Method of obtaining docosahexamic and docosapentaenic acids

Description of the invention

The invention relates to a method of producing lipids containing docosahexaenoic acid (hereinafter also referred to as DHA) and / or docosapentaenoic acid (hereinafter also referred to as DPA) by culturing a microorganism, as well as a method of producing DHA and / or DPA from lipids. The invention further relates to a microorganism belonging to the genus Ulkenia, capable of producing lipids and a lipid extract, nutritional supplements for humans and animals, and the use of the microorganism as nutritional supplements.

DHA is a component of the oil of fish belonging to the group of blue fish. DHA in particular is part of sardine and tuna oil, with a content of approximately 20%.

Recently, thanks to the discovery of the sources of raw material derived from fish with high DHA content, the fat body of the eye socket of tuna, and thanks to advances in the production of highly purified fatty acids, a lot of effort has been put into explaining the physiological functions of DHA and investigating its practical application. The physiological functions of DHA have been found to include cholesterol-lowering, anticoagulant and cancer-inhibitory effects. In the case of metabolic changes in the brain, it has been found that DHA is effective in improving memory and learning ability, prevents senile dementia and has a therapeutic effect in the treatment of Alzheimer's disease. Moreover, it has been proven that DHA is an essential fatty acid for the growth of fry. For these reasons, DHA is used in a variety of foods, feeds, and baits.

DPA is also known to be an ingredient in fish oil, although its content is extremely low. The physiological functions of DPA remain largely unknown. The only known function of DPA is its utility as a vehicle for delivering pharmacological agents to the brain [Japanese Patent Publication (Kokai) No. 61-204136 (1986)]. It should be assumed that DPA may play a physiological role in the animal organism due to the known phenomenon of an increase in the amount of DPA in the organism compensating for DHA deficiency [Homayoun et al., J.Neurochem., 51:45 (1988); Hamm et al., Biochem. J., 245: 907 (1987) and Rebhung et al., Biosci. Biotech. Biochem. 58: 314 (1994)].

Obtaining DHA and / or DPA from fish oil has numerous disadvantages, such as the low concentration of the desired fatty acids, the inability to provide a stable source of fish oil due to fish migration, and the intense odor inherent in fish oil. Moreover, it is difficult to obtain lipids of reliable quality because the fish oil also contains unsaturated fatty acids such as arachidonic acid (ARA) and eicosapentaenoic acid (EPA), which make the lipids more susceptible to oxidation.

In addition to fish oil, the source of DHA and / or DPA may also be lipids accumulated in cultured cells of a microorganism capable of producing DHA and / or DPA. For example, the following DHA and / or DPA producing microorganisms are known: Vibrio marinus ATCC 15381, bacterium isolated from the deep sea; the Vibrio bacterium isolated from the insides of deep-sea fish; flagellate fungi such as Thraustochytrium aureum ATCC 34304, Thraustrochytrium sp. ATCC 28211, ATCC 20890 and ATC 20891, Schizochytrium sp. ATCC 20888 and 20889 (US Patent No. 5340742), Thraustochytrium SR21 strain (Nippon Nogei Kagaku Kaishi, vol. 69, additional edition, July 5, 1995) and Japanochytrium sp. ATCC 28207 (Japanese Patent Publication (Kokai) No. 1-199588 (1989)]; microalgae such as Cyclotella Cryptica, Crypthecodinium cohnii (Japanese Patent Publication (Kohyo) No. 5-503425 (1993)] and Emiliania sp. (Japanese Patent Publication (Kokai) No. 5-308978 (1993)].

The use of each of the above-mentioned microorganisms is associated with a number of problems, such as, for example, low DHA and / or DPA yield, or the requirements of a specific medium or culture conditions. When used in the production of algae such as Emiliania sp., High DHA yield can be obtained, but with the disadvantage that its cultivation requires light. This process is not suitable for industrial production.

The present invention enables the production of both DHA and / or DPA and DHA and / or DPA containing lipids using inexpensive conventional medium and simple production steps in short time and with high yield.

The invention relates to a process for the production of DHA and / or DPA containing lipids comprising culturing a microorganism belonging to the genus Ulkenia in a nutrient medium, capable of producing DHA and / or DPA, and isolating the lipids from the culture.

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The invention further relates to a process for the production of DHA and / or DPA comprising culturing a microorganism belonging to the genus Ulkenia on a nutrient medium, capable of producing DHA and / or DPA, isolating lipids from the culture and isolating DHA and / or DPA from a lipid mixture.

The invention relates to a process for the production of docosahexaenoic acid and / or docosapentaenoic acid containing lipids which comprises culturing in a medium a microorganism belonging to the Ulkenia genus capable of producing docosahexaenoic acid and / or docosapentaenoic acid containing lipids and recovering the lipids from the culture.

The method for producing docohexaenoic acid and / or docosapentaenoic acid further comprises separating the docosahexaenoic acid and / or docosapentaenoic acid from the lipids.

The method of producing structured lipids containing docosahexaenoic acid and / or docosapentaenoic acid further comprises treating the lipids with a fungal lipase to convert the 1 and 3 fatty acids into C8-12 fatty acids.

The microorganism used in the method according to the invention is Ulkenia sp. SAM 2179 (FERM BP-5601).

The invention also relates to cells of the microorganism Ulkenia sp. SAM 2179 (FERM BP-5601) containing lipids including neutral lipids containing 30-38% palmitic acid, 40-48% docosahexaenoic acid, 8-13% docosapentaenoic acid, 0-1% acid eicosapentaenoic acid, 0-0.6% arachidonic acid and 10-20% other fatty acids by weight percent based on total fatty acid content.

Cells of the Ulkenia sp. SAM 2179 microorganism strain (FERM BP-5601) contain lipids including docosahexaenoic acid and / or docosapentaenoic acid, which cells include neutral lipids containing triacylglycerols consisting only of polyunsaturated fatty acids.

In cells, a neutral lipid contains at least 85% by weight, preferably 90% by weight, of triacylglycerols.

The subject of the invention is also Ulkenia sp. SAM 2179 (FERM BP-5601) strain capable of producing lipids including neutral lipids containing 30-38% palmitic acid, 40-48% docosahexaenoic acid, 8-13% docosapentaenoic acid, 0-1% acid eicosapentaenoic acid, 0-0.6% arachidonic acid and 10-20% other fatty acids by weight percent based on total fatty acid content.

The lipid extract obtained from the microorganism belonging to the genus Ulkenia as defined above includes neutral lipids containing 30-38% palmitic acid, 40-48% docosahexaenoic acid, 8-13% docosapentaenoic acid, 0-1% eicosapentaenoic acid, 0-0.6% arachidonic acid and 10-20% other fatty acids on a weight percent basis based on the total fatty acid content.

The lipid extract comprises docosahexaenoic acid and / or docosapentaenoic acid, the lipid extract comprising neutral lipids containing triacylglycerols consisting only of polyunsaturated fatty acids, and the neutral lipid containing at least 85% by weight, preferably 90% by weight of triacylglycerols.

A lipid extract obtained from a microorganism belonging to the genus Ulkenia containing lipids that include docosahexaenoic acid and / or docosapentaenoic acid as defined above includes neutral lipids containing about 20% triacylglycerols consisting only of polyunsaturated fatty acids.

The preparation of the structural lipids is prepared by treating the lipid extract defined above with a fungal lipase to convert the fatty acids at the 1 and 3 positions into C8-12 fatty acids.

The lipid extract or structured lipid preparation is characterized in that the microorganism is the strain Ulkenia sp. SAM 2179 (FERM BP-5601).

The invention also relates to:

a food-supplementing nutritional product containing the lipid extract as defined above; infant formula for infant milk suitable for the nutrition of infants containing a lipid extract as defined above;

substitute artificial mother's milk for infants suitable for the nutrition of immature infants containing a lipid extract as defined above;

baby food containing the lipid extract as defined above;

food for old people containing the lipid extract as defined above;

an enteral extract for the aid of nutrition containing the lipid extract as defined above;

Animal feed containing the lipid extract as defined above or cells as defined above; an animal feed additive containing the lipid extract as defined above or cells as defined above;

a zooplankton feed bait containing the lipid extract as defined above or cells as defined above;

food for expectant babies or nursing mothers containing the lipid extract specified above.

The invention also relates to the use of a microorganism belonging to the genus Ulkenia having the ability to produce docosahexaenoic acid and / or docosapentaenoic acid containing lipids, preferably the Ulkenia sp. SAM 2179 (FERM BP-5601) strain as a nutritional supplement food supplement;

use of a lipid extract as defined above as a nutritional supplement food supplement;

use of a structured lipid preparation as defined above as a nutritional supplement food supplement;

a use as defined above, wherein the food additive is incorporated into a food product for expectant and nursing mothers, infants, immature infants or old people;

the use as defined above as an enteric nutritional aid;

the use defined above as animal feed;

the use as defined above as zooplankton bait for feed, and the use as defined above wherein the microorganism belonging to the genus Ulkenia is applied after drying or sterilization.

The method of producing docosahexaenoic and docosapentaenoic acid-containing lipids according to the invention comprises culturing on a nutrient medium a microorganism belonging to the genus Ulkenia having the ability to produce docosahexaenoic and docosapentaenoic acid-containing lipids and isolating the lipids from the culture.

The method of producing docosahexaenoic acid according to the invention comprises culturing on a medium a microorganism belonging to the genus Ulkenia having the ability to produce docosahexaenoic acid containing lipids, isolating lipids from the culture and isolating docosahexaenoic acid from the obtained lipids.

The method of producing docosapentaenoic acid according to the invention comprises culturing on a nutrient a microorganism belonging to the genus Ulkenia having the ability to produce lipids containing docosapentaenoic acid, isolating lipids from the culture (culture) and isolating docosapentaenoic acid from the obtained lipids.

The invention discloses the cells of a microorganism belonging to the genus Ulkenia, containing lipids composed of docosaheptaenoic acid and / or docosaheptaenoic acid.

The invention further discloses the Ulkenia sp. SAM 2179 strain having the ability to produce docosahexaenoic and docosapentaenoic acid containing lipids.

The invention further comprises lipids obtained by one of the aforementioned methods: a nutritional supplement for food, a formula suitable for feeding an infant, a formula suitable for feeding preterm infants, a baby formula, a formula for pregnant and lactating women, a nutrition for the elderly, an enteral aid to aid infants. nutrition, pet food, pet food additive, bait medium for microorganisms.

The method of producing the structural lipids containing docosahexaenoic acid and docosapentaenoic acid according to the invention comprises the cultivation on a medium of a microorganism belonging to the species Ulkenia having the ability to produce lipids containing docosahexaenoic acid and docosapentaenoic acid, isolating these lipids from the medium and treating the lipids obtained with lipase from the fatty acids in order to convert positions 1 and 3 in medium chain fatty acids (C: 8-12, see "SEIKAGAKUJITEN" (2nd Ed.), Pp. 834, TOKYO KAGAKUDOJ1N (1990).

Brief explanation of the drawings

Fig. 1 shows the liquid chromatogram of triacylglycerols in neutral lipid obtained according to the invention.

PL 193 818 B1

The most preferred embodiment of the invention

The present invention will be described in more detail below. The term "docosahexaenoic acid" or "DHA" as used herein refers to the series (n-3) docosahexaenoic acid. The term "docosapentaenoic acid" or "DPA" as used herein refers to the series (n-3) and / or (n-6) docosapentaenoic acid. The terms "fats", "lipids" and "oil" are used herein with the same meaning.

Any microorganism belonging to the genus Ulkenia can be used in the method of the invention, as long as it is capable of producing DHA and / or DPA. For example, the strains Ulkenia sp. SAM 2180 and SAM 2179, isolated from sea water by the inventors, can be used. The SAM 2179 strain is more effective in producing DHA and DPA. This strain was deposited with the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology (Address: 1-3, Higashi 1 chome Sukuba-shi Baraki-ken 305, Japan) on July 23, 1996 and received a deposit number FERM BP-5601.

The mycological characteristics of the Ulkenia sp. SAM 2179 and SAM 2180 strains are as follows. When culturing these microorganisms in KMV liquid medium (edited by Fuller M. I Jaworski A; Zoosporic Fungi in Teaching & Research VII, 303 pp., Southeastern Publishing Corporation, Athens 1987) at 20 ° C in the dark, spherical or spherical cells were observed. oval and double-stranded zoospores. However, the ectoplasm filament network was not observed. On this basis, according to the "Icons of the Japanese Water Mold with Precise Explanation" acc. Yosio Kobayasi and Kazuko Konno (private publication sponsored by the authors, pp. 169, 1986), these microorganisms were classified as fungi belonging to Thraustochytriales. Moreover, these microorganisms formed rhizoids in the KMV liquid medium, without suckers and amoeboid cells. Hence, they were identified as fungi belonging to the genus Ulkenia, and the two isolated strains were designated Ulkenia sp. SAM 2179 and SAM 2180, respectively.

The microorganisms belonging to the genus Ulkenia used in the method of the invention are not limited to a wild-type strain, but may also include mutant or recombinant strains. Thus, the use of a mutant or recombinant strain designed to produce DHA and / or DPA in an efficient manner is also within the scope of the present invention. Such mutant or recombinant strains include microorganisms designed to produce lipids with a higher percentage of DHA and / or DPA, a higher total lipid amount, or both, compared to the percentage or amount produced by the original wild-type strains using the same starting materials . The wild strains according to the invention contain in their lipids at least 25% DHA and / or 5% DPA, preferably 40-48% DHA and 8-13% DPA. These strains contain at least 3 g of DHA and / or 0.5 g of DPA, preferably 5 g of DHA and / or 1 g of DPA per liter of medium. Furthermore, microorganisms designed to produce a comparable amount of DHA and / or DPA to that obtained with corresponding wild-type strains, efficiently using the starting materials with higher economic benefits, are contemplated.

The microorganisms of the invention are cultivated in the solid or liquid medium by inoculating the solid or liquid medium with microorganism preculture. The medium may or may not contain natural or artificial seawater.

The carbon sources added to the medium are, but are not limited to, any known carbon sources, for example, carbohydrates such as glucose, fructose, xylose, sucrose, maltose, soluble starch, fucose, glucosamine and dextran, and furthermore oleic acid, fats such as like soybean oil, glutamic acid, molasses, glycerol, mannitol, and sodium acetate.

Nitrogen sources are natural nitrogen sources such as peptone, yeast extract, malt extract, meat extract, casamic acid and corn steep extract, soybean cake, organic nitrogen sources such as monosodium glutamate and urea, or inorganic nitrogen sources such as ammonium acetate, sulfate ammonium, ammonium chloride and ammonium nitrate.

Moreover, if desired, 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 and calcium chloride, and vitamins can be added as micronutrients.

The amount of these components in the medium is not particularly precise as long as the concentrations of the components do not adversely affect the growth of the microorganisms. Generally, the carbon sources are added to achieve a concentration of 20 to 180 g per liter of medium, and the nitrogen sources are added to the

In order to obtain a concentration of 0.6 to 6 g per liter of medium. Preferably, the amount of the nitrogen source increases with the increasing amount of the carbon source.

After the medium has been prepared, its pH is adjusted to between 3.0 and 8.0, preferably between 3.5 and 5, and even more preferably between 3.5 and 4.5, using an appropriate acid or base, and then the medium is subjected to autoclaving or other sterilization. The cultivation of the microorganism is usually carried out for 2 to 7 days, preferably for 2 to 5 days, at a temperature of 10 to 35 ° C, preferably 17 to 30 ° C, using agitation by aeration or shaking or in stationary culture conditions.

Moreover, precursors thereof can be added to the medium to accelerate the production of DHA and / or DPA. Examples of such precursors are hydrocarbons such as tetradecane, hexadecane and octadecane, fatty acids such as oleic, linoleic and α-linolenic acids, or salts (e.g. sodium or potassium salts) or esters thereof. In addition, fats containing these fatty acids as an ingredient (e.g. olive oil, soybean oil, cottonseed oil, palm oil) may be added. These ingredients can be added singly or as a mixture.

The sources of carbon, nitrogen, precursors, and other ingredients may be added to the medium before or during cultivation. The addition of these ingredients may take place once, periodically or continuously.

In order to isolate the lipids containing DHA and / or DPA in practical amounts, it is preferable to use a liquid medium and to cultivate the culture under agitation with aeration. A conventional fermentation mixer or bubble column fermenter may be used.

By culturing as described, lipids containing DHA and / or DPA accumulated in the cells are obtained. Using liquid medium, DPA and / or DHA containing lipids are recovered from the culture or from sterilized culture during the cultivation process, after completion of cultivation, or from cultured cells or from dried cells harvested from any of the aforementioned cultures. The term "culture" as used herein includes both cultured cells, dried cultured cells, and processed cultured cells, as well as the culture broth containing the cells and culture supernatants.

DHA and / or DPA are isolated from lipids containing them separated from the culture cells as follows. After completion of the culture, the cells are separated from the culture by a known solid / liquid separation method such as, for example, centrifugation or filtration. The cells are washed extensively with water and then preferably dried. Drying the cells is accomplished by a freeze-drying method, air-drying method or other method. The dried cells are then subjected to destruction, e.g. in a dynamometer or by ultrasound, and the lipids are then extracted from the cells with an organic solvent, preferably under a nitrogen stream. The organic solvent used is ether, hexane, methanol, ethanol, chloroform, dichloromethane or petroleum ether. An alternative method is extraction with methanol and petroleum ether, or extraction with a mixed solvent system of chloroform / methanol / water. A high concentration of DHA and / or DPA containing lipids is obtained by evaporating the organic solvent from the extract under reduced pressure.

Alternatively, wet cells are extracted. In this case, a solvent with an affinity for water, such as methanol or ethanol, or a mixed solvent with an affinity for water, consisting of alcohol (s) and water, and / or other solvents is used. The rest of the procedure is the same as previously described.

The method described above makes it possible to obtain lipids with a DHA content of at least 3 g per liter of medium, preferably 5 g per liter of medium. The amount of DPA in the lipids is at least 0.7 g per liter of medium, preferably 1.0 g per liter of medium.

In the lipids obtained as described above, DHA and / or DPA is present in the form of neutral lipids (e.g. triacylglycerol) or polar lipids (e.g. phosphatidylcholine, phosphatidylethanolamine or phosphatidylinositol). The triacylglycerols containing DHA and / or DPA are purified from the lipids containing them obtained from the culture using known separation techniques such as separation by cooling or column chromatography.

Typically, a large percentage of the total amount of lipids according to the invention (greater than 90% of the total amount of lipids) is neutral lipids. A representative fatty acid composition in neutral lipids is as follows: palmitic acid - 30-38%; (n-3) DHA - 40-48%; (n-6) DPA - 8-13%; (n-3) EPA 0-1%; ARA - 0-0.6%; other fatty acids -10-20%.

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The neutral lipids obtained from the lipids of the invention contain at least 85% triacylglycerols, preferably at least 90% triacylglycerols. The content of diacylglycerols and monoacylglycerols in neutral lipids is very low. Free sterol and / or sterol ester constitute 1-3% of their total amount. Typically, the following molecules are found in triacylglycerols: 16: 0-16: 0-22: 5, 16: 0-16: 0-22: 6, 16: 0-22: 5-22: 6, 16: 0-22: 6-22: 6, 22: 5-22: 6-22: 6 and 22: 6-22: 6-22: 6, where for example "16: 0" means a fatty acid having 16 carbon atoms and no (0 ) double bond. Interestingly, the lipids according to the invention contain triacylglycerols containing only polyunsaturated fatty acids.

The isolation of DHA and / or DPA from lipids containing them is carried out by hydrolyzing the lipids and then concentrating and separating the resulting mixture of fatty acids or esters derived therefrom, using known methods such as addition of urea, separation by cooling or column chromatography.

The DHA and / or DPA obtained as described above, as well as the lipids containing them, can be added to a variety of nutrients, feeds and baits to compensate for DHA and / or DPA deficiency. Examples of such nutrients include, for example, food supplement nutrients, formulations suitable for the feeding of infants or preterm infants, health food, baby formula, maternity and nursing nutrition, and nutrition for the elderly. The feed materials include food for domestic animals such as pigs and cows, food for birds such as chickens, food for dogs, cats and the like, and food for breeding fish. The baits include, for example, a medium for microorganisms (so-called zooplankton) used as bait in fish and shellfish farming.

In particular for feed and baits, it is advantageous and economical to use cultures of the microorganism according to the invention, cells harvested from the culture, or cell debris after lipid isolation. For example, cells of DHA and / or DPA producing microorganisms can be used directly to feed fry (young fish) instead of indirectly feeding them with zooplankton or the like. These materials can be used after drying or when necessary sterilized.

The lipids according to the invention can be used to produce poultry eggs enriched in DHA and / or DPA which are obtained by feeding poultry intended for laying eggs (especially chickens) with lipid containing foods according to the invention. By extracting oil from such poultry eggs or egg yolk in a manner known per se, egg yolk oil enriched with DHA and / or DPA can be obtained. You should also take into account preparations containing egg yolk oil suitable for feeding infants and premature infants, baby food, supplements for pregnant and lactating women.

Efforts have been made for a long time to obtain powdered milk for children with a composition similar to that of human milk. In particular, it is important to obtain a composition of the basic components of human milk (i.e. protein, fat and sugar) in powdered milk similar to that of human milk. However, a problem has arisen that known powdered milks are low in polyunsaturated fatty acids inherent in human milk. Several reports have been published on the composition of unsaturated fatty acids in human milk (on unsaturated fatty acids in breast milk in America, Europe and Africa see INFORM, 6 (8): 940-946 (1995), on polyunsaturated fatty acids in breast milk in Japan see JJPEN, 13 (9): 765-772 (1991)).

Recently, ARA and DHA, in human milk, have been shown to influence the growth of infants (Advances in Polyunsaturated Fatty Acid Research ", Elsevier Science Publishers, p.161164 (1993)). A large effect of ARA and DHA on brain growth and development has also been reported (Proc. Natl. Acad. Sci. USA, 90: 1073-1077 (1993); Lancet, 344: 1319-1322 (1994)).

This fact results in an increased interest in adding ARA and DHA to modified milk. There is a formula on the market that contains fish oil as a source of DHA. Fish oil, however, also contains EPA, present in small amounts in human milk, which has been reported to have an adverse effect on the growth of immature infants ("Advances in Polyunsaturated Fatty Acid Research", Elsevier Science Publishers, pp. 261-264 (1993)). The inventive lipids are useful as an additive to modified milk due to their particularly low EPA content. The lipids according to the invention may furthermore be added to baby nutrition.

The lipids of the invention may be added to provide DHA and / or DPA or to maintain good health in foods such as nutritional supplements, nutrition for the elderly, health foods. Food can be solid or liquid, or else

PL 193 818 B1 in a form containing oils. Preferably the lipid content of the food is between 0.001 and 50% by weight, depending on the type of food to which the lipids are added.

Examples of food containing oils are natural products inherently containing oils (meat, fish, nuts), products to which lipids are added during cooking (such as soup), products where lipids are used as a heating medium (donuts), edible fats (butter) , processed foods to which lipids are added during processing (cookies) or foods to which lipids are applied by splashing or after processing (hard cookies). The lipids according to the invention (or the isolated DHA and / or DPA) can also be added to plant products, fermentation products, pet products, marine products and fat-free beverages.

Alternatively, the lipids according to the invention may be added to foods that exhibit the physiological functional action of DHA and / or DPA to enhance or prevent impaired functioning of the body. The functional food of the invention may be in the form of a pharmaceutical preparation or in a processed form (such as enteral nutrition aid, powder, granules, troches, internal solutions, suspensions, emulsions, syrups and the like) in which the lipids of the invention are combined with proteins , sugars, lipids, trace elements, vitamins, emulsifying agents or fragrances.

Furthermore, the lipids according to the invention can be used as additives to cosmetics and cleansing agents, or as starting materials for the preparation of their derivatives for use as medicaments.

Next, the invention will be illustrated by means of examples. The examples are not intended to limit the scope of the invention in any way.

Example 1.

Lipid production using microorganisms belonging to the genus Ulkenia (1)

The Ulkenia sp. SAM 2180 and SAM 2179 strains were grown in a 5 L fermentor (shake fermenter) containing 3 L of medium with the following composition and culture conditions.

(1) Composition of the composition:

1. Glucose (g / l) 60

2. Potassium phosphate (g / l) 3

3. Ammonium sulphate (g / l) 2

4. Corn steep extract (g / l) 0.7

5. 50% artificial sea water (1) 1

6. pH: 4.0 (2) Culture conditions

1. Culture temperature (° C) 28

2. Air entrainment (vol / vol / min) 0.5

3. Mixing speed (rpm) 300

4. PH adjustment: maintaining the pH at 4 with 10% (w / v) sodium hydroxide solution and 1M sulfuric acid

After completion of the culture, cells were harvested by ultracentrifugation and freeze-dried, and the number (weight) of cells per liter of medium was determined. Cells were destroyed and lipids were extracted by adding chloroform / methanol (2: 1, v / v) to dry cells at a ratio of 100 volumes per cell weight, and then homogenizing the mixture in the presence of glass beads. After washing the extract according to the Folch method, the solvent was evaporated to obtain purified lipids, and then their weight was determined.

In order to determine the composition of the obtained purified lipids, methyl esters of fatty acids were prepared by dissolving the lipid fraction in a mixed solvent consisting of equal volumes of a methanol solution containing 10% HCl and dichloromethane and heating the mixture at 50 ° C for 2 hours. The esters were then subjected to gas chromatography analyzing the fatty acid composition. The separation conditions for gas liquid chromatography were as follows.

(3) Separation conditions

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

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

3. Carrier gas: nitrogen

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4. Column temperature: growing system, 170-220 ° C (4 C / min)

5. Detection: FID

The results are summarized in Table 1 and Table 2.

Table 1.

<td>Strain</td><td>Time breeding (days)</td><td>Dry Cell Weight (g) * 1</td><td>Total amount of lipids (g) * 1</td><td>Percentage of lipids (wt%) * 2</td><td>Quantity DHA (g) * 1</td><td>Quantity DPA (g) * 1</td>

<td>SAM 2180</td><td> 3</td><td> 23,2</td><td> 14,1</td><td> 61</td><td> 4,0</td><td> 0,9</td>

<td>SAM 2179</td><td> 3</td><td> 19,5</td><td> 11,9</td><td> 61</td><td> 5,5</td><td> 1,3</td>

* 1) Weight per liter of medium * 2) Percentage of dry cells

Table 2.

<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 (AA)</td><td>20: 5 (EPA)</td><td>22: 5 (DPA)</td><td>22: 6 (DHA)</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>

Example 2.

Lipid production using a microorganism belonging to the genus Ulkenia (2)

The Ulkenia sp. SAM 2179 strain was grown in a 5 L fermentor (shake fermenter) containing 3 L of medium with the following composition and culture conditions.

(1) Composition of the composition:

1. Glucose (g / l) 60

2. Potassium phosphate (g / l) 3

3. Ammonium sulphate (g / l) 2

4. Magnesium chloride (g / l) 1.3

5. Sodium Sulphate (g / L) 1

6. Calcium chloride (g / l) 0.3

7. Corn steep extract (g / l) 0.7

8. pH: 4.0 (2) Culture conditions

1. Culture temperature (° C) 28

2. Air entrainment (vol / vol / min) 0.5

3. Mixing speed (rpm) 300

4. PH adjustment: maintaining the pH at 4 with 10% (w / v) sodium hydroxide solution and 1M sulfuric acid

After completion of the culture, cells were harvested by ultracentrifugation and freeze-dried, and the number (weight) of cells per liter of medium was determined. Cells were destroyed and lipids were extracted by adding chloroform / methanol (2: 1, v / v) to dry cells at a ratio of 100 volumes per cell weight, and then homogenizing the mixture in the presence of glass beads. After washing the extract according to the Folch method, the solvent was evaporated to obtain purified lipids, and then their weight was determined.

In order to determine the composition of the obtained purified lipids, methyl esters of fatty acids were prepared by dissolving the lipid fraction in a mixed solvent consisting of equal volumes of methanol solution containing 10% HCl and dichloromethane and heating the mixture at 60 ° C for 2 hours. The esters were then subjected to gas chromatography, analyzing the fatty acid composition. The separation conditions for gas liquid chromatography are described in Example 1.

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The results are shown in Tables 3 and 4.

Table 3.

<td>Strain</td><td>Time breeding (days)</td><td>Dry Cell Weight (g) * 1</td><td>Total amount of lipids (g) * 1</td><td>Percentage of lipids (wt%) * 2</td><td>Quantity DHA (g) * 1</td><td>Quantity DPA (g) * 1</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) Weight per liter of medium * 2) Percentage of dry cells

Table 4.

<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 (AA)</td><td>20: 5 (EPA)</td><td>22: 5 (DPA)</td><td>22: 6 (DHA)</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>

Example 3.

Analysis of lipids obtained from Ulkenia SAM 2179.

Neutral and polar lipids were separated from the lipids obtained in Example 1 by a known liquid-liquid partition technique using hexane and 90% methanol. From 1 g of lipids, 0.92 g of neutral lipids and 0.05 g of polar lipids were obtained, respectively. The obtained neutral and polar lipids were analyzed by thin layer chromatography. The chromatograph was developed using sulfuric acid and the obtained spots were identified by comparing their Rf values ​​with those of the reference lipids.

Over 90% of neutral lipids were triacylglycerols. The polar lipids included phosphatidyl dicholine (60-80%), phosphatidylethanolamine (5-20%) and phosphatidylinositol (2-8%).

Triacylglycerols in neutral lipids were analyzed by separating the molecules by liquid chromatography (column: ODS; mobile phase: acetone / acetonitrile (3: 2); detection: differential refractometer) (see Fig. 1). The obtained fractions were separated and then, after hydrolysis, converted into methyl esters. The fatty acid residues were identified by gas liquid chromatography.

The 5 major peaks collected in Table 5 were identified. The triacylglycerols were composed of 12.8% 1,2,3-tridocosahexaenooyl-triacylglycerol and 8.0% 1-docosapentaenooyl-2,3-didocosahexaenooyl-triacylglycerol. About 20% of the total amount of triacylglycerols was made up of the tri-polyunsaturated fatty acid composition.

Table 5

<td>Peak</td><td>Molecule</td><td>Ratio (%)</td>

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

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

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

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

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

Example 4.

Determination of the position of attachment of fatty acid residues in triacylglycerols

The attachment positions of the fatty acid residues in the triacylglycerols obtained in Example 3 were analyzed as follows. The triacylglycerols obtained in Example 3 (molecules: 16: 0-16: 0-22: 6) were dried and treated with a lipase (from Rhizopus japonicus) specific for the 1,3 position. The resulting 2-monoacylglycerols were trimethylsilylated and the remainder of the fatty acid identified using gas chromatography / mass spectrography. The reaction with lipase was carried out in 2 ml of 50 nM acetate buffer (pH 5.5) with 1000 units. lipase at 35 ° C for 30 minutes.

PL 193 818 B1

The reaction products were extracted with ether and trimethylsilylated using a commercially available trimethylsilylating agent.

A partial peak corresponding to the molecular weight of the monoacylglycerols to which 22: 6 is attached was observed, indicating that the triacylglycerol to which the 22: 6 acid residue is attached at the 2-position of the glycerol backbone is 16: 0-22: 6-16: 0 triacylglycerol.

Example 5

Preparation of modified milk containing DHA and DPA

Modified milk containing DHA and DPA was prepared by adding 0.44 g of the lipids from Example 1 containing 46.2% DHA and 10.6% DPA to 100 g of powdered milk.

DHA and DPA in the powdered milk accounted for 0.80% and 0.19% of the total amount of lipids, respectively, a value similar to that of human milk.

Example 6

Preparation of structural lipids containing DHA and DPA ml of lipase solution (5600 U / ml, 1,3-position specific lipase from Rhizopus delemar) was mixed with 2.5 g of CaCO3 as the immobilized carrier. The enzyme was immobilized and precipitated by adding 40 ml of acetone to the mixture, and then the immobilized enzyme was dried. The specific activity of the obtained immobilized enzyme was 9.3 U / ml. 120 mg of immobilized enzyme was mixed with 1 g of DHA and DPA containing lipids obtained from SAM 2180 in Example 1, 2 g of heptanecarboxylic acid-1 and 60 mg of water, with stirring at 30 ° C for 8 hours. The triacylglycerols were separated from the reaction mixture in a known manner, and the fatty acid composition of the triacylglycerols was determined. The results are summarized in Table 6.

Table 6.

<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 (AA)</td><td>20: 5 (EPA)</td><td>22: 5 (DPA)</td><td>22: 6 (DHA)</td>

<td>Not treated with lipase</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 lipase action</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 triacylglycerols have polyunsaturated fatty acids in the SN2 position of the glycerols. Thus, these lipids are useful for the production of structural lipids containing medium chain fatty acids in the SN1 and SN3 position of glycerols and polyunsaturated fatty acids in the SN2 position of glycerols.

Industrial use

The method according to the invention makes it possible to obtain lipids with a high DHA and / or DPA content with a low EPA content. From these lipids, DHA and / or DPA can then be separated.

The lipids containing DHA and / or DPA, isolated DHA and isolated DPA according to the invention are useful as additives in food, feed, bait, medicaments and the like. Cells containing DHA and / or DPA can be used in the feed and baits. By feeding poultry with the feed according to the invention, poultry eggs and poultry egg yolks enriched with DHA and / or DPA can be obtained.