td>E-138</td>

<td>g 118025801 | gb | AY046625.1 | Uncultured eukaryote isolate C1_E024 ...</td><td>502</td><td>E-138</td>

<td>gi | 67624915 | dbj | AB191426.1 | Uncultured eukaryote gene for small ...</td><td>502</td><td>E-138</td>

<td>gi | 67624913 | dbj | AB191424.1 | Uncultured eukaryote gene for small ...</td><td>502</td><td>E-138</td>

<td>gi | 67624912 | dbj | AB191423.1 | Uncultured eukaryote gene for small ...</td><td>502</td><td>E-138</td>

<td>gi 39981861 | gb | AY381208.1 | Uncultured eukaryote clone BL010320,1 ...</td><td>500</td><td>E-138</td>

<td>g 114349249 | dbj | AB052556.1 | Thraustochytrium sp. KK17-3 gene for ...</td><td>500</td><td>E-138</td>

<td>gi | 20218962 | dbj | AB073307.1 | Thraustochytriidae sp. M4-103 gene f ...</td><td>498</td><td>E-137</td>

<td>gi | 59709960 | gb | AY916582.1 | Uncultured eukaryote clone Zeuk76 18S ...</td><td>496</td><td>E-136</td>

<td>g 118025960 | gb | AY046784.1 | Uncultured eukaryote isolate A3 E043 ...</td><td>496</td><td>E-136</td>

<td>g 118025789 | gb | AY046613.1 | Uncultured eukaryote isolate C1 E009 ...</td><td>496</td><td>E-136</td>

<td>gi | 30144548 | gb | AY256336.1 | Uncultured eukaryote isolate D278 fried ...</td><td>496</td><td>E-136</td>

<td>gi | 2138106 | gb U59933,1 U59933 Scybalium jamaicense 18S ribosomal ...</td><td>496</td><td>E-136</td>

<td>gi 53828186 | gb | AY744948.1 | Phytophthora palmivora isolate 88108 ...</td><td>494</td><td>E-136</td>

<td>gi | 60687349 | gb | AY821976.1 | Uncultured oomycete clone CV1 B2 5 sm ...</td><td>494</td><td>E-136</td>

<td>gi | 60687347 | gb | AY821974.1 | Uncultured Phytophthora-like oomycete ...</td><td>494</td><td>E-136</td>

<td>gi | 60687342 | gb | AY821969.1 | Uncultured oomycete clone CV1 B149 s ...</td><td>494</td><td>E-136</td>

<td>gi 39981870 | gb | AY381217.1 | Uncultured eukaryote clone BL010625,3 ...</td><td>494</td><td>E-136</td>

<td>gi 39981864 | gb | AY381211.1 | Uncultured eukaryote clone BL010320,2 ...</td><td>494</td><td>E-136</td>

<td colspan="2">Sequences that give significant matches:</td><td>Punctation (Bits)</td><td>Value E</td>

<td>g^and</td><td>39981860 | gb | AY381207.1 | Uncultured eukaryote clone BL010320,6 ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>39981844 | gb | AY381191.1 | Uncultured eukaryote clone BL000921,1 ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>18026046 | gb | AY046870.1 | Uncultured eukaryote isolate C3 E044 ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>18026039 | gb | AY046863.1 | Uncultured eukaryote isolate C3 E035 ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>18026031 | gb | AY046855.1 | Uncultured eukaryote isolate C3 E026 ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>42412527 | gb | AY486144.1 | Pythium insidiosum 18S ribosomal RNA ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>73533425 | gb | DQ103828.1 | Uncultured marine eukaryote clone M2 ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>34576227 | gb | AY129064.1 | Uncultured marine eukaryote UEPAC45p4 ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>30144522 | gb | AY256310.1 | Uncultured eukaryote isolate D85 smal ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>30144521 | gb | AY256309.1 | Uncultured eukaryote isolate D84 smal ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>30144518 | gb | AY256306.1 | Uncultured eukaryote isolate D79 smal ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>30144475 | gb | AY256263.1 | Uncultured eukaryote isolate E106 fried ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>30144473 | gb | AY256261.1 | Uncultured eukaryote isolate E94 smal ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>21954246 | gb | AY116220.1 | Uncultured eukaryote clone ANT12-26 1 ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>41393027 | emb | AJ535176.1 | LMI535176 Leptocylindrus minimum 18S ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>53693111 | gb | AY742743.1 | Phytophthora tropicalis isolate 129F -...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>53693108 | gb | AY742759.1 | Pythium vexans isolate Pyv6-2 18S rib ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>53693105 | gb | AY742756.1 | Pythium splendens isolate 117 18S rib ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>53693104 | gb | AY742755.1 | Pythium aphanidermatum 18S ribosomal ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>53693097 | gb | AY742748.1 | Phytophthora capsici isolate 98110 18 ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>53693096 | gb | AY742747.1 | Phytophthora tropicalis isolate 23047 ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>53693094 | gb | AY742745.1 | Phytophthora palmivora isolate 8829 1 ...</td><td>494</td><td>E-136</td>

<td>g^and</td><td>58531862 | gb | AY882508.1 | Uncultured marine eukaryote clone T53 ...</td><td>494</td><td>E-136</td>

3. Example 3: Optimized biomass production using the ONC-T18 strain [0279] 295. The production of oils derived from microorganisms (or individual cells) depends on various process parameters, such as the initial inoculum amount, type

- media, medium composition, temperature and pH. Specifically, the production of highly unsaturated fatty acids by microorganisms using strains of the Thraustochytridae subgroup shows a direct correlation between the production of biomass and fatty acids. As a result, understanding basic needs or optimizing parameters is an important factor for achieving the highest performance. Therefore, in order to determine the best medium for the production of increased amounts of fatty acids, initial biomass optimization experiments were undertaken. In particular, the recently developed method of Taguchi (Joseph J and Piganatiells JR, IIE Trans 20: 247-254, 1998), based on orthogonal systems, was used to determine the optimal medium configuration for increased optical density (directly related to the production of biomass). In this case, the Taguchi method was used to gain an understanding of the collective influences of variables that affect the production of biomass. Influences of changes in the amount of nitrogen (yeast extract, peptone, L-glutamate), carbon (glucose) and salt concentration (artificial sea salt) on the production of biomass. Thus, various liquid media were prepared with varying amounts of yeast extract, peptone and L15 glutamate (0, 4, 10, 20, 50 g L^-1) for variable amounts of glucose and sea salt solution (5, 40, 100, 160, 200 g L respectively^-1 and 0, 6, 20, 30, 40 g L^-1). Concentrations were calculated according to the orthogonal table L²⁵ in such a way that the chosen nitrogenous medium can be distinguished by applying the signal to noise ratio analysis (SNL) after 48 and 120 hours, using the following formula:

where n = number of levels and y = efficiency (mean ODD600 of three replicate experiments).

[0280] 296. The results of these experiments (which specifically address biomass issues), as shown in Figure 2 below, showed that the ONC-T18 nitrogen utilization rate with respect to optical density (OD600) had peptone, then yeast extract, and then L-glutamate. However, based on the growth maxima, the best source of nitrogen for increasing biomass production was the yeast extract, then peptone followed by L-glutamate. In addition, by using similar experiments with changes in glucose and salinity (sea salt concentration), the optimal and the cheapest composition of the ONC-T18 biomass production medium was the medium containing 2 g L^-1 yeast extract, 8 g L^-1 MSG, 60 g L^-1 glucose and 6 g L^-1 sea ​​salt.

<img file="PL2447356T3_D0001.tif" />

4. Example 4: Optimized production of docosahexaenoic acid (DHA) by strain ONC-T18 [0281] 297. Mediums produced from a nitrogen source (peptone, yeast extract, Lglutamate (MSG) or a combination thereof) and a carbon (glucose) source were produced. , in a saline solution (artificial sea water), to determine the best medium composition for optimal biomass and DHA production in a similar manner to that described in Example 3 (shown in Table 4). After culturing at 25 ° C at 130 rpm for 3 days, biomass, total fatty acids per liter of nutrient solution, percentage of fatty acids by weight, percentage of DHA in fatty acids together and amount of DHA per liter medium was determined by gas chromatography as in the method described in Example 1, and hereby, and shown in

Table 4 below.

[0282] 298. In this case, DHA was confirmed by comparison with known DHA standards using gas chromatography, mass spectrometry and peak blocking methods. Findings from the O-package of experiments, where changes in both natural and organic forms of nitrogen were investigated, showed that the optimal composition of the medium should contain

4.0 and 6.0 g L^-1 both yeast extract and L-glutamate for optimal biomass and DNA production. On the other hand, the package of experiments, which studied the changes in the composition added to the sodium medium, showed optimal DHA production and biomass production when using artificial sea salt. In addition, the pakiet trial package in which the sodium concentration in the medium was changed represented maxima for the production of DHA and biomass between 5 and 15% artificial seawater L^-1 dH2O. The results from the O-package of experiences, where changes in glucose levels were assessed, showed that the range was from 40 to less than 160 g L^-1 glucose translated into the optimal production of biomass and DHA. Finally, the results of the experimental package Θ indicate that the ONC-T18 strain produced equivalent values ​​for both cellular biomass and DHA concentrations when glucose or glycerol was used as the carbon source.

- Table 4: Results of DHA production optimization experiments with respect to changes in nutrient compositions.

<td rowspan="2">-</td><td rowspan="2">No. exp in.</td><td colspan="2">Coal sources</td><td colspan="2">Types of salt</td><td colspan="2">Sources of nitrogen</td><td rowspan="2">Total fatty acids (g L-1)</td><td rowspan="2">Percentage of fatty acids (% by weight)</td><td rowspan="2">Percentage of DHA (% by weight)</td><td rowspan="2">Amount of DHA (g L-1)</td>

<td>Used source coal</td><td>Added amount (g L-1)</td><td>Used blend salt</td><td>added quantity (% salinity)</td><td>Yeast extract (g L-1)</td><td>MSG (g L-1)</td>

<td>ο</td><td>401</td><td>Glucose</td><td>60.0</td><td>Sea salt</td><td>15.0</td><td>10.0</td><td>0.0</td><td>7.7</td><td>34.53</td><td>20.20</td><td>1.5579</td>

<td></td><td>402</td><td>Glucose</td><td>60.0</td><td>Sea salt</td><td>15.0</td><td>8.0</td><td>2.0</td><td>10.0</td><td>44,01</td><td>17,49</td><td>1.7156</td>

<td></td><td>403</td><td>Glucose</td><td>60.0</td><td>Sea salt</td><td>15.0</td><td>6.0</td><td>4.0</td><td>11.5</td><td>50.69</td><td>16.23</td><td>1.8624</td>

<td></td><td>404</td><td>Glucose</td><td>66.0</td><td>Sea salt</td><td>15.0</td><td>4.0</td><td>6.0</td><td>16.9</td><td>69.07</td><td>24.19</td><td>4.0877</td>

<td></td><td>405</td><td>Glucose</td><td>60.0</td><td>Sea salt</td><td>15.0</td><td>2.0</td><td>8.0</td><td>21.3</td><td>81.73</td><td>20.99</td><td>4.4752</td>

<td></td><td>406</td><td>Glucose</td><td>60.0</td><td>Sea salt</td><td>15.0</td><td>0.0</td><td>10.0</td><td>0.15</td><td>1.97</td><td>28.81</td><td>0.0426</td>

<td rowspan="3">θ</td><td>407</td><td>Glucose</td><td>60.0</td><td>Sea salt</td><td>15.0</td><td>10.0</td><td>0.0</td><td>13.2</td><td>58.92</td><td>31.44</td><td>4.1362</td>

<td>408</td><td>Glucose</td><td>60.0</td><td>NaCl</td><td>15.0</td><td>10.0</td><td>0.0</td><td>9.6</td><td>63.75</td><td>38.45</td><td>3.7009</td>

<td>409</td><td>Glucose</td><td>60.0</td><td>NaSO 4</td><td>15.0</td><td>10.0</td><td>0.0</td><td>0.05</td><td>1.41</td><td>20.14</td><td>0.0109</td>

<td rowspan="2">©</td><td>410</td><td>Glucose</td><td>60.0</td><td>Sea salt</td><td>5.0</td><td>10.0</td><td>0.0</td><td>14.6</td><td>59.23</td><td>31.44</td><td>4.6004</td>

<td>411</td><td>Glucose</td><td>60.0</td><td>Sea salt</td><td>15.0</td><td>10.0</td><td>0.0</td><td>10.8</td><td>51.01</td><td>26.17</td><td>2.8144</td>

<td rowspan="2"></td><td rowspan="2">No. exp in.</td><td colspan="2">Coal sources</td><td colspan="2">Types of salt</td><td colspan="2">Sources of nitrogen</td><td rowspan="2">Total fatty acids (g L-1)</td><td rowspan="2">Percent acids fatty (wt%)</td><td rowspan="2">Content percentage DHA (wt%)</td><td rowspan="2">Amount of DHA (g L-1)</td>

<td>Used source coal</td><td>added quantity (g L-1)</td><td>Used blend salt</td><td>added quantity (% salinity)</td><td>Extract yeast (g L-1)</td><td>MSG (g L-1)</td>

<td rowspan="4"></td><td>412</td><td>Glucose</td><td>60.0</td><td>Sea salt</td><td>37.5</td><td>10.0</td><td>0.0</td><td>15.9</td><td>69.32</td><td>25.32</td><td>4.0194</td>

<td>413</td><td>Glucose</td><td>60.0</td><td>Sea salt</td><td>75.0</td><td>10.0</td><td>0.0</td><td>10.8</td><td>61.02</td><td>25.25</td><td>2.7356</td>

<td>414</td><td>Glucose</td><td>60.0</td><td>Sea salt</td><td>100.0</td><td>10.0</td><td>0.0</td><td>11.8</td><td>68.21</td><td>24.02</td><td>2.8290</td>

<td>415</td><td>Glucose</td><td>60.0</td><td>Sea salt</td><td>125.0</td><td>10.0</td><td>0.0</td><td>11.2</td><td>59.63</td><td>22,56</td><td>2.5256</td>

<td rowspan="3">Θ</td><td>416</td><td>Glucose</td><td>5.0</td><td>Sea salt</td><td>15.0</td><td>10.0</td><td>0.0</td><td>0.63</td><td>5.21</td><td>29.18</td><td>.1844</td>

<td>417</td><td>Glucose</td><td>20.0</td><td>Sea salt</td><td>15.0</td><td>10.0</td><td>0.0</td><td>4.06</td><td>29,59</td><td>24.01</td><td>.9752</td>

<td>418</td><td>Glucose</td><td>40.0</td><td>Sea salt</td><td>15.0</td><td>10.0</td><td>0.0</td><td>9.91</td><td>59.39</td><td>23.88</td><td>2.3665</td>

<td rowspan="3"></td><td>419</td><td>Glucose</td><td>60.0</td><td>Sea salt</td><td>15.0</td><td>10.0</td><td>0.0</td><td>10.76</td><td>51.01</td><td>26.17</td><td>2.8144</td>

<td>420</td><td>Glucose</td><td>100.0</td><td>Sea salt</td><td>15.0</td><td>10.0</td><td>0.0</td><td>12.79</td><td>69,50</td><td>31.55</td><td>4.0344</td>

<td>421</td><td>Glucose</td><td>160.0</td><td>Sea salt</td><td>15.0</td><td>10.0</td><td>0.0</td><td>1.00</td><td>9.40</td><td>30,01</td><td>.3013</td>

<td rowspan="2">θ</td><td>422</td><td>Glucose</td><td>5.0</td><td>Sea salt</td><td>15.0</td><td>10.0</td><td>0.0</td><td>0.62</td><td>12.74</td><td>29.86</td><td>.1866</td>

<td>423</td><td>Glycerol</td><td>5.0</td><td>Sea salt</td><td>15.0</td><td>10.0</td><td>0.0</td><td>0.52</td><td>18.84</td><td>35.07</td><td>.1836</td>

5. EXAMPLE 5: Optimal collection time ONC-T18 for maximum DHA production [0283] 299. ONC-T18 was grown with medium composition and same conditions as shown in Example 1. In this particular case, the time when ONC- T18 should be collected to obtain maximum amounts of DHA, DPA and EPA, also taking into account the time necessary for the amounts mentioned (see Figure 3).

[0284] 300. Experimental results with respect to time showed that the optimal time to collect ONC-T18 for optimal DHA production in the flask and bioreactor varied between 3 and 5 days, respectively.

6. Example 6: Analysis of lipids derived from ONC-T18 [0285] 301. With ONC-T18, the lipid fraction was extracted together using the modified method of Bligha and Dyera. Specifically 2.0 g of dried cellular biomass was irrigated overnight at 4 ° C in 8 ml distilled H2O. To the mixture was added 30 ml of methanol: chloroform (2: 1 v) and shaken gently at 120 rpm for 20 min, and the obtained supernatant was decanted. The pellet was then resuspended in methanol: chloroform: H2O (2: 1: 0.8 vol.) And the process repeated, the supernatants pooled together and transferred to a separatory funnel. Then, 5 ml of chloroform and 5 ml of H2O were added to the separator, resulting in the formation of a two-phase liquid system. After vigorous stirring in the separating funnel, the chloroform layer was removed, concentrated under a N2 gas shield,

A plurality of 1 μl of the lipid fraction was plotted onto several Chromarods chromatography bars together, separated and analyzed using the Iatroscan MK6 TLC / FID instrument.

[0286] 302. Analysis of the results shows that the fatty acid component that the ONC-T18 strain produces under heterotrophic fermentation conditions is almost the triglyceride compound (at least 95%). In addition to the neutral fatty acid fraction mentioned above, ONC-T18 also produces a distinct fraction of carotenoids and phospholipids. At the subsequent isolation of the phospholipid fraction, first by burning 50%, and then 75% followed by separation with solvents, it was established that a large and complex fraction of phospholipids was present. The results showed in the sample the presence of components of phosphatidylcholine, phosphatidylserine and phosphatidic acid.

7. Example 7: Preparation of antioxidants using the ONC-T18 strain [0287] 303. Eucarionta ONC-T18 was cultured using the conditions and medium as mentioned previously. The obtained cell biomass after heterotrophic fermentation is harvested by centrifugation, filtration or sedimentation. Cells were collected by centrifugation

- 87 at 3800 xg and washed with phosphate buffered saline. The cell biomass (fresh or lyophilized) was resuspended in 10 volumes of acetone, stirred for 5 minutes at 200 rpm, centrifuged at 3,800 xg for 5 minutes and concentrated to dryness by evaporation under a cover of N2. The pigments were then directly resuspended in the least amount of 10% acetone in hexane and stored at -20 ° C until analysis by HPLC. Then, the identification of carotenoid extracts was carried out on an Agilent 1100 HPLC instrument (Agilent, Palo Alto, CA, USA) equipped with a variable wavelength detector set at 470 nm. Samples were injected through a Symmetry C18 pre-column (Waters, Milford, IA, USA) onto a Bondclone C18 reversed phase column (Phenomenex, Torrance, CA, USA, 10 Lim particles, 3.9 x 300 mm internal diameter). The injection volume was 10 μl, and a flow rate of 1.00 ml / min of 10% acetone in hexane over a period of 25 minutes was used. The quantitative data of carotenoids was based on a comparison of the peak field with known standards (in this case: astaxanthin, canthaxanthin, β-cryptoxanthin, zeaxanthin, echinenone and β-carotene, ChromaDex, Santa Ana, CA, USA). In the absence of a known standard, as in the case of the carotenoid of phycixxanthin, the astaxanthin peak field was used to calculate its concentrations. The identity of the carotenoids was further confirmed using HPLC-MS, using a Waters HPLC chromatograph equipped with a photodiodes kit (Waters model 996), leading to a Micromass ESI-Q-Tof mass spectrometer (Waters, Milford, MA, USA). HPLC analysis of ONC-T18 in turn revealed the presence of several antioxidant compounds (between 50 to 1250 mg kg 00 ml / min 10% acetone in hexane over a period of 25 minutes. The quantitative data of carotenoids was based on a comparison of the peak field with known standards (in this case: astaxanthin, canthaxanthin, β-cryptoxanthin, zeaxanthin, echinenone and β-carotene, ChromaDex, Santa Ana, CA, USA). In the absence of a known standard, as in the case of the carotenoid of phycixxanthin, the astaxanthin peak field was used to calculate its concentrations. The identity of the carotenoids was further confirmed using HPLC-MS, using a Waters HPLC chromatograph equipped with a photodiodes kit (Waters model 996), leading to a Micromass ESI-Q-Tof mass spectrometer (Waters, Milford, MA, USA). HPLC analysis of ONC-T18 in turn revealed the presence of several antioxidant compounds (between 50 to 1250 mg kg 00 ml / min 10% acetone in hexane over a period of 25 minutes. The quantitative data of carotenoids was based on a comparison of the peak field with known standards (in this case: astaxanthin, canthaxanthin, β-cryptoxanthin, zeaxanthin, echinenone and β-carotene, ChromaDex, Santa Ana, CA, USA). In the absence of a known standard, as in the case of the carotenoid of phycixxanthin, the astaxanthin peak field was used to calculate its concentrations. The identity of the carotenoids was further confirmed using HPLC-MS, using a Waters HPLC chromatograph equipped with a photodiodes kit (Waters model 996), leading to a Micromass ESI-Q-Tof mass spectrometer (Waters, Milford, MA, USA). HPLC analysis of ONC-T18 in turn revealed the presence of several antioxidant compounds (between 50 to 1250 mg kg The quantitative data of carotenoids was based on a comparison of the peak field with known standards (in this case: astaxanthin, canthaxanthin, β-cryptoxanthin, zeaxanthin, echinenone and β-carotene, ChromaDex, Santa Ana, CA, USA). In the absence of a known standard, as in the case of the carotenoid of phycixxanthin, the astaxanthin peak field was used to calculate its concentrations. The identity of the carotenoids was further confirmed using HPLC-MS, using a Waters HPLC chromatograph equipped with a photodiodes kit (Waters model 996), leading to a Micromass ESI-Q-Tof mass spectrometer (Waters, Milford, MA, USA). HPLC analysis of ONC-T18 in turn revealed the presence of several antioxidant compounds (between 50 to 1250 mg kg The quantitative data of carotenoids was based on a comparison of the peak field with known standards (in this case: astaxanthin, canthaxanthin, β-cryptoxanthin, zeaxanthin, echinenone and β-carotene, ChromaDex, Santa Ana, CA, USA). In the absence of a known standard, as in the case of the carotenoid of phycixxanthin, the astaxanthin peak field was used to calculate its concentrations. The identity of the carotenoids was further confirmed using HPLC-MS, using a Waters HPLC chromatograph equipped with a photodiodes kit (Waters model 996), leading to a Micromass ESI-Q-Tof mass spectrometer (Waters, Milford, MA, USA). HPLC analysis of ONC-T18 in turn revealed the presence of several antioxidant compounds (between 50 to 1250 mg kg as in the case of the phycoxanthin carotenoid, the astaxanthin peak field was used to calculate its concentrations. The identity of the carotenoids was further confirmed using HPLC-MS, using a Waters HPLC chromatograph equipped with a photodiodes kit (Waters model 996), leading to a Micromass ESI-Q-Tof mass spectrometer (Waters, Milford, MA, USA). HPLC analysis of ONC-T18 in turn revealed the presence of several antioxidant compounds (between 50 to 1250 mg kg as in the case of the phycoxanthin carotenoid, the astaxanthin peak field was used to calculate its concentrations. The identity of the carotenoids was further confirmed using HPLC-MS, using a Waters HPLC chromatograph equipped with a photodiodes kit (Waters model 996), leading to a Micromass ESI-Q-Tof mass spectrometer (Waters, Milford, MA, USA). HPLC analysis of ONC-T18 in turn revealed the presence of several antioxidant compounds (between 50 to 1250 mg kg^-1) in cellular biomass. These compounds included antioxidant carotenoids, astaxanthin, zeaxanthin, canthaxanthin, echineone and beta-carotene in the range of 1 to 20 mg kg, respectively.^-10.25 to 10 mg kg^-1, 1 to 20 mg kg^-1 1 to 20 mg kg^-1 and 1 to 200 mg kg^-1as well as several unidentified flavonoid polyphenol compounds.

8. Example 8: Comparison with known microorganisms [0288] 304. The ability of ONC-T18 to produce DHA, EPA and DPA was compared to that of known microorganisms. The amount of cellular biomass per liter of nutrient solution, percentage of fat or fatty acids per dried cellular biomass, percentage of DHA, EPA and DPA in fatty acids together, and amount of DHA, EPA and DPA, when DHA, EPA and DPA were produced by Thraustochytrium was obtained. aureum ATCC 34304, Thraustochytrium sp. ATCC 20891, Thraustochytrium sp. ATCC 20892, Thraustochytrium roseum ATCC 28210, Thraustochytrium sp. ATCC 26185, Schizochytrium sp. ATCC 20888, Schizochytrium aggregatum ATCC 28209 and Schizochytrium limacinum MYA1381, as well as when DHA, EPA and DPA are produced by the ONC-T18 culture according to the present invention.

- 88 Table 5. Comparison of lipid production and biomass characteristics for several representative strains of the Thraustochytridae subgroup.

<td>Microorganism</td><td>The amount of cellular biomass (gL-1)</td><td>Content percentage lipids (% g-1)</td><td>Content percentage DHA (% g-1)</td><td>Content percentage EPA (% g-1)</td><td>Content percentage DPA (% g-1)</td><td>DHA together (gL-1)</td><td>EPA together (mg l-1)</td><td>DPA together (mg l-1)</td>

<td>Thraustochytrium sp ATCC 20891</td><td>1.8</td><td>no data</td><td>12</td><td>no data</td><td>no data</td><td>lack data</td><td>lack data</td><td>lack data</td>

<td>Thraustochytrium sp ATCC 20892</td><td>3</td><td>7</td><td>35</td><td>no data</td><td>no data</td><td>0.07</td><td>lack data</td><td>lack data</td>

<td>Thraustochytrium sp. ATCC 26185</td><td>2.3</td><td>no data</td><td>41.9</td><td>3.1</td><td>10</td><td>lack data</td><td>lack data</td><td>lack data</td>

<td>T. aureum ATCC 34304</td><td>4-5</td><td>8-20</td><td>24-51</td><td>3,6-9,3</td><td>no data</td><td>0.1-0.5</td><td>0.0001</td><td>lack data</td>

<td>T. roseum ATCC 28210</td><td>8-17</td><td>18-25</td><td>50</td><td>no data</td><td>no data</td><td>0,6-2,1</td><td>lack data</td><td>lack data</td>

<td>Schizochytrium sp. ATCC 20888</td><td>10.5</td><td>50</td><td>25-37</td><td>no data</td><td>no data</td><td>1.95</td><td>lack data</td><td>lack data</td>

<td>S. aggregatum ATCC 28209</td><td>1.4</td><td>1.7</td><td>6.0</td><td>6.1</td><td>no data</td><td>1</td><td>1</td><td>lack data</td>

<td>Microorganism</td><td>The amount of cellular biomass (gL-1)</td><td>Content percentage lipids (% g-1)</td><td>Content percentage DHA (% g-1)</td><td>Content percentage EPA (% g-1)</td><td>Content percentage DPA (% g-1)</td><td>DHA together (gL-1)</td><td>EPA together (mg l-1)</td><td>DPA together (mg l-1)</td>

<td>S. limacinum SR21 MYA-1381</td><td>23-40</td><td>40-53,5</td><td>29,7-34</td><td>0.2-0.4</td><td>no data</td><td>3,0-7,2</td><td>0.08</td><td>lack data</td>

<td>ONC-T18</td><td>25-55</td><td>45-80</td><td>24-34,2</td><td>0.1-2</td><td>6-10</td><td>4,6-13</td><td>0.2-0.8</td><td>0,9-3,8</td>

[0289] 305. As shown in Table 5, it is apparent that when culturing was carried out using the ONC-T18 strain of the present invention, the cellular biomass per liter of nutrient medium was extremely high compared to the other strains tested. In addition, according to the present invention, ONC-T18 has a very high percentage of lipids compared to the other strains mentioned above. Furthermore, according to the present invention, the percentage of DHA and DPA in ONC-T18 is extremely high, whereby EPA levels have been shown to be comparable for all tested strains. Thus, it appears that ONC-T18 has the ability to produce large amounts of DHA, EPA and DPA under fermentation conditions as mentioned in Example 1.

9. Example 9: Information on alternative carbon sources [0290] 306. It has been shown that ONC-T18 grows selectively on media, where the main nitrogen sources are yeast extract, monosodium glutamate and / or peptone, and the main carbon source is D-glucose. As a result of this detailed study of the ONC-T18 metabolic profile, it was observed that glycerol (a carbon source) was also an acceptable alternative. In addition, waste streams from fish oil processing containing glycerol have also been tested for suitability as cheap alternative nutrients. Experiments were carried out using 200 ml of media in 500 ml flasks, cultured at 25 ° C for 3 days, 120 rpm for glycerol. The content of glycerol in two waste products from fish oil processing, GWW (leaching of glycerol with water) and GAW (leaching of glycerol with acid), accounted for 40% by volume.

Table 6. Fatty acids, biomass and glycerol content when testing alternative carbon sources.

<td rowspan="2"></td><td colspan="5">Percentage (%) of fatty acids to total lipid, by weight</td><td rowspan="2">TFA (mg g-1)</td><td rowspan="2">Glycerol (g L-1)</td><td rowspan="2">Biomass (g L-1)</td>

<td>AA</td><td>EPA</td><td>DHA</td><td>DPA n-3</td><td>DPA n-6</td>

<td>6% glycerol (Wag.:obj).</td><td>029</td><td>0.52</td><td>26.31</td><td>0.24</td><td>9.49</td><td>426.12</td><td>76,00</td><td>9.13</td>

<td>40% GAW ^(about· bj)</td><td>0.37</td><td>1.32</td><td>19.69</td><td>0.42</td><td>6.36</td><td>294.55</td><td>68.59</td><td>5.94</td>

<td>40%</td><td>0.46</td><td>5.55</td><td>12.46</td><td>1.01</td><td>3.82</td><td>274.33</td><td>2.70</td><td>3.08</td>

<td rowspan="2"></td><td colspan="5">Percentage (%) of fatty acids to total lipid, by weight</td><td rowspan="2">TFA (mg g-1)</td><td rowspan="2">Glycerol (g L-1)</td><td rowspan="2">Biomass (g L-1)</td>

<td>AA</td><td>EPA</td><td>DHA</td><td>DPA n-3</td><td>DPA n-6</td>

<td>GWW (^obj ·)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

[0291] 307. Analysis of these results found that the use of components of a fish waste stream, such as glycerol by-products as carbon sources in the large-scale ONC-T18 fermentation, resulting in a decreased amount of fatty acids together, represented the maintained DHA content in microbial cells. (Figure 10).

10. Example 10: Dry cell weight multiplier [0292] 308. Thraustochytrium sp. ONC-T18 can be grown for the production of omega 3 oils in various reactor configurations up to 100,000 L. All fermentations start with the preparation of inoculum in 10-20% volume which is used to establish a fermentation culture. Initial nutrient configurations include up to 6 g / L sea salt, 10 g / L nitrogen source and 60 g / L carbon source, with periodic addition of a further 75 g / L carbon source after 24 to 36 hours of initial fermentation for an additional 72 to 96 hours and temperature range 18-25 ° C. For example, using 6 g / L nutrient salt, 2 g / L yeast extract, 8 g / L L-glutamate and 60 g / L D-glucose (with the addition of

75 g / L added after 36 hours), cultured at 25 ° C for 96 hours, ONCT18 was able to produce 40 g / L cell dry weight (smk), 80% (Smk) of fatty acids together (TFA) in the lipid fraction ( between C14: 0 and C24: 0) and 30% (TFA) DHA. Similarly, by multiplying nitrogen and carbon as nutrient components, the dry cell mass can be increased to exactly the same biomass multiplication effect without affecting the content of either TFA or DHA. For example, using 24 g / L nutrient salt, 8 g / L yeast extract, 32 g / L L-glutamate and 300 g / L D-glucose, grown at 25 ° C for 312 hours, ONC-T18 was capable of producing 80 g / L cell dry weight (SMK), 60% (SMK) total fatty acids (TFA) in the lipid fraction (between C14: 0 and C24: 0) and 38% (TFA) DHA.

11. Example 11: Growth of Thraustochytrium sp. ONC-T18 on various alternative carbon sources (C) and nitrogen (N) and effect on dry cell mass and lipids [0293] 309. Growth of Thraustochytrium sp. ONC-T18 on various cheap sources was investigated. nitrogen and carbon. In detail, 50 ml of ONC-T18 were grown in 250 ml flasks containing 6

- 92 g / L of artificial sea salt, for 72 hours at 25 ° C. The concentrations of carbon and nitrogen sources are shown below, where 2 g / L of each of the mentioned nitrogen sources was used in combination with 8 g / L of L-glutamate (except for fishmeal, where 4 g were used). The carbon sources were changed as indicated. All experiments were carried out in three versions;

all extractions for the analysis of fatty acid methyl esters were carried out in three versions together with three GC injections.

[0294] 310. The results indicate that Thraustochytrium sp. ONC-T18 produces optimal dry biomass of cells (i.e., more than twice on control medium) when grown on yeast extract EMD and fish meal as nitrogen sources. Conversely, the lipid was found to be less than in the comparative test, and the DHA content was optimal using the EMD cornpet and peptone extract. Finally, it was found that dextrose as a carbon source increases lipid content, and fructose and dextrose produce high DHA content relative to comparative trials.

- 93 Table 7: Growth of Thraustochytrium sp. ONC-T18

<td colspan="2">Nutrient (salt 6 g / l, increase 72 h,</td><td rowspan="2">C (G / l)</td><td rowspan="2">N (G / l)</td><td rowspan="2">SMK / l nutrient (g)</td><td rowspan="2">lipids (Mg / g)</td><td rowspan="2">lipids (G / l)</td><td rowspan="2">DHA (Mg / g)</td><td rowspan="2">DHA (G / l)</td><td rowspan="2">DHA (% lipids)</td>

<td></td><td>50 ml of breeding)</td>

<td rowspan="3">Nitrogen sources</td><td>Soil extract of maize-MSG</td><td>60</td><td>10</td><td>11.36</td><td>371.97</td><td>4.33</td><td>111.64</td><td>1,244</td><td>29.72</td>

<td>Cotton seeds-MSG</td><td>60</td><td>10</td><td>9.99</td><td>297.08</td><td>2.70</td><td>45,20</td><td>0,500</td><td>16,94</td>

<td>emd ™ ye-msg</td><td>60</td><td>10</td><td>15.94</td><td>343.96</td><td>5.02</td><td>70.68</td><td>0,786</td><td>20,70</td>

<td></td><td>emd ™ ye</td><td>60</td><td>10</td><td>35.79</td><td>189.01</td><td>6.76</td><td>37.14</td><td>0.448</td><td>19.65</td>

<td></td><td>emd ™ Pepton-MSG</td><td>60</td><td>10</td><td>11.70</td><td>379.48</td><td>4.19</td><td>83.50</td><td>0.926</td><td>23.33</td>

<td></td><td>Sigma ™ YE-MSG</td><td>60</td><td>10</td><td>9.77</td><td>257.86</td><td>3.28</td><td>54.60</td><td>0.618</td><td>19.69</td>

<td></td><td>Sigma ™ YE</td><td>60</td><td>10</td><td>10.39</td><td>341.98</td><td>3.53</td><td>58.30</td><td>0.629</td><td>17.01</td>

<td></td><td>Fermtech ™ YE-MSG</td><td>60</td><td>10</td><td>13.99</td><td>269.53</td><td>3.82</td><td>56,97</td><td>0.664</td><td>21.10</td>

<td></td><td>Fermtech ™ YE</td><td>60</td><td>10</td><td>17.07</td><td>243.23</td><td>4.15</td><td>48.01</td><td>0.530</td><td>19.74</td>

<td></td><td>Fish meal (62% protein)</td><td>60</td><td>12</td><td>19.53</td><td>290.72</td><td>5.68</td><td>73.59</td><td>0.828</td><td>25.31</td>

<td rowspan="4">Coal sources</td><td>Fructose</td><td>60</td><td>10</td><td>14.57</td><td>498.54</td><td>8.09</td><td>96.97</td><td>1,070</td><td>21.55</td>

<td>dextrose</td><td>60</td><td>10</td><td>14.98</td><td>623.91</td><td>9.87</td><td>113.69</td><td>1,232</td><td>18.94</td>

<td>Corn dextrin</td><td>60</td><td>10</td><td>4.65</td><td>89.69</td><td>0.39</td><td>25.69</td><td>0.278</td><td>26.75</td>

<td>Gelatine</td><td>60</td><td>10</td><td>7.09</td><td>31,87</td><td>0.13</td><td>11.86</td><td>0,127</td><td>27,70</td>

<td></td><td>Corn starch)</td><td>5</td><td>10</td><td>4.85</td><td>94.04</td><td>0.46</td><td>19.49</td><td>0.206</td><td>20.72</td>

<td></td><td></td><td>thirty</td><td>10</td><td>3.13</td><td>90,07</td><td>0.28</td><td>23.78</td><td>0.256</td><td>26.40</td>

<td></td><td>Wheat starch)</td><td>5</td><td>10</td><td>8.03</td><td>86.96</td><td>0.47</td><td>17.62</td><td>6.185</td><td>17.76</td>

<td></td><td></td><td>thirty</td><td>10</td><td>18.16</td><td>18.59</td><td>0.34</td><td>3.83</td><td>0.042</td><td>20.58</td>

<td></td><td>Comparative medium (1)</td><td>60</td><td>10</td><td>16.92</td><td>487.59</td><td>8.25</td><td>70.87</td><td>0.768</td><td>13.25</td>

<td></td><td>Comparative medium (2)</td><td>60</td><td>10</td><td>10.88</td><td>483.06</td><td>6.06</td><td>74.64</td><td>0.818</td><td>16:19</td>

abbreviations:

MSG = Sodium L-glutamate

YE = Smk yeast extract = dry mass of cells

12. Example 12: Extraction techniques for secretion of total lipids and fractions [0295] 311. Various methods of isolation of selected omega-3 oils have been tested to determine the optimal efficiency of secretion. These methods included: the normal Bligh and Dyer method (Bligh and Dyer, Can J. Biochem. Physiol, 37: 912-917, 1959); a method of combined extraction and transesterification, used specifically for Thraustochytridae species, permitting the processing of samples for rapid GC FAME analysis (Lewis et al., J Microbiol. Methods, 43: 107-116, 2000); extraction by concurrent saponification (Cartens et al., J. Am. Oil Chem. Soc. 73: 1025-1031, 1996); and solid phase extraction using silica gel columns that can selectively secrete triglycerides; diglycerides and monoglycerides (Pinkart et al., J. Microbiol. Methods, 34: 9-15, 1998; Bateman &amp; Jenkins, J. Agric. Food Chem., 45: 132-135, 1997).

[0296] 312. In detail, 40 grams of dry Thraustochytrium sp. ONCT18 cell biomass produced in a single fermentation course (see example 1) were divided into 0.44 g lots and used for each of these techniques. All techniques were performed in three versions, analyzing the yields using the fatty acid methyl esters by FID-GC method, again in three versions with three chromatograms per sample. The results show that the total fatty acid content can vary between individual methods, the fluctuations most likely associated with solvent saturation: compound, biomass disruption issues and other issues related to physical conditions (e.g. temperature and time).

Table 8: Extraction techniques for secretion of total lipids and fractions.

<td colspan="13">Bligh and Dyer's way</td>

<td></td><td>DHA</td><td>EP AND</td><td>C14: 0</td><td>C14: 1</td><td>C15: 0</td><td>C16: 0</td><td>C16: 1</td><td>C18: 1</td><td>C20: 0</td><td>C20: 4</td><td>C22: 5</td><td>TFA</td>

<td colspan="13">mg omega-3 per gram of biomass (mg / g)</td>

<td>1</td><td>104.39</td><td>4.25</td><td>36.28</td><td>5.82</td><td>113.94</td><td>77.27</td><td>4.92</td><td>44,01</td><td>1.13</td><td>1.67</td><td>28.86</td><td>430.99</td>

<td>2</td><td>136.75</td><td>5.45</td><td>46.51</td><td>7.40</td><td>142.96</td><td>98,38</td><td>6.07</td><td>56.49</td><td>1.40</td><td>2.19</td><td>37.92</td><td>552.98</td>

<td>3</td><td>134.59</td><td>4.78</td><td>42.51</td><td>6.91</td><td>128.54</td><td>87.01</td><td>5.20</td><td>51.10</td><td>1.30</td><td>2.10</td><td>35.98</td><td>532.91</td>

<td>Av.</td><td>125.24</td><td>4.83</td><td>41.77</td><td>6.71</td><td>128.48</td><td>87.55</td><td>5.40</td><td>50.53</td><td>1.28</td><td>1.99</td><td>34.25</td><td>505.63</td>

<td colspan="13"></td>

<td colspan="13">Direct transesterification</td>

<td></td><td>DHA</td><td>EPA</td><td>C14: 0</td><td>C15: 0</td><td>C16: 0</td><td>C16: 1</td><td>C18: 0</td><td>C18: 1</td><td>about about cj</td><td>C20: 4</td><td>C22: 5</td><td>TFA</td>

<td colspan="13">mg omega-3 per gram of biomass (mg / g)</td>

<td>1</td><td>104.39</td><td>4.24</td><td>36.39</td><td>5.42</td><td>112.94</td><td>75.27</td><td>5.42</td><td>44,01</td><td>1.13</td><td>1.67</td><td>28.86</td><td>420.99</td>

<td>2</td><td>89,83</td><td>4.54</td><td>34,81</td><td>5.60</td><td>103,04</td><td>73.43</td><td>5.56</td><td>42.85</td><td>0.98</td><td>1.87</td><td>25.35</td><td>392.88</td>

<td>3</td><td>101.64</td><td>4.25</td><td>37.16</td><td>5.98</td><td>106.94</td><td>75.98</td><td>5.35</td><td>43,95</td><td>1.11</td><td>1.78</td><td>26.46</td><td>410.65</td>

<td>AND v.</td><td>98.65</td><td>4.34</td><td>36,12</td><td>5.67</td><td>107.64</td><td>74.89</td><td>5.44</td><td>43.60</td><td>1.07</td><td>1.77</td><td>26.89</td><td>408.17</td>

<td colspan="13"></td>

<td colspan="13">Simultaneous saponification</td>

<td></td><td>DHA</td><td>EP</td><td>C14:</td><td>C15:</td><td>C16:</td><td>C16:</td><td>C18:</td><td>C18:</td><td>C20:</td><td>C20:</td><td>C22:</td><td>TFA</td>

<td></td><td></td><td>AND</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>1</td><td>0</td><td>4</td><td>5</td><td></td>

<td colspan="13">mg omega-3 per gram of biomass (mg / g)</td>

<td>1</td><td>204.85</td><td>6.75</td><td>46.55</td><td>8.56</td><td>182.26</td><td>134.81</td><td>8.73</td><td>105.04</td><td>2.16</td><td>3.20</td><td>66.68</td><td>785.25</td>

<td>2</td><td>188.51</td><td>6.17</td><td>47.64</td><td>9.32</td><td>208.29</td><td>121.25</td><td>10.35</td><td>95.80</td><td>2.53</td><td>2.89</td><td>61.41</td><td>770.14</td>

<td>3</td><td>198.25</td><td>6.12</td><td>47.21</td><td>9.65</td><td>207.71</td><td>136.51</td><td>9.58</td><td>98.50</td><td>2.41</td><td>3.10</td><td>63.58</td><td>782.54</td>

<td>Av.</td><td>197.20</td><td>6.35</td><td>47.13</td><td>9.18</td><td>199.42</td><td>130.86</td><td>9.55</td><td>99.78</td><td>2.37</td><td>3.06</td><td>63.89</td><td>779.31</td>

<td colspan="13"></td>

<td colspan="13">Solid phase extraction</td>

<td></td><td>DHA</td><td>EPA</td><td>C14: 0</td><td>C15: 0</td><td>C16: 0</td><td>C16: 1</td><td>C18: 0</td><td>C18: 1</td><td>C20: 0</td><td>C20: 4</td><td>C22: 5</td><td>TFA</td>

<td colspan="13">mg omega-3 per gram of biomass (mg / g)</td>

<td>1</td><td>169.17</td><td>0.42</td><td>68.41</td><td>10.83</td><td>204.43</td><td>140.14</td><td>8.09</td><td>76.97</td><td>1.75</td><td>3.00</td><td>47.05</td><td>748.33</td>

<td>2</td><td>172.26</td><td>0.44</td><td>69.59</td><td>11.01</td><td>207.01</td><td>143.74</td><td>8.11</td><td>78,72</td><td>1.74</td><td>3.27</td><td>47.86</td><td>819.04</td>

<td>3</td><td>173.65</td><td>0.43</td><td>69.21</td><td>11.31</td><td>208.97</td><td>146.64</td><td>8.16</td><td>77.64</td><td>1.73</td><td>3.64</td><td>46.98</td><td>785.64</td>

<td>Av.</td><td>171.69</td><td>0.43</td><td>69.07</td><td>11.05</td><td>206.80</td><td>143.51</td><td>8.12</td><td>77.78</td><td>1.74</td><td>3.30</td><td>47.30</td><td>784.34</td>

<td colspan="13">Note: Each value given above is the average of three runs using FID-GC to</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td colspan="2">FAME analysis</td><td></td><td></td><td></td><td></td><td></td>

- 96 13. Example 13: a) Materials and methods (1) Separation and maintenance Thraustochytridae [317] 313. Seventy marine samples including: Spartina alterniflora, Zostera marina and sediment were collected at locations on the east coast of Canada in Nova Scotia, on the Isle Prince Edward, New Brunswick, Newfoundland and Labrador between July and August 2002. The samples were placed in 20 mL vials containing 10 mL sterile filtered through 0.2 μm filter of natural seawater and 300 mg

L^-1 penicillin and 500 mg L^-1 streptomycin. A bait from sterile pollen (Acer sp.) Was added to the suspensions and incubated for 48 hours at 18 ° C, according to (Bremer,

Marine Mycology - A Practical Approach. Fungal Diversity Press, Hong Kong, pp. 49-61 (2000)). Next, the pollen grains were transferred with the ese and streaked on B1 agar plates (1 g

L^-1 yeast extract, 1 g L^-1 peptone, 10 g L^-1 agar to 1 L natural sea water) containing antibiotics and incubated. Single, irregular, glassy colonies composed of spherical or cochlear cells were collected and untypical for yeast or bacterial colonies and grown further at least three times on B1 plates to ensure purity.

(2) Preparation of biomass for fatty acid screening [0298] 314. A liquid medium was prepared for screening isolates to determine the growth and production of fatty acids using 0.2 μm-filtered natural seawater containing 2 g L^-1 peptone (BD, Franklin Lakes, NJ, USA) and 2 g L¹ yeast extract (BD, Franklin Lanes, NJ, USA), which was sterilized in an autoclave, then sterilized by filtration through a 0.2 μm filter, 5 g L solution was added^-1glucose (Sigma-Aldrich, St. Louis, MO, USA) (Bowles et al., J Biotechnol 70: 193-202 (1999)).

A 30-mL culture was inoculated with the agar plate and cultured for 4 days at 18 ° C on a shaker at 100 rpm. Then, 5 mL of this culture was used to inoculate 95 mL cultures incubated for a further 4 days (stationary phase). The cells were harvested by centrifugation at 4500 rpm, washed with 5 mL of distilled water and centrifuged again. The cell pellets are lyophilized, weighed and stored at -80 ° C before being subjected to fatty acid analysis.

(3) Preparation of fatty acid methyl esters (FAME)

[0299] 315. Extraction of fatty acid methyl esters (FAME) was performed by direct transesterification, modified from Lewis et al. (J Microbiol Metah. 43: 107-116 (2000)). Specifically, 20 mg of lyophilised material and 3 ml of the transesterification reaction mixture (methanol: hydrochloric acid: chloroform (10: 1: 1 v)) were added. The cells were vortexed for 10 seconds to ensure an even distribution of biomass and placed at 90 ° C for 120 minutes. When the transesterification was complete, the samples were removed and allowed to cool to room temperature. Water (1 ml) was then added and vortexed for 10 seconds. The FAME was then extracted by adding 3 x 2 ml portions of hexane: chloroform (4: 1),

(4) FAME analysis by gas chromatography (GC) [0300] 316. GAME FAME analysis was carried out using two internal standards (200 μl each). One, hexacose acid (C23: 0) was added before the transesterification and the second, acid Nonaddecanoic (C19: 0) is added immediately prior to analysis The analyzes were carried out using an Agilent 6890 GC instrument (Agilent Technologies, Palo Alto, CA, USA) equipped with a capillary column 30 mx 0.32 m internal diameter (0.25 μm layer thickness) OMEGAWAX 320 with fused silica (Sigma-Aldrich, St. Louis, MO, USA) and flame ionization detector (injection volume 1 g, H2O carrier gas at constant flow 5.0 ml min^-1 and set at 250 ° C, a 50: 1 division ratio to the FID detector at 275 ° C). Confirmation of FAME identity was performed using a Trace GC-DSQ mass spectrometer (Thermo Electron, Boston, MA, USA) and a comparison of retention times for laboratory standards.

(5) Genetic identification [0301] 317. Genomic DNA was extracted using the MoBio UltraClean Microbial DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA) according to the manufacturer's instructions. The oligonucleotide primers used in the 18S rRNA gene amplification were modified from Vinda et al. (J Eukaryot Microbiol. 46: 637-647 (1999)) namely T18S1F 5'CAACCTGGTTGATCCTGCCAGTA-3 'and T18S5R 5'-TCACTACGGAAACCTTGTTACGAC30 3'. The volume of 20 μl of the PCR reaction mixture contained 2U Biolase ™ DNA polymerase (Biolin, Boston, MA, USA), 1 x NH4 reaction buffer, 3 mM MgCl2, 1M betaine (SigmaAldrich, St. Louis, MO), 200 μΜ nucleotide mix for PCR (Promega, Madison, WI, USA), 1 μΜ of each of the forward and reverse primers (MWG Biotech., High

- 98 Point, NC, USA) and 100 ng genomic DNA template. After an initial denaturation of fork minutes at 94 ° C, PCR amplification was performed using an Eppendorf Master Cycle Gradient thermal cycler (Eppendorf, Westbury, NY, USA) using a 45 second program at 94 ° C, 30 seconds at 64 ° C and 2 minutes at 72 ° C for 30 cycles followed by a 10 minute extension at 72 ° C. The PCR product was purified using the MoBio UltraClean PCR Clean-up Kit (MoBio Laboratories Inc., Carlsbad, CA, USA) for direct sequencing (MWG Biotech., High Point, NC, USA) using the FA2, FA3, RA1, R primers ( Mo et al., Mar Biol 140: 883-889 2002), T18S1F and T18S5R. The resulting sequences were assembled and compared to the nucleotide sequences of similar microorganisms stored in GenBank (Benson et al., Nucleic Acids Res 33: D34-38 (2005)), using DS Gene (Accelrys, San Diego, CA, USA). In turn, a phylogenetic tree was generated using the method of joining neighbors (Saito and Nei, Mol Biol Evol 4: 406-425 (1987)), with statistical significance assessed using 1000-fold sampling with returning the sample.

bootstrap resamplings) (Felsenstein, Evolution 39: 783-791 (1985)).

(6) Identification of carotenoids [0302] 318. Cells were harvested by centrifugation at 3,800 xg and washed with phosphate buffered saline. It was then resuspended in 10x volume of acetone (Sigma-Aldrich, St. Louis, MO, USA), mixed for 5 minutes at 200 rpm, centrifuged at 3800 xg for 5 min and concentrated to dryness by evaporation with N2. It was then resuspended in the least amount of 10% acetone in hexane before HPLC analysis. The identifications were performed on an Agilent 1100 HPLC instrument (Agilent, Palo Alto, CA, USA) equipped with a variable wavelength detector set at 470 nm. Samples were injected through the Symmetry C18 pre-column (Waters, Milford, MA, USA) per column

Bondclone C18 with reverse phase (Phenomenex, Torrance, CA, USA, 10 μm particles, 3.9 x 300 mm internal diameter). The injection volume was 10 ml, and a flow rate of 1 ml was used^-1 10% acetone in hexane for 25 minutes. The identity of carotenoids was further confirmed by mass spectrometry analysis (Micromass ESI-QTof MS, Waters, Milford, MA, USA). The quantitative data for each of the carotenoids was based on the development of a calibration curve at

Using standards (astaxanthin, zeaxanthin, canthaxanthin, echinenone and β-carotene) and a comparison of peak areas with defined concentrations.

(7) Fermentation optimization

[0303] 319. The effect of carbon, nitrogen and sea salt on the production of fatty acids and DHA was checked using batch cultures in 250 ml Erlenmeyer flasks shaken at 130 rpm for 3 days at 25 ° C. Further culturing tests were carried out using a Biostat reactor^® Bplus Twin SL Bioreactor (Sartorius BBI Systems Inc., Betlehem, PA, USA).

A 100 ml inoculum was used to inoculate 4.9 L medium in the bioreactor. The glucose concentration was measured using a Glucose (HK) Assay Kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. The nutrient components and conditions used in the bioreactor are detailed with the relevant results.

b) Results [0304] 320. A collection and screening method was developed that allowed the members of the Labyrinthulida family of protists, especially the Schizochytrium and Thraustochytrium species, to be isolated using pollen treatment and selective bacteriological media. This study, including 20 unique collection sites distributed in the Atlantic area of ​​Canada, provided 68 pure strains, identified microscopically. The choice of oil strains having more than 20% of their cell dry cell content as fatty acids was based on the results of the PUFA profile testing by GC, biomass yield, maximum concentrations of TFA, DHA and to a lesser extent EPA (Fig. 11), according to the method of ( Lewis et al., J Microbiol Meth 43,107-116 (2000)). Biomass values, TFA,^-127.1 to 321.14, 5.18

83.63 and 2.97 to 21.25 mg g^-1 (Fig. 11).

[0305] 321. All isolates that grew in liquid medium (54 out of 68) produced large amounts of omega-3 polyunsaturated fatty acids, particularly DHA, which represented between 22 and 80% of the total C20 to C22 content in these cells (Fig. 11). This confirms the previous findings according to which Thraustochytridae isolated from moderately cold environments have fatty acid profiles with DHA constituting up to 53% of the fatty acids present together (Bowles et al., J Biotechnol 70: 193-202 (1999) and Huang et al. Mar Biotechnol 5450-457 (2003)). Particularly notable is ONC-T18, which produces up to 90% of its C20 to C22 content as DHA, which accounts for approximately 35% of intracellular fatty acids together. It was shown that this DHA content was equivalent to the content for several strains for commercial production, such as Schizochytrium sp. ATCC 20888 (32%) and S. limacinum MYA 1381/8821 (34%) (Barclay et al., J Appl Phycol 6: 123-129 (1994) and Yokochi et al., Appl Microbiol Biotechnol 49 : 72-76, (2003)). In addition, all isolates synthesized eicosapentaenoic acid (EPA) in an amount that changes

- 100 between 2 and 20 wt.% identified PUFAs together (Fig. 11). In addition to the omega-3 oils produced, approximately 80% of all isolates synthesized omega-6 PUFA, arachidonic acid (AA) or docosapentaenoic acid (DPA), at concentrations varying between 1 and 18% and 3 to 7% by weight, respectively. (Fig. 11).

[0306] 322. Huang et al. (Mar Biotechnol 5: 450-457 (2003)) suggested that for Thraustochytridae isolated from the tropical coastal waters of Japan and Fiji one can describe five profiles of polyunsaturated fatty acids, namely DHA / DPA (n-6), DHA / DPA / EPA, DHA / EPA, DHA / DPA / EPA / AA and DHA / DPA / EPA / AA / docosatetraenic acid (Huang et al., Mar Biotechnol 5: 450-457 (2003)). In this collection of 10 Thraustochytridae, separated from the moderate waters of Atlantic Canada, four PUFA profiles could be identified, three of which are identical to those mentioned above, namely DHA / DPA / EPA for 7.4% of the collection, DHA / EPA for 13% of the collection and DHA / DPA / EPA / AA, 74%, and the fourth included a DHA / EPA / AA mixture of 5.6%.

[0307] 323. By direct sequencing of the 188 rDNA gene, ONC-T 18 was positively identified as a member of the Thraustochytridae family (GenBank, accession number: DQ374149). Phylogenetic analysis showed that ONC-T18 formed a unique group (97.5% identity) with Thraustochytrium striatum T91-6 (Fig. 12) (Leander and Porter, Mycologia 93: 459-464 (2001)). However, Thraustochytriidae sp. MBIC 11093, N1-27 and Thraustochytrium sp. CHN-1 were found to be collected from tropical coastal waters.

Japan, and being major DHA producers (Carmona et al., Biosci Biotechnol Biochem 67: 884-888 (2003) and Huang et al., Mar Biotechnol 5: 450-457 (2003)), were 96, respectively,

95.5 and 94.5% similarity. Genetic diversity between all Thraustochytriidae members shown in Figure 12 is quite low, in terms of similarity in the whole group of 97.5-91.0%. However, these species are widespread globally, with two-thirds of them separated from the tropical coastal waters of Japan, China and Israel, and the rest from temperate waters of America, Europe and Canada.

[0308] 324. ONC-T18 fatty acid profile included high levels of C22 PUFA, very low levels of C18 and C20 fatty acids, and the occurrence of saturated chain fatty acids with an odd number of carbon atoms (15: 0 and 17: 0), as well as Schizochytrium sp. KH105 or S. limacinum SR21. In addition, the analysis of carbon and nitrogen utilization profiles for ONC-T18, SR21 and KH105 strains showed a similar pattern of absorption. The n-6 DPA content in the ONC-T18 strain was in the range of 6-10%, which appears to be extremely high when considering the limited occurrence of n-6 DPA in the biosphere.

- 101 Similar levels of n-6 DPA were described by Nakahara et al. (J Am Oil Chem Soc. 73: 1421-1426 (1996)) in Schizochytrium sp. SR21 (6-10%) and Ellenbogen et al. (Comp Biochem Physiol 29: 805-81 (1969)) in T. aureum (9.5%) and T. roseum (6.6%).

[0309] 325. Analysis of the ONC-T18 fatty acid profile in three different culturing configurations: (1) agar plate; (2) a conical flask and (3) bioreactor and grown on the same medium (Figure 13), shows a decrease in the diversity of the PUFAs present and a general increase in TFA from the agar plate to the bioreactor. In detail, agar plates showed a PUFA kit, and cultures in flasks and bioreactors were dominated by one or two intermediates (Figure 13). In comparison with Thraustochytrium aureum, which in breeding in a flask grew better than in a stirred tank fermenter (Ilda et al., J Ferment Bioeng 81: 76-78 (1996)), ONC-T18 grew better in the bioreactor. This result is consistent with the result of Nakahary et al., J Am Oil Chem Soc 73: 1421-1426 (1996), who found that Schizochytrium sp. SR21 showed high resistance to mechanical mixing,

[0310] 326. Furthermore, it was found that carotenoid pigments were produced in the Thraustochytrium sp. ONC-T18 fermentations on the plates, in the flask and in the bioreactor, giving a pale orange color. The production of these antioxidants is maximal in fermentations in the bioreactor, along with the production of fatty acids. In addition, by using HPLC and mass spectrometry, these antioxidant compounds have been identified as astaxanthin, zeaxanthin, canthaxanthin, echinenone and β-carotene (Fig.

14), conjugated with various PUFAs. Similar results have been reported for members of the Thraustochytridae protista group. Specifically, Schizochytrium aggregatum has been shown to produce echinenone and canthaxanthin (Valadon, Trans Br Mycol Soc 67: 1-15 (1976)), and Carmona et al. (Biosci Biotechnol Biochem 67: 884-888 (2003) and Huang et al. (Mar Biotechnol 5: 450457 (2003)) showed the production of astaxanthin, echinenone, canthaxanthin, phycikoxanthin (and not zeaxanthin as in ONC-T18) and β-carotene by Thraustochytrium sp. CHN-1, a close relative of ONC-T18 (Fig. 12). In this study, the concentrations of these carotenoids were an order of magnitude smaller than those of CHN-1, with the main compound being β-carotene instead of astaxanthin. Therefore, among the Thraustochytrium species, the production of PUFA and the production of carotenoids can be combined,

[0311] 327. It has previously been found that the relative amounts of essential fatty acid components (myristic, palmitic and oleic acids) can be altered to some extent by changing the growth conditions of the culture (Ilda et al., J Ferment Bioeng 81: 76-78

102 (1996)). In this way, during the fermentation, the final fatty acid composition and thus the physical properties of the desired PUFAs can be manipulated in a regulated manner (Sijtsma et al., Recent Res Devel Microbiol 2: 219-232 (1998)). In an attempt to reduce the inhibitory factors for the production of both biomass and omega-3 PUFA in ONC-T18, the components of carbon, nitrogen and sea salt were manipulated in nutrient media (Table 9), along with the duration of the culture (Figure 15).

Table 9: Average biomass production (SD &lt;15%), total fatty acids (TEA) and DHA content for Thraustochytrium sp. ONC-T18.

<td colspan="2">Glucose (g L^-1)</td><td colspan="3">Biomass (g L^-1)</td><td colspan="3">TFA (% biomass)</td><td>DHA (% TFA)</td><td>DHA (g L^-1)</td>

<td colspan="2">5</td><td colspan="3">12.13</td><td colspan="3">5.21</td><td>29.18</td><td>0.18</td>

<td colspan="2">20</td><td colspan="3">13.73</td><td colspan="3">29,59</td><td>24.01</td><td>0.98</td>

<td colspan="2">40</td><td colspan="3">16.69</td><td colspan="3">59.39</td><td>23.88</td><td>2.37</td>

<td colspan="2">60</td><td colspan="3">21.08</td><td colspan="3">51.01</td><td>26.17</td><td>2.81</td>

<td colspan="2">100</td><td colspan="3">18.40</td><td colspan="3">69.49</td><td>31.55</td><td>4.03</td>

<td colspan="2">160</td><td colspan="3">10.68</td><td colspan="3">9.40</td><td>30,01</td><td>0.30</td>

<td>YE (g L^-1)</td><td>MSG (</td><td colspan="2">II)</td><td>Biomass (g 1</td><td colspan="2">^¹)</td><td>TFA (% biomass)</td><td>DHA (% TFA)</td><td>DHA (g L^-1)</td>

<td>10</td><td colspan="3">0</td><td colspan="3">22.33</td><td>34.53</td><td>20.20</td><td>1.56</td>

<td>8</td><td colspan="3">2</td><td colspan="3">22.81</td><td>44,00</td><td>17.52</td><td>1.72</td>

<td>6</td><td colspan="3">4</td><td colspan="3">22.64</td><td>50.69</td><td>16.23</td><td>1.86</td>

<td>4</td><td colspan="3">6</td><td colspan="3">24.46</td><td>69.07</td><td>24.19</td><td>4.09</td>

<td>2</td><td colspan="3">8</td><td colspan="3">26.09</td><td>81.73</td><td>20.99</td><td>4.47</td>

<td>0</td><td colspan="3">10</td><td colspan="3">7.50</td><td>1.97</td><td>28.81</td><td>0.04</td>

<td colspan="3">Sea salt (g L^-1)</td><td colspan="3">Biomass (g L^-1)</td><td colspan="2">TFA (% biomass)</td><td>DHA (% TFA)</td><td>DHA (g L^-1)</td>

<td colspan="3">2</td><td colspan="3">24.70</td><td colspan="2">59.23</td><td>31.44</td><td>4.60</td>

<td colspan="3">6</td><td colspan="3">21.08</td><td colspan="2">51.01</td><td>26.17</td><td>2.81</td>

<td colspan="3">15</td><td colspan="3">22.90</td><td colspan="2">69.32</td><td>25.32</td><td>4.02</td>

<td colspan="3">thirty</td><td colspan="3">17.76</td><td colspan="2">61.02</td><td>25.25</td><td>2.74</td>

- 103 -

<td>Sea salt (g L^-1)</td><td>Biomass (g L^-1)</td><td>TFA (% biomass)</td><td>DHA (% TFA)</td><td>DHA (g L^-1)</td>

<td>40</td><td>17.27</td><td>68.21</td><td>24.02</td><td>2.83</td>

<td>50</td><td>18.77</td><td>59.63</td><td>22,56</td><td>2.53</td>

[0312] 328. In this study, as the concentration of nitrogen decreased, the fatty acid content was increased together, with the highest total fatty acid content (approximately 80%) obtained at 1% concentration of yeast extract and / or monosodium glutamate (at ./obj.). However, cultures with low nitrogen concentration also limited cell growth and hence the production of fatty acids together. In this experiment, optimal production was obtained using 8 gL^-1 monosodium glutamate and 2 g L^-1 yeast extract, producing 26.1 g L^-1 biomass and 4.5 g L^-1 DHA (Table 9). In addition, increasing carbon up to 100 g L^-1 effectively increased the DHA yield, which is in accordance with the results obtained for Schizochytrium sp. SR21 (Yokochi et al., Appl Microbiol Biotechnol 49: 72-76, (2003)) and contrary to those shown for T. aureum, where glucose concentrations above 10 g L^-1 they were inhibitory (Ilda et al., J Ferment Bioeng 81: 76-78 (1996)). Maximum DHA yields of more than 4.0 g L^-1 obtained on a glucose medium, at a yield greater than five times greater than the yield of T. aureum (Bajpai et al., J Am Oil Chem Soc. 15: 509-514 (1991)) and T. roseum (Li and Ward, Ind. Microbiol 13: 238 -241 (1994)) and comparable with the yields of Schizochytrium sp. SR21 and KH105 (Aki et al., J Am Oil Chem Soc 80: 789-794 (2003)). Finally, ONC-T18 exhibited classical euryhalic properties, withstanding salinity in the range of 2.0 to 50.0 g L^-1, which yielded biomass yields with a variability of 25-30% (Table 9). In the same experiment, it was found that the DOA values ​​according to L^-1 changed to 45%, between optimal at 4.6 g L^-1 and a minimum of 2.5 g L^-1(Table 9).

[0313] 329. The biomass, TFA and DHA produced by ONC-T18 over a period of 168 h in the L-bioreactor are shown in Figure 15. The drawn growth curve is typical of several obtained under identical conditions. The maximum biomass production was achieved after

120 h, close to the point of exhaustion of the carbon source (ie glucose). It was also the point where the content of fatty acids together in biomass reached a maximum at around 70% biomass. Interestingly, just after 24 h of cultivation, the DHA content jumped to 30% of fatty acids together, after which it remained constant at 20-25%. These results are consistent with

- 104 results for other fatty-producing Thraustochytridae strains, but there is a discrepancy regarding the rate at which these reactions occur.

c) Discussion [0314] 330. To date, most Labyrinturomycota studies have identified strains that are capable of storing fatty acids together in amounts of no more than 20% biomass. For example, before isolating Schizochytrium sp. SR 21, which has the ability to accumulate up to 50% biomass as fat, the best organism accumulating 20% ​​biomass was T. aureum (Bajpai et al., J Am Oil Chem Soc 68: 509- 514 (1991)). On the other hand

ONC-T18 has the ability to accumulate up to 80% of its biomass in the form of a lipid.

[0315] 331. For oily microorganisms such as ONC-T18 to accumulate oil, typically they should be cultured in a culture medium with a limited amount of nitrogen (typically depleted after 24 to 36 h) and abundant carbon sources. When the nitrogen is depleted, the microorganisms assimilate oil still a carbon source, but can no longer undergo cell division due to a lack of nitrogen (to prevent the synthesis of proteins and nucleic acids). The result is the conversion of these carbon sources (i.e., sugars, such as glucose) into reserve oils. In this regard, it is believed that ONC-T18 growing more slowly than other strains Thraustochytridae such as G13 (Bowles et al., J Biotechnol 70: 193-202 (1999) and Huang et al., Mar Biotechnol 5: 450-457 ( 2003), yet it produces DHA at higher rates and has the unique ability to incorporate elevated amounts of fatty acids together. Finally, the ability of ONC-T18 to grow at very low salt concentrations with high yields of both biomass and fatty acids together is noteworthy. What is suitable for enlarging the scale by disregarding the corrosive effect of salt water on industrial fermentation equipment.

Representative of DSM Nutritional Products AG:

- EP 547476 B1