Process for preparing docosahexaenoic acid and docosapentaenoic acid

The present invention relates to a process for the preparation of lipids containing docosahexaenoic acid (hereinafter referred to as "DHA") and / or docosapentaenoic acid (hereinafter referred to as "DPR") by culturing the microorganism, as well as a method for obtaining DHA and / or DPR from lipids. The invention also describes a microorganism belonging to the Ulkenia genes and having the ability to synthesize lipids.

State of the art

DHA is present in fish oil made from fish belonging to the blue fish group. Extreme amounts of DHA are found in sardine and tuna fish oils, which contain approximately 20% DHA.

Recently, the discovery of fish tissues with high levels of DHA, such as tuna loins, or technological advances in the production of highly purified fatty acids have led to intense exploration of the physiological functions of DHA and its practical application. The physiological functions of DHA include cholesterol lowering, anticoagulant, and carcinostatic effects. In connection with the metabolic brain system, DHR improves memory, learning, stops dementia in the elderly, and cures Alzheimer's disease. The supplement has proven that DHR is a valuable fatty acid that stimulates fry growth. Due to the properties listed above, DHR is used in a variety of foods, feeds and bait.

DPR is present in fish oil, although it is present in very small amounts. Most

The physiological functions of DPR are still unknown. The only known physiological function of DPR is its ability to deliver pharmaceutical agents to the brain [Japanese Patent Publication (Kokai) No. 61: 204136 (1986)]. But it is expected that DPR can affect physiological processes in the body of an animal as it is known that

DPR compensates for the lack of DHR in the animal body [Homayou et al., J. Neurochem., 51:45 (1988); Hamm et al., Biochem. J., 245: 907 (1987); and Rebhung et ak, Biosci. Biotech. Biochem., 58: 314 (1994)].

There are some disadvantages in isolating DHA and / or DPR from fish oil, such as low levels of desired fatty acids, and it is not possible to maintain a constant source of fish oil due to fish migration or the odor inherent in fish oil. In addition, good quality lipids are difficult to isolate because fish oil contains other fatty acid impurities such as arachidonic (ARR) and eicosapentaenoic (EPR) acids which oxidize lipids.

In addition to fish oil, lipids accumulate in cultured cells of a microorganism capable of synthesizing DHR and / or DPR and are a source of stored DHR and / or DPR. For example, the following microorganisms synthesizing DHR and / or DPR are known: Vibrio marinus ATCC 15381, a bacterium isolated from deep water; Vibrio bacterium isolated from the gut of deep-sea fish; flagellate fungi such as Thraustochytrium aureum 34304, Thraustochvtrium sp.

ATCC 28211, ATCC 20890 and ATCC 20891, Schizochytrium sp., ATCC 20888 and ATCC 20889 (U.S. Patent No. 5,340,742), Traustochytrium SR21 strain (Nippon Nogei Kagaku Kaishi, vol. 69, extra edition. July 5, 1995), and Japonochytrium. m.p. ATCC 28207 [Japanese Patent Publication (Kokai) no. 1199588 (1989)]; microscopic seaweed such as Cvclotella cryptica,

2.5 Crypthecodinium cohnii [Japanese Patent Publication (Kohyo) no. 5-503425 (1993)], and Emiliania sp. [Japanese Patent Publication (Kokai) no. 5-308978 (1993)].

The use of any of the microorganisms listed above raises several issues, such as low yields of DHA and / or DPR, since an extended culture period, specific nutrient media, or special culture conditions are required to obtain sufficient amounts of DHA and / or DPR. When seaweed, such as Emiliania sp., Is used for synthesis, DHR can be obtained with good yields, although it can be difficult because cultivation is hindered by the light necessary for this process. This means that this process is inconvenient for industrial use.

The present invention describes the possibility of synthesizing DHR and / or DPR as well as lipids containing DHR. and / or DPR using inexpensive and convenient nutrient media and simple manufacturing techniques in a short time and with good yields.

The present invention also relates to the production of lipids containing DHR and / or DPR by culturing in a nutrient medium a microorganism belonging to the genes Ulkenia, which is capable of synthesizing DHR and / or DPR and isolating lipids from the culture.

The invention also provides a method of producing DHR and / or DPR by culturing in a nutrient medium a microorganism belonging to the genes Ulkenia, which is capable of synthesizing DHR and / or DPR by isolating lipids from the culture and separating DHR and / or DPR from lipids.

THE SUBSTANCE OF THE INVENTION

According to the present invention, the process for the preparation of lipids containing docosahexaenoic acid and docosapentaenoic acid consists in the cultivation of a microorganism belonging to the Ulkenia genes and capable of producing lipids containing docosahexaenoic acid and docosapentaenoic acid in culture medium.

The method of producing the docosahexaenoic acid according to the present invention comprises isolating said lipids from said culture with a microorganism belonging to the Ulkenia genes having the ability to produce lipids containing docosahexaenoic acid in culture medium, and isolating said docosahexaenoic acid from said lipids.

According to the present invention, the method of producing docosapentaenoic acid consists of isolating said liposomes from said culture with a microorganism belonging to the Ulkenia genes having the ability to produce lipids containing docosapentaenoic acid in culture medium, and isolating said docosapentaenoic acid from said lipids.

The invention relates to cells of a microorganism belonging to the genes Ulkenia containing lipids containing docosahexaenoic acid and / or docosapentaenoic acid.

The present invention describes Ulkenia sp. Strain SAM 2179, which has the property of synthesizing lipids containing docosahexaenoic acid and docosapentaenoic acid.

The present invention relates to nutritional supplements, a composition suitable for infant nutrition, a composition suitable for premature infants, a baby food, a food for pregnant and pregnant women, a food for the elderly, an enteral agent for feeding, livestock feed, livestock feed supplement, and bait. for the nutrition of microorganisms containing lipids obtained by any of the above methods.

The method of producing the structured peptides comprising docosahexaenoic acid and docosapentaenoic acid according to the present invention comprises the step of culturing the microorganism belonging to the Ulkenia genes and having the ability to produce lipids containing docosahexaenoic acid and docosapentaenoic acid by culturing the lipids from the culture. to convert fatty acids at positions 1 and 3 to medium-chain fatty acids (C:

8-12, cf. "SECOND EDITIONS" pp. 834, ΤΟΚΥΟ KAGAKUDOJIN (1990)).

BRIEF DESCRIPTION OF THE FIGURES

FIG. Figure 1 is a liquid chromatogram of triacylglycerols in a neutral lipid synthesized using the process of the present invention.

METHOD OF IMPLEMENTING THE INVENTION

The present invention is further described below. As used herein, the term "docosahexaenoic acid" or "DHR" is assigned to the docosahexaenoic acid (n-3) series. As used herein, the term "docosapentaenoic acid" or "DPR" is assigned to the docosapentaenoic acid (n-3) and / or (n-6) series. The terms "fats", "lipids" and "oils" are used interchangeably.

Any microorganism belonging to the Ulkenia genes may be used in the present invention for the production of DHA and / or DPR as long as they are capable of synthesizing DHA and / or DPR. For example, Ulkenia sp. S AM 2180 and SAM 2179 strains isolated from seawater by the present inventors. Among these strains, SAM 2179 has the ability to synthesize both acids, DHR and DPR, in much larger amounts. This strain was deposited with the National Institute of Biology and Human Technology, Industrial Science and Technology Agency (Address: 1-3, Higashi 1 chome Tsukuba-shi Ibaraki-ken 305, JAPAN), July 23, 1996 under receipt number FERM BP-5601 .

Ulkenia sp. Mycological characteristics of SAM 2179 and SAM 2180 strains are as follows. When these microorganisms were cultured in liquid KMV medium (Fuller. M. and A. Jaworski eds .; Zoosporic Fungi in Teaching & Research VII, 303 pp., 1987. Southern Publishing Corporation, Athens) at 20 ° C and dark, observations were made. spherical or round cells, and biliary zoospores were also seen. However, the ectoplasmic filament network was not visible. Thus, these microorganisms were classified as fungi and classified as Thraustochytriales, according to Yosio, author of Icons of the Japanese Water Mold with Precise Explanation. Kobayasi and Kazuko Konno (published personally by the authors, p. 169, 1986). In addition, these microorganisms formed risoids in the absence of apophyses and formed cells similar to amebas in KMV liquid medium. Therefore, they (microorganisms) were identified as fungi and assigned to the genes Ulkenia, and two isolated as Ulkenia sp. SAM 2179 and SAM 2180, respectively.

The microorganism assigned to the Ulkenia genes used in the method of the present invention is not limited to the wild-type strain, but may also include mutant or recombinant strains. Thus, the use of mutant or recombinant strains to obtain a sufficient amount of DHR and / or DPR is within the scope of the invention. Such mutant or recombinant genera include microorganisms having a higher percentage of DHA and / or DPR in lipids, higher total lipid content or both (both lipid and acid content) in comparison to the percentage concentration of DHA and / or DPR or lipid produced in the original wild-type strain using the same substrates. According to this wild-type strain, it has at least 25% DHA and / or 5% DPR, preferably 40-48% DHA and / or 8-13% DPR in lipids. Further, the wild type strain according to the present invention has less than 3 g of DHR and / or 0.5 g of DPR, preferably 5 g of DHR and / or 1 g of DPR per liter of nutrient medium. In addition, microorganisms for the synthesis of a comparative amount of DHR and / or DPR with an appropriate wild-type strain and especially with substrates at the highest production costs are also described.

The microorganisms of the present invention are cultured by inoculating a liquid or solid nutrient medium with a microorganism inoculum. The nutrient medium may or may not contain natural or artificial seawater.

Any source of carbon including, but not limited to, such as glucose, fructose, xylose, sucrose, maltose, soluble starch, fucose, glucosamine and dextran, as well as oleic acid, fats such as soybean oil, glutamic acid, molasses , glycerol, mannitol and sodium acetate, can be used as a carbon source and added to the nutrient medium.

Natural sources of nitrogen such as peptone, yeast extract, malt extract, meat extract, cassamic acid and corn extract, dried soybeans, organic nitrogen sources such as glutamate and urea sodium and inorganic nitrogen sources such as ammonium acetate, ammonium sulfate, ammonium chloride and ammonium nitrate can be used as a source of nitrogen.

In addition, phosphates such as potassium phosphate and potassium dihydrogen phosphate, inorganic salts such as ammonium sulfate, sodium sulfate, magnesium sulfate, iron sulfate, copper sulfate, magnesium chloride, calcium chloride and vitamins may be added as trace elements, as appropriate.

The amount of these components in the nutrient medium is not specifically determined and must be such that the concentrations of the components do not affect the growth of the micro-organism. Generally, carbon sources can be added in a concentration of 20 to 180 g per liter of nutrient medium and nitrogen sources can be added in concentrations of 0.6 to 6 g per liter of nutrient medium. It is desirable that the amount of nitrogen source increases with increasing carbon source content.

After the nutrient medium has been prepared, its pH is maintained between 3.0 and 8.0, preferably between 3.5 and 5.0, and ideally between 3.5 and 4.5, with a suitable acid or alkali. the medium is sterilized by autoclaving or the like. The cultivation of the microorganism generally takes from 2 to 7 days, preferably from 2 to 5 days, from 10 to 35 ° C, but most preferably from 17 to 30 ° C, and aeration, mixing, or stationary culture is also used.

Additionally, DHR and / or DPR precursors can be added to the culture medium to accelerate the synthesis of DHR and / or DPR. For example, hydrocarbons such as tetradecane, hexadecane and octadecane, fatty acids such as oleic acid, linoleic acid and α-linolenic acid, or salts such as sodium or potassium salts or their esters are used as precursors. fats containing these fatty acids as an ingredient (for example, olive oil, soybean oil, cottonseed oil or palm oil). These components can be used alone or in combination with one another.

Carbon sources, nitrogen sources, precursors or similar materials may be added to the culture medium before or during cultivation. These components can be added once, repeatedly or permanently.

Except for a sufficient amount of lipids containing DHR and / or DPR for practical use , it is best to use liquid media and cultivate with aeration-mixing. A conventional fermenter mixer or fermenter barbecue column may be used.

By culturing as described above, lipids containing DHR and / or DPR are obtained and accumulated in cells. Where a liquid medium is used, lipids containing DHR and / or DPR may be isolated from the culture either from the sterilized culture, from the culture at the end of the culture or from the culture at the end of the culture, either from the cultured cells or from the dried cells. of the cultures described above. The term "culture" as used above includes cultured cells, dried cultured cells, and cultured cultured cells; as well as cell culture and supematant culture of meat broth.

DHA and / or DPR can be distinguished from lipids containing DHR and / or DPR isolated from cultured cells as listed below. After culturing, the cells are harvested from the culture by a commonly accepted solid / liquid separation such as centrifugation and filtration. The cells are washed extensively with water and preferably when they are subsequently dried. Cold drying, air drying or the like may be used to dry the cells. The dried cells are then disrupted, for example, using a press or ultrasound and the lipids are released from the cells using an organic solvent, preferably in a nitrogen atmosphere. Organic solvents such as ether, hexane, methanol, ethanol, chloroform, dichloromethane or petroleum ether may be used. Alternative methanol-petroleum ether extraction or solvent mixed solvent system consisting of chloroform / methanol / water may also be used. High lipid content containing DHR and / or DPR is obtained by evaporation of the organic solvent from the extract under reduced pressure.

... v

Alternatively, wet cell extraction may be performed. In this case, a solvent such as methanol and ethanol in water, or a mixed solvent consisting of alcohol (s) and water, and / or other solvents in water, may be used. Other steps are performed as described above.

The amount of DHA in the lipids obtained as described above is at least 3 g per liter of culture, preferably 5 g per liter of culture.

The amount of DPR in the lipids is at least 0.7 g per liter of culture, most preferably

1.0 g per liter of culture.

In lipids prepared as described above, DHR and / or DPR are provided in the form of neutral lipids (e.g. triacylglycerol) or polar lipids (e.g. phosphatidylcholine, phosphatidylethanolamine or phosphatidylinositol). Purification of triacylglycerols containing DHR and / or DPR from lipids containing DHR and / or DPR isolated from the culture is accomplished by standard methods such as cooling separation or column chromatography.

Typically, the amount of neutral lipids in the lipids of the present invention, as described in the present invention, is very high (more than 90% of the total lipid content). An example of the composition of fatty acids in neutral lipids is as follows palmitic acid: 30-38%; (n-3) DHR: 40-48%; (n-6) DPR: 8-13%; (n-3) EPR: 0-1%; ARR: 0-0.6%; other fatty acids: 10-20%.

Neutral lipids, containing at least 85% of the triacylglycerols of the total lipids of the present invention, preferably at least 90% of the triacylglycerols. The content of diacylglycerols and monoacylglycerols in neutral lipids is very low. The free sterol and / or sterol ester content is also obtained in 1-3%. The following typical molecular species are found in triacylglycerols:

: 0-16: 0-22: 5, 16: 0-16: 0-22: 6, 16: 0-22: 5-22: 6,: 0-22: 6-22: 6, 22: 5- 22: 6-22: 6 and 22: 6-22: 6-22: 6, where, for example, "16: 0" refers to a fatty acid having "16" carbon atoms and having ("0") double bonds. Interestingly, in the lipids described in our invention, triacylglycerols consist only of polyunsaturated fatty acids.

The DHR and / or DPR is separated from the lipids containing DHR and / or DPR by hydrolysis of the lipids followed by concentration and separation of the resulting mixed fatty acids or mixed fatty acid esters prepared by standard methods such as urea addition, cooling separation or column chromatography.

DHA and / or DPR, as well as lipids containing DHA and / or DPR prepared as described above, may be added to a variety of food, feed or bait to increase the lack of DHA and / or DPR. Examples of such foods include, for example, nutritional supplements that are suitable for infant or preterm infants, healthy foods, functional foods (such as enteric factor to induce digestion), baby foods, foods for pregnant or breastfeeding women, and the elderly. Feed includes pet food such as pigs and cows, poultry food such as chickens and food for dogs, cats and the like, for fish growth. Bait includes, for example, microorganisms (so-called zooplankton) that are given as bait in the cultivation of fish and molluscs.

For feed and bait, it is more advantageous and economical to use the microorganism culture of the invention, cells isolated from culture, or cellular residues after lipid extraction. For example, microorganism cells that produce DHR and / or DPR can be used directly to feed fry (juvenile fish), instead of indirectly feeding on zooplankton, or the like. These materials can be used as needed after drying or sterilization.

The lipids described in the present invention can be used to correct DHR and / or DPR deficiency in, for example, poultry eggs, which are deficient in these acids and are therefore suitable for feeding poultry, preferably chick eggs, to a lipid-containing food of the invention. Egg yolk oil enriched with DHR and / or DPR may also be produced by extracting oil from such poultry eggs or egg yolk using a generally accepted method. Ingredients that contain egg yolk oil are suitable for children, premature infants, infants, pregnant and nursing women.

It has long been attempted to produce a powdered milk composition for children that is similar in composition to that of a woman. It is especially important that the main ingredients of a woman's milk (proteins, fats and sugars) are the same as the ingredients in powdered milk. This is very problematic precisely because of the lipid content because plain powdered milk does not contain enough polyunsaturated fatty acids, which are enough in breast milk. Several reports of polyunsaturated fatty acids in breast milk have been published (polyunsaturated fatty acids in American, European and African women's milk, see INFORM 6 (8): 940-946 (1995); polyunsaturated fatty acids in Japanese women's milk, see JJPEN, 13 (9) : 765-772 (1991).

Recently, ARR and DHA, both of which are included in the milk of a woman, have been shown to be effective for growth in children (Advances in Polyunsaturated Fatty Acid Research, Elsevier Science Publishers, pp. 261-264 (1993)). The importance of ARR and DHR in height gain and brain development has also been described (Proc. Natl. Acad. Sci. USA, 90: 1073-1077 (1993); Lancet, 344: 1319-1322 (1994)).

Therefore, there is an increased focus on the addition of DHR and ARR to modified milk. Modified milk containing fish oil as a source of DHA is already marketed. Fish oil also has EPR, which is hardly present in breast milk, and which has been reported to adversely affect the growth of underdeveloped infants, Advs in Polyunsaturated Fatty Acid Research, Elsevier Science Publishers, p. 261-264 (1993). The lipids of the invention are suitable as modified milk supplements because they have a particularly low EPR content and can be added to baby food.

The lipids of the invention may be added to foods, such foods with nutrient additives, to foods for the elderly, or to healthy foods for correcting DHA and / or DPR deficiency or improving snack. The food composition may be in liquid, solid or oily form. Preferably, the lipid content of the food is from 0.001% to 50% by weight, depending on the type of food to which the lipid is added.

Examples of foods containing oils include natural foods (such as meat, fish or nuts), foods to which lipids are added during cooking (such as soup), foods that use lipids as a cooking medium (such as donuts), fatty foods (such as donuts) butter), processed food into which lipids are added during cooking (such as biscuits), food into which lipids are injected or used in the final stage of cooking (such as chips). The lipids (or isolated DHA and / or DPR) of the invention may also be added to agro-culture food, enzymatic food, pet food, seafood or non-fat beverage.

Alternatively, the lipids of the invention may also be added to a functional food that exhibits the physiological action of DHA and / or DPR to prevent the restoration of impaired bodily functions or the reduction of physiological activity. The functional food of the present invention may be in a pharmaceutical or processed form (such as an enteral agent for nutrition, powders, granules, tablets, internal solution, suspension, emulsion, syrup or the like) in which the lipids of the invention are combined with proteins, saccharides. , lipids, rare elements, vitamins, emulsifiers or flavorings.

In addition, the lipids of the invention may be used as an additive in cosmetics or as a detergent, as a starting material for the preparation of their derivatives, which can be used as a medicament.

The invention will now be described by way of example but not by way of limitation.

An example

Method of lipid production using genes belonging to microorganisms

Ulkenia (1)

Ulkenia sp. SAM 2180 and SAM 2179 strains were cultured in a 5-liter (1) volume fermenter (pitcher fermenter) containing 3 L of nutrient medium under the following culture conditions:

(1) The composition of the nutrient medium

<td>(1) Glucose (g / 1):</td><td> 60</td>

<td>(2) Potassium phosphate (g / l):</td><td> 3</td>

<td>(3) Ammonium sulphate (g / l):</td><td> 2</td>

<td>4) Aqueous solution of maize (g / 1):</td><td> 0,7</td>

<td>5. 50% artificial sea water (1):</td><td> 1</td>

<td>6) pH:</td><td> 4,0</td>

(2) Cultural conditions

(1) Cultivation temperature (° C): 28

Aeration Volume (VVM): 0.5

(3) Stirring speed (revolutions per minute): 300

4) pH adjustment: pH 4 is maintained with 10% (g / l) sodium hydroxide and 1M sulfuric acid.

After culturing, cells are harvested by centrifugation and freeze-dried, followed by measurement of the cell content (by weight) per liter of culture medium. Subsequently, cell destruction and lipid extraction were performed by adding the chloroform / methanol mixture (2: 1 by volume) to the dried cells at a ratio of 100 cell volumes by cell weight and homogenizing the mixture using vitreous humor. After washing the extract with the Folch method, the solvent was evaporated to give the purified lipids, and the lipid mass was then measured.

To evaluate the fatty acid composition after lipid purification, methyl esters of fatty acids were prepared by dissolving a portion of the lipids in a mixed solution of an equal volume of methanol solution containing 10% HCl and dichloromethane at 60 ° C for two hours. The esters were subjected to gas chromatography to analyze the fatty acid composition. The gas-liquid chromatography separation conditions were as follows:

(3) Conditions for Separation

1) Column: Capillary column TC-70 (GL Science Co., LTD.), 0.25 mm x 30 m internal diameter

2) Flow rate: 0.8 ml / min, 100 kPa (column head pressure)

3) Carrier gas: Nitrogen gas

4) Column temperature: Method of rise, 170-220 ° C (4 ° C / min)

5) Registration: FID

Data are presented in Tables 1 and 2

TABLE

<td>Strain</td><td>Cultivation time (days)</td><td>Dried cells mass (g) * l</td><td>Total lipid content (g) * i</td><td>Lipids percentage quantity (% by weight) * 2</td><td>DHR quantity (g) * l</td><td>DPR quantity (g) * l</td>

<td>SAM2180</td><td> 3</td><td> 23,2</td><td> 14,1</td><td> 61</td><td> 4,0</td><td> 0,9</td>

<td>S AM 2179</td><td>J</td><td> 19,5</td><td> 11,9</td><td> 61</td><td> 5,5</td><td> 1,3</td>

* 1) Mass per liter of nutrient medium * 2) Percentage relative to dried cells

TABLE

<td>Strain</td><td> 14 : 0</td><td> 15 : 0</td><td> 16 : 0</td><td> 17 : 0</td><td> 18 : 0</td><td>20: 4 (ARR)</td><td>20: 5 (EPR)</td><td>22: 5 (DPR)</td><td>22: 6 (DHR)</td>

<td>SAM 2180</td><td> 2,7</td><td> 2,4</td><td> 55,0</td><td> 1,0</td><td> 1,4</td><td> •</td><td> 0,2</td><td> 6,7</td><td> 28,7</td>

<td>SAM 2179</td><td> 2,4</td><td> 0,9</td><td> 37,2</td><td> 0,3</td><td> 0,8</td><td> 0,4</td><td> 0,6</td><td> 10,6</td><td> 46,2</td>

An example

Method of lipid production using genes belonging to microorganisms

Ulkenia (2)

Method for the preparation of lipids using microorganisms belonging to Ulkenia sp. S AM 2179 strain was cultured in a 5 L fermenter (fermenter as pitcher) containing 3 L of nutrient medium containing this composition under the following culture conditions.

1) The composition of the nutrient medium

<td></td><td>(1) Glucose (g / 1):</td><td> 60</td>

<td></td><td>(2) Potassium phosphate (g / l):</td><td> 3</td>

<td> 30</td><td>(3) Ammonium sulphate (g / l):</td><td> 2</td>

<td></td><td>(4) Magnesium chloride (g / l):</td><td> 1,3</td>

<td></td><td>(5) Sodium sulphate (g / l):</td><td> 1</td>

<td></td><td>6. Calcium chloride (g / l):</td><td> 0,3</td>

<td></td><td>7. Corn solution in water (g / l):</td><td> 0,7</td>

<td> 35</td><td>8) pH: 2) Cultural conditions</td><td> 4,0</td>

<td></td><td>1. Cultivation temperature (° C):</td><td> 28</td>

<td></td><td>(2) Aeration Volume (VMM):</td><td> 0,5</td>

<td> 40</td><td>(3) Stirring speed (revolutions per minute):</td><td> 300</td>

<td></td><td>4) pH adjustment: maintain pH 4 with 10</td><td>% (g / l) of sodium</td>

hydroxide and 1 M sulfuric acid.

After culturing, the cells were harvested by centrifugation and freeze-dried, and the cell weight (wt) per liter of culture medium was weighed. This is followed by cell destruction and lipid extraction by addition of chloroform / methanol mixture (v / v 2T) to dried cells at 100 v / v cell homogenisation,. 10 using glassware. After washing the extract with the Folch method, the solvent was evaporated to give the purified lipids and the lipid mass was then measured.

To evaluate the composition of fatty species after lipid purification, methyl esters of fatty acids were prepared by dissolving a portion of the lipids in a mixed solution of an equal volume of methanolic solution containing 10% HCl and dichloromethane at 60 ° C for two hours. The esters were subjected to gas chromatography to analyze the fatty acid composition. Gaseous liquid chromotography separation conditions were as described in the first example.

Data are presented in Tables 3 and 4.

TABLE

<td>Strain</td><td>Cultivation time (days)</td><td>Dried cells mass (g) * l</td><td>Total lipid content (g) * i</td><td>Lipids Percentage Quantity (wt%) * 2</td><td>DHR quantity (g) * l</td><td>DPR quantity (g) * l</td>

<td>SAM 2179</td><td> 3</td><td> 21,5</td><td> 12,0</td><td> 56</td><td> 5,5</td><td> 1,5</td>

* 1) Mass per liter of nutrient medium * 2) Percentage relative to dried cells

4 TABLE

<td>Strain</td><td> 14 : 0</td><td> 15 :0</td><td> 16 : 0</td><td> 17 : 0</td><td> 18 : 0</td><td>20: 4 (ARR)</td><td>20: 5 (EPR)</td><td>22: 5 (DPR)</td><td>22: 6 (DHR)</td>

<td>SAM 2179</td><td> 2,0</td><td> 1,5</td><td> 34,3</td><td> 0,5</td><td> 0,9</td><td> 0,7</td><td> 0,7</td><td> 12,4</td><td> 45,8</td>

An example

Lipid belonging to Ulkenia sp. S AM 2179. analysis

Neutral and polar lipids were separated from the lipids obtained in the first example by the commonly used liquid-liquid partitioning method using the appropriate hexane and 90% methanol. 0.92 g of neutral lipids and 0.05 g of polar lipids were prepared from 1 g of lipids. Neutral and polar lipid results were analyzed using thin layer chromatography. A color test was performed using sulfuric acid. Identification of the resulting spots was confirmed by comparing their Rf values ​​with standard lipids.

More than 90% of the neutral lipids were triacylglycerols. Polar lipids consist of phosphatidylcholine (60-80%), phosphatidylethanolamine (5-20%) and phosphatidylinositol (2-8%).

Triacylglycerols in neutral lipids were further analyzed by molecular species separation using liquid chromatography (column; ODS column; mobile phase; acetone / acetonitrile (3: 2); registration: differential refractometer) (see Table 1). The resulting peaks were isolated and converted to methyl esters after hydrolysis. The fatty acid residues were determined using gas-liquid chromatography.

Five major vertices were identified as shown in Table 5. Triacylglycerols were composed of 12.8% 1,2,3-tri-docosahexaenolotriacylglycerol and 8.0% l-docosapentaenol-2,3-di-docosahexaenolotriacylglycerol. About 20% of all triacylglycerols were composed of tripol unsaturated fatty acids.

TABLE

<td>Peak</td><td colspan="4">Types of molecules</td><td>Ratio (%)</td>

<td> 1</td><td> 22</td><td> 6-22</td><td> 6-22</td><td> 6</td><td> 12,8</td>

<td> 2</td><td> 22</td><td> 5-22</td><td> 6-22</td><td> 6</td><td> 8,0</td>

<td> 3</td><td> 16</td><td> 0-22</td><td> 6-22</td><td> 6</td><td> 18,3</td>

<td> 4</td><td> 16</td><td> 0-22</td><td> 5-22</td><td> 6</td><td> 8,1</td>

<td> 5</td><td> 16</td><td> 0-16</td><td> 0-22</td><td> 6</td><td> 10,8</td>

An example

Determination of fatty acid residues depending on their position in triacylglycerols

The position dependence of fatty acid residue positions on triacylglycerols is described in the third example and was analyzed as follows. The triacylglycerols described in the third example (molecular species; 16: 0-16: 0-22; 6) were dried and treated with lipase (from Rhizopus japonicus), which is specific for position 1.3. The fatty acid residues were determined using GC / MS and were trimethylsilylated upon receipt of 2monoacylglycerols. Lipase treatment was performed in 2 mL of 50 mM acetate buffer (pH 5.5) with 1000 units of lipase at 35 ° C for 30 minutes. The reaction products were extracted with ether and trimethylsilylated using a commercially available trimethylsilylation agent.

The peak of the fragment corresponding to the molecular weight of the triacylglycerols to which the 22: 6 is attached indicated that the triacylglycerols were 16: 0-22: 6-16: 0 to which the 22: 6 fatty acid residues were attached at the 2 position of the glycerol chains .

An example

Production of modified milk containing DHR and / or DPR

Modified milk containing DHR and / or DPR was prepared by adding 0.44 g of lipid from Example 1 containing 46.2% of DHR and 10.6% of DPR to 100 g of milk powder.

The composition of DHA and DPR in the final product was 0.80% and 0.19% of the total fatty acids, respectively, which was similar to that found in human milk.

An example

Preparation of structured lipid containing DHR and DPR in ml of lipase solution (5,600 v / ml, lipase specific for position 1.3 from Rhizopus delemar) was mixed with 2.5 g of calcium carbonate as an immobilization carrier. The enzyme was immobilized and precipitated by addition of 40 ml of acetone to the mixture and then the immobilized enzyme was dried. The specific activity of the resulting immobilized enzyme was 9.3 v / mg. 120 mg of immobilized enzyme was. mixed with 1 g of DHR and DPR-containing lipids prepared from S AM 2180, Example 1, 2 g of caproic acid and 60 mg of water, shaking at 30 ° C for 8 hours. The triacylglycerols were then isolated from the reaction mixture using a generally accepted method and the fatty acids contained in the triacylglycerols were determined. The data are shown in Table 6.

TABLE

<td></td><td> 8:0</td><td> 14:0</td><td> 15:0</td><td> 16:0</td><td> 17:0</td><td> 18:0</td><td>20: 4 (ARR)</td><td>20: 5 (EPR)</td><td>22: 5 (DPR)</td><td>22: 6 (DHR)</td>

<td>Yes processing</td><td> 0,0</td><td> 2,7</td><td> 2,4</td><td> 55,0</td><td> 1,0</td><td> 1,4</td><td> 0,0</td><td> 0,2</td><td> 6,7</td><td> 28,7</td>

<td>After processing lipase</td><td> 36,2</td><td> 1,8</td><td> 1,2</td><td> 17,6</td><td> 1,0</td><td> 1,8</td><td> 0,0</td><td> 0,2</td><td> 7,3</td><td> 30,4</td>

More than 30% of the triacylglycerols contained polyunsaturated fatty acids at the SN 2 position of the glycerols. Thus, these lipids are suitable for the production of structured lipids containing mid-chain fatty acids at the SN 1 and SN 3 positions of glycerols and polyunsaturated fatty acids at the SN 2 position of glycerols.

INDUSTRIAL APPLICABILITY

Lipids with high levels of DHR and / or DPR and low levels of EPR can be prepared using the preparation method described in the invention. DHA and / or DPR can also be prepared by further isolating them from lipids.

The lipids containing DHR and / or DPR isolated by the invention and used in food, feed, bait, pharmaceuticals and the like are described herein. Cells containing DHR and / or DPR as described in the invention may be used as feed or bait. Poultry eggs or poultry egg yolks may be enriched with DHR and / or DPR by feeding the poultry with the feed described in the invention.

DEFINITION OF INVENTION