Method for production of pure phosphides and uses thereof in cosmetic, pharmaceutics, and food

FIELD: biotechnology.

SUBSTANCE: claimed method includes interaction of mixtures of natural phosphatides or components thereof, for instance, soybean or egg lecithin or animal phospholipids, or synthetic phosphatides, by reaction thereof with D phospholipase having transphosphatidylase activity in water medium.

EFFECT: simplified method for isolation of pure phosphatides of improved purity with increased yield.

28 cl, 13 ex

 

The technical FIELD

The present invention relates to a method of obtaining pure phosphatides from mixtures of natural phosphatides or their individual components, such as soy or egg lecithin, or animal phospholipids or synthetic phosphatides, through their reaction with phospholipase D with transpeptidase activity in a single aqueous phase in the presence of certain substrates containing a primary or secondary alcohol group.

The invention also relates to the production, purification and characterization of phospholipase D, which is used in the method.

PRIOR art

The synthesis of pure phospholipids, especially on an industrial scale, is a very widespread problem. Indeed, there are numerous scientific publications and patents, including some very recent, which describes various methodologies. Typically in these methods use transpeptidase properties of phospholipase D to obtain optically active phosphatides. One of the main problems is that each of these methods are suitable only for one specific phosphatide and cannot be adapted for the synthesis of the entire class of compounds. In General, the most widely studied phospholipid is phosphatidylserine (FS), because it is widely used in isoamsa to obtain pharmaceutical compositions for the preparation of formulations containing liposomes, and food additives. Relatively little or nothing published regarding the synthesis of sphingophospholipids.

One of the limitations of all methods described in the scientific and patent literature, due to the fact that the reaction transpeptidation is in two-phase systems consisting of water and an organic solvent. This creates a number of technical problems associated with the use of large quantities of solvent, especially if the industrial method has a chemical nature, and its purpose is to obtain high-quality product. In the patent application DE 19917249 A1 describes a way in which is actually used one aqueous phase, but neither the output nor the degree of purity of the obtained FS, neither the type of the used enzyme. In addition, there is no information about whether it is using the same methods and the same substrates to receive other phospholipids, in addition to the Federal Assembly, and can described in terms other phospholipids to act as substrates of the reaction. In Japanese Patent Publication No. 5/42917 (JP 2130088) also described the way in which the environment is composed solely of water or of a mixture of water and an organic solvent. However, in this patent the conclusion that in order to prevent adverse reactions preferably the water content is 10% or less. Thus, this work allows pre is put, what is the use of one of the aquatic environment adversely. In fact, in the examples contained in it only describes the ways in which used a two-phase mixture of water and ethyl ether.

The General nature of the information and the lack of these studies prior art information about the applicability of the reaction transpeptidation with phospholipase D in one aqueous phase did not allow the specialists in the art to believe that this could be a problem. In addition, the importance of removing contaminants arising from the use of organic solvents in the production methods of products used in the fields of nutrition and pharmaceutical, was known only in recent years after the limits set by the Pharmacopoeia of the United States (USP) and the European Community Directive (CPMP/CN/283/95).

Another critical question that has not been deeply investigated neither in the scientific literature, nor on the sick, concerns peroxidation products formed during use of heterogeneous phases "water/solvent in the emulsion during the reaction of transpeptidation used the reaction conditions and the subsequent need to perform multiple steps of the method (re-deposition, leaching and possibly chromatography) to obtain products of the highest level of purity. Many of the solvents used for these reactions in heterogeneous phase does not guarantee the absence of free radical precursors (intermediate products), typical for the initiation of peroxidation. In addition, the shaking/stirring is necessary to perform the reaction in heterogeneous phase, increase the likelihood of contact with atmospheric oxygen and the subsequent launch of oxidative phenomena. It peroxidation acts like a chain reaction, when even minor primary primary stage may over time to give good results, despite the fact that the triggering conditions have been eliminated or minimized. Peroxidation "fat" substances such as triglycerides (oils or fats), as well as phospholipids, leads to the fact that fatty acids become rancid with subsequent formation of unpleasant odors and taste. It is particularly important that the products should have a high level of appatichnosti (smell and taste), if they, especially FS, used for cooking food supplements (nutraceuticals, dietary supplements) with special formulations such as granules or so-called "functional foods", to which is added the product to enrich their composition. It is therefore important that the products obtained, especially FS, could be well Oh what bacteriology chemical terms as regards the chemical composition of fatty acids, and according to the degree of lipid peroxidation and the resulting appatichnosti.

Finally, it should be noted that the preparations of the enzyme phospholipase D, available on the market usually are transpeptidase activity against phosphatidylcholine fraction of phospholipid mixtures. As a result, other components, such as phosphatidylethanolamine (PV), are subjected to hydrolysis to phosphatidic acid, which is reduced as the output, and the degree of purity of the final product.

The INVENTION

The authors of the present invention it has been unexpectedly discovered that the use of purified fractions of phospholipase D, isolated from Streptoverticillium hachijoense, it is possible to conduct the reaction transpeptidation with different alcohol receptors, starting from a mixture of natural phosphatides or their purified fractions, 100% of the aquatic environment with high yield and peroxide number (level peroxidation) less than About 5 mEq2/kg, established in the European Pharmacopoeia Supplement 2000, page 41 (method A)by one-stage reaction and one deposition.

The reaction transpeptidation can be performed under different conditions in respect of temperature and substrate concentration, settling first in the range from 20 to 60°C, preferably 45°and the second is in the range from 10 to 500 mg/ml, prefer the Ino - 150 mg/ml, depending on the degree of dispersion of substrates.

The second aspect of the present invention is that this particular enzyme preparation has transpeptidase activity with high yields of product, even when using substrates that differ from phosphatidylcholine (lecithin), which allows to use this method for inexpensive raw materials.

The value of the strain used to produce Phospholipase D and its fractionation, can be understood from the following table, in which the comparison of purified fractions of the enzyme obtained according to Example 1, with various commercially available enzyme preparations.

Getting phosphatidylserine

StrainCompanyProductFSFCPF
Streptomyces sp.SigmaP4912 Type VI50%45%5%
Streptomyces sp.Asahi Chemical Industry Co., LtdPL-DP50%45%5%
Streptomyces chromofuscusSigmaP8023 Type VI40%40%20%
Actinomadura sp.Meito Sangyo Co., Ltd.Code no 45%40%15%
Streptoverticillium hachijoenseFidiaFRS95%5%-

(pure lecithin containing phosphatidylcholine, equal to 95%, as a substrate; reaction conditions as in Example 1).

Another advantage of this enzymatic preparation and reaction conditions described in the present invention, is the almost complete conversion of the substrate in FS compared with the degree of conversion, not exceeding 70%, achieved in the two-phase system water/organic solvent described in JP 213008.

The third aspect of the present invention relates to pharmaceutical and cosmetic compositions and food and dietary supplements based on phospholipids obtained in accordance with the above method and having a degree of peroxidation of less than 5 mEq O2/kg and a high level of appatichnosti, in order to make them more preferable than other similar products available on the market, but do not have these characteristics.

Pharmaceutical compositions and nutritional and dietary supplements especially indicated for the treatment of States of mental and physical stress with impaired attention, concentration and memory, often associated with aging, and they can be manufactured by Garrett, Borg is Bered, for example, in the form of capsules, tablets and granules.

Cosmetic compositions primarily can be used in the treatment of disorders in the physiological functions of the skin and as an aid in the treatment of eczematous dermatitis and/or inflammatory type, and they can be produced, for example, in the form of creams or gels.

From a purely illustrative purposes, below are a few examples of the preparation of Phospholipase D and phospholipids obtained with its use according to the present invention.

DESCRIPTION of embodiments of the INVENTION

Example 1: Getting Phospholipase D

We used a strain of Streptoverticillium hachijoense ATCC 19769.

Preparation of inoculum for use in the test fermentation begins with colonies on solid medium in a Petri dish. The composition of the solid medium is as follows: 21,0 g/liter yeast extract, 2% agar. Bacteria taken from the Petri dishes, used for system start-up "scaling" in Erlenmeyer flasks as follows:

- bacteria taken from the solid medium with a sterile platinum loop and inoculant in a flask with a volume of 100 ml, containing 20 ml of medium of the following composition: 21,0 g/l yeast broth;

the flask was placed in an incubator-shaker with installations on 30°C, 150 rpm for 48 hours;

to 20 ml culture is transferred into a flask of 5.0 liters, containing 2.5 l of environment described above;

After that, the culture is ready for inoculation into the bioreactor. This operation is possible due to the fact that culture is a flask equipped with a system consisting of silicone tubing and needle. Used bioreactor has a capacity of 50 liters (Braun patch BIOSTAT U). The composition of the medium following: yeast broth - 21,0 g/l, pH 6.5 (Wednesday sterilized directly in the bioreactor adding anti-foam funds); the parameters of fermentation: an agitation of 200 rpm and a temperature of 30°With air flow of 0.5 vvm.

After 72 hours fermentation is interrupted. Maximum received the amount of enzyme is 5000 IU/l, and the enzymatic activity determined according to the modified method of Kato et al., described in the literature (.Shimbo et al., Agric. Biol. Chem. 54(5), 1189-93, 1990).

The culture broth is filtered to remove biomass supernatant concentrated using a spiral cartridge cellulose acetate with a cutoff molecular weight corresponding to 10000 D, and deleteroute against Tris-HCl buffer at a concentration of 50 mm, pH 8.0. Thus obtained sample is loaded into a chromatographic column (inner diameter = 10 cm, h=50 cm)filled with 500 g of anion exchange resin Watman DE-52, pre-equilibrated with the same buffer used for dialysis. The enzyme is adsorbed and eluted from the column, it dialyzer is against Na-phosphate buffer with a pH of 5.4, and then loaded into a chromatographic column (inner diameter = 10 cm, h=50 cm)filled with 50 g of cation-exchange resin CM-Pharmacia Sephadex C-50, pre-balanced with the same buffer and the enzyme. The enzyme elute in a gradient of pH from 5.5 to 7.0 using Na-phosphate buffer (20 mm, pH 7.0). The fraction that eluted at pH of 6.2, collect, concentrate, and deleteroute against Tris-HCl buffer (50 mm, pH 8.0) to a concentration of 100 U/ml, and then lyophilizer.

Total activity (Units × 103)The total protein content (mg × 103)Specific activity (Units × mg protein)Allocation (%)
Raw broth2502501100
Concentration and dialysis237,5118,8295
DE-52 Whatman212,52,29785
CM-Sephadex C-50, Pharmacia1500,2657760
Concentration and dialysis1450,2460458
Lyophilization112,50,24469 45

Example 2: Obtain FS (phosphatidyl-L-serine) of PC (phosphatidylcholine)

In jacketed reactors, equipped with agitator and reflux condenser, 272 g of sodium acetate of three-hydrate and 59 g of the hydrochloride sodium three-hydrate dissolved in 10 liters of water. the pH was adjusted to 5.6 (acetate buffer, 0.2 M + calcium chloride, 0.04 M, pH 5,6)add 5,0 kg L-serine and solubilizers by heating to 45°C. the Entire process is carried out in nitrogen atmosphere.

After complete solubilization add 1.5 kg of purified soybean lecithin following composition: PC (phosphatidylcholine) 95%, FC (phosphatidic acid) 4%, Lisa-PF (effective absorption with added) 1%. After ten minutes add 16100 Units of phospholipase D from Example 1 and the mixture is left for reaction for 24 hours at 45°C.

After completion of the reaction the reactor was unloaded by adding 10 liters of a mixture of n-hexane/isopropanol/water (60/80/15); the mass is dissolved by shaking, and then the shaking stopped and leaving the reaction mixture for phase separation.

The lower phase was collected and emit unreacted L-serine by cooling in the mould.

The upper phase is washed with 6950 ml of 1N HCl and 1113 ml isopropanol at 5°C. an Acidic phase re-extracted with 10 liters of a mixture of hexane/isopropanol/water (60/80/15), after which two of the organic phase in a cascade installation is ke washed with a mixture of 7 liters of water and 6 liters of isopropanol.

The organic phase concentrated under vacuum and precipitated by slow addition of a mixture of 450 ml of 4.5 M aqueous solution of sodium acetate and 33.2 liters of ethanol.

The precipitate is filtered and dried.

Get 1.14 kg phosphatidylserine with a titer of more than 95%, the content of phosphatidic acid less than 5% and a peroxide number of less than 5.

After crystallization and drying allocate 3.25 kg of L-serine.

Example 3: Getting FS (phosphatidyl-L-serine) from egg lecithin

180 ml of a mixture of 0.2 M acetate buffer and 0.04 M calcium chloride with a pH of 5.6 is placed in a jacketed reactors, equipped with agitator and reflux condenser, and then add 90 g of L-serine and solubilizers by heating to 45°C. the Entire process is carried out in nitrogen atmosphere.

After complete solubilization added 27 g of purified egg lecithin (contents PF≥95%); after 10 minutes, add 290 Units of phospholipase D from Example 1 and left for reaction for 24 hours at 45°C.

After completion of the reaction the reactor was unloaded by adding 200 ml of a mixture n-hexane/isopropanol/water (60/80/15) and dissolved mass by shaking, then shake out and leave the two phases to separate.

The lower phase is discarded.

The upper phase is washed with 150 ml of 1N HCl and 30 ml of isopropanol at 5°C.

Acidic phase re-extracted with 180 ml of a mixture of the-hexane/isopropanol/water (60/80/15), then two organic phase cascaded washed with a mixture of 100 ml of water and 130 ml of isopropanol.

The organic phase concentrated under vacuum and precipitated by slow addition of a solution of 8 ml of 4.5 M sodium acetate in water 600 ml of ethanol. The precipitate is filtered and dried.

This gives 20,8 g phosphatidylserine with a titer of more than 95% with the content of phosphatidic acid less than 5% and a peroxide number of less than 5.

Example 4: Obtaining the phosphatidyl-L-serine from crude soybean lecithin

180 ml of a mixture of 0.2 M acetate buffer and 0.04 M calcium chloride with a pH of 5.6 is placed in a jacketed reactors, equipped with agitator and reflux condenser, and then add 90 g of L-serine and solubilizers by heating to 45°C. the Entire process is carried out in nitrogen atmosphere.

After complete solubilization added 27 g of crude soybean lecithin (total content of phospholipids 75%, the percentage of PF 60,6/Feh 29,5/FC 3,4/Lisa-PF 2,5/other 3,9); after 10 minutes, add 290 Units of phospholipase D from Example 1 and left for reaction for 24 hours at 45°C.

After completion of the reaction the reactor was unloaded by adding 200 ml of a mixture n-hexane/isopropanol/water (60/80/15) and dissolved mass by shaking, then shake out and leave the two phases to separate.

The lower phase is discarded.

Top the phase is washed with 150 ml of 1N HCl and 30 ml of isopropanol at 5° C.

Acidic phase re-extracted with 180 ml of a mixture n-hexane/isopropanol/water (60/80/15), then the two organic phases are in cascaded washed with a mixture of 100 ml of water and 130 ml of isopropanol.

The organic phase concentrated under vacuum and precipitated by slow addition of a mixture of 8 ml of 4.5 M aqueous solution of sodium acetate and 600 ml of ethanol. The precipitate is filtered and dried.

This gives 19 g of phosphatidylserine with a title more to 83.5% (FC 7,7%, lyso-FS 2,3%, PV of 1.9%, other 4.5%) and peroxide number of less than 5.

Example 5: Receiving the phosphatidyl-D-serine

180 ml of a mixture of 0.2 M acetate buffer and 0.04 M calcium chloride with a pH of 5.6 is placed in a jacketed reactors, equipped with agitator and reflux condenser, and then add 90 g of D-serine and solubilizers by heating to 45°C. the Entire process is carried out in nitrogen atmosphere.

After complete solubilization added 27 g of purified soybean lecithin as in Example 2; after 10 minutes, add 290 Units of phospholipase D from Example 1 and left for reaction for 24 hours at 45°C.

After completion of the reaction the reactor was unloaded by adding 200 ml of a mixture n-hexane/isopropanol/water (60/80/15) and dissolved mass by shaking, then shake out and leave the two phases to separate.

The lower phase is discarded.

The upper phase is washed with 150 ml of 1 N HCl and 0 ml isopropanol at 5° C.

Acidic phase re-extracted with 180 ml of a mixture n-hexane/isopropanol/water (60/80/15), then the two organic phases are in cascaded washed with a mixture of 100 ml of water and 130 ml of isopropanol.

The organic phase concentrated under vacuum and precipitated by slow addition of a mixture of 8 ml of 4.5 M aqueous solution of sodium acetate and 600 ml of ethanol. The precipitate is filtered and dried.

This gives to 18.7 g of phosphatidyl-D-serine with a titer of more than 95%, with the content of phosphatidic acid less than 5% and the peroxide number of less than 5.

Example 6: Getting phosphatidylethanolamine (PV) of phosphatidylcholine (PC)

200 ml of a mixture of 0.2 M acetate buffer and 0.04 M calcium chloride with a pH of 5.6 is placed in a jacketed reactors, equipped with agitator and reflux condenser. The whole process is carried out in nitrogen atmosphere.

Added 58 g of ethanolamine and bring the pH to 5.6 glacial acetic acid at a given temperature 30°C.

At the end of the operation, the mixture is heated to 45°and add 30 g of purified soybean lecithin as in Example 2. After 10 minutes add 325 Units of phospholipase D from Example 1, and then left for reaction for 24 hours at 45°C.

After completion of the reaction the reactor was unloaded by adding 400 ml of a mixture n-hexane/isopropanol/water (60/80/15), then shake out and leave the two phases to separate.

The lower phase Abrasiv the Ute.

The upper phase is washed with 150 ml of 1N HCl and 30 ml of isopropanol at 5°C.

Acidic phase re-extracted with 180 ml of a mixture n-hexane/isopropanol/water (60/80/15), then the two organic phases are in cascaded washed with a mixture of 100 ml of water and 130 ml of isopropanol, and then 100 ml of 1N sodium acetate solution and 130 ml of isopropanol.

The organic phase concentrated under vacuum.

They purify by chromatography on silica gel using chromatograph axial pressure with a column of 1 l, a balanced mixture of chloroform and methanol (80/20); perform gradient elution to the ratio of chloroform/methanol/water, equal 70/30/3.

The pure fractions are evaporated, dissolved in 250 ml of cyclohexane and lyophilizer to obtain 24 g of a light yellow solid product, not containing phosphatidic acid and isoprostane and having a purity of more than 99%.

Example 7: Getting the phosphatidyl-homoserine from PF

180 ml of a mixture of 0.2 M acetate buffer and 0.04 M calcium chloride with a pH of 5.6 is placed in a jacketed reactors, equipped with agitator and reflux condenser, and then added 102 g of homoserine and solubilizers by heating to 45°C. the Entire process is carried out in nitrogen atmosphere.

After complete solubilization added 27 g of purified soybean lecithin as in Example 2; after 10 minutes, add 290 Units of phospholipase D from the Use of the 1 and left for reaction for 24 hours at 45° C.

After completion of the reaction the reactor was unloaded by adding 200 ml of a mixture n-hexane/isopropanol/water (60/80/15). The mass is dissolved by shaking, then shake out and leave the two phases to separate.

The lower phase is discarded.

The upper phase is washed with 150 ml of 1N HCl and 30 ml of isopropanol at 5°C.

Acidic phase re-extracted with 180 ml of a mixture n-hexane/isopropanol/water (60/80/15), then the two organic phases are in cascaded washed with a mixture of 100 ml of water and 130 ml of isopropanol.

The organic phase concentrated under vacuum and precipitated by slow addition of a mixture of 8 ml of 4.5 M aqueous solution of sodium acetate and 600 ml of ethanol. The precipitate is filtered and dried.

Get to 22.6 g of phosphatidyl-homoserine with a titer of more than 95%, with the content of phosphatidic acid less than 5% and the peroxide number of less than 5.

Example 8: Getting the phosphatidyl-hydroxyproline from PC (phosphatidylcholine)

180 ml of a mixture of 0.2 M acetate buffer and 0.04 M calcium chloride with a pH of 5.6 is placed in a jacketed reactors, equipped with agitator and reflux condenser, and then add 112 g hydroxyproline and solubilizers by heating to 45°C. the Entire process is carried out in nitrogen atmosphere.

After complete solubilization added 27 g of purified soybean lecithin as in Example 2; after 10 minutes, add 20 Units of phospholipase D from Example 1 and left for reaction for 24 hours at 45° C.

After completion of the reaction the reactor was unloaded by adding 200 ml of a mixture n-hexane/isopropanol/water (60/80/15) and dissolved mass by shaking, then shake out and leave the two phases to separate.

The lower phase is discarded.

The upper phase is washed with 150 ml of 1N HCl and 30 ml of isopropanol at 5°C.

Acidic phase re-extracted with 180 ml of a mixture n-hexane/isopropanol/water (60/80/15), then the two organic phases are in cascaded washed with a mixture of 100 ml of water and 30 ml of isopropanol.

The organic phase concentrated under vacuum and precipitated by slow addition of a mixture of 8 ml of 4.5 M aqueous solution of sodium acetate and 600 ml of ethanol. The precipitate is filtered and dried.

This gives an 18.4 g of phosphatidyl-hydroxyproline with a titer of more than 95%, with the content of phosphatidic acid less than 5% and the peroxide number of less than 5.

Example 9: Getting phosphatidylglycerol of PC (phosphatidylcholine)

200 ml of a mixture of 0.2 M acetate buffer and 0.04 M calcium chloride with a pH of 5.6 is placed in a jacketed reactors, equipped with agitator and reflux condenser. The whole process is carried out in nitrogen atmosphere.

Add 80 g of glycerin, it is heated up to 45°With, then add 30 g of purified soybean lecithin as in Example 2; after 10 minutes, add 325 Units of phospholipase D from Example 1 and left for reaction for 24 cha is and at 45° C.

After completion of the reaction the reactor was unloaded by adding 400 ml of a mixture n-hexane/isopropanol/water (60/80/15), then shake out and leave the two phases to separate.

The lower phase is discarded.

The upper phase is washed with 150 ml of 1N HCl and 30 ml of isopropanol at 5°C.

Acidic phase re-extracted with 180 ml of a mixture n-hexane/isopropanol/water (60/80/15), then the two organic phases are washed in cascaded with a mixture of 100 ml of water and 130 ml of isopropanol, and then 100 ml of 1N sodium acetate solution and 130 ml of isopropanol.

The organic phase concentrated under vacuum.

Cleaning produce by chromatography on silica gel using chromatograph axial pressure with a column of 1 l, a balanced mixture of chloroform and methanol (80/20). Perform gradient elution to the ratio of chloroform/methanol/water, equal 70/30/3.

The pure fractions are evaporated, dissolved in 250 ml of cyclohexane and lyophilizers with the receipt of 22.2 g of white solid product, not containing phosphatidic acid and isoprostane and having a purity of more than 95%.

It is filtered and dried.

Example 10: Separation and reuse of L-serine

Uterine water from the first distribution of the enzymatic reaction as in Example 2, is maintained at a temperature of 0°within 24 hours, then kristallitov is, which product is filtered. It is washed with ethanol and dried to obtain white needle-shaped crystals of the pure product, identical to the original.

Fill in the missing number (about 20%), after which the product can be used as normal.

Example 11: Examples of pharmaceutical compositions

(a) Each gelatin capsule contains:

phosphatidylserine100.0 mg
vegetable oil270,0 mg
soy lecithin30.0 mg

(b) Each ampoule for injection contains:

phosphatidylserine50.0 mg
mannitol100.0 mg
soy lecithin (suitable for injection)7.5 mg
phosphate buffer2.2 mg
water as needed to2.0 ml

Example 12: Examples of food and dietary supplements

(a) Each gelatin capsule contains:

phosphatidylserine100.0 mg
vitamin E5.0 mg
vegetable oil295,0 mg

(b) Each package contains:

phosphatidylserine100 mg
creatine1.5 g
beta carotene0.6 mg
vitamin E5.0 mg
vitamin C30.0 mg
vitamin B10.7 mg
vitamin B61.0 mg
vitamin B120.5 mg
folic acid0.1 mg

(C) Each chewable tablet contains:

phosphatidylserine75,0 mg
vitamin E5.0 mg
vitamin C30.0 mg
vitamin B10.7 mg
vitamin B61.0 mg
vitamin B120.5 mg
folic acid0.1 mg

18 Example 13: Examples of cosmetic compositions.

(a) Each tube of body cream contains:

phosphatidylserine4.0 g
macadamia ternifolia (nut Australian)2.0 g
of sodium hyaluronate10.0 mg
tetereluctant8.0 g
the triglyceride of Kapralov the th/Caproic acid 7.0 g
sorbitol5.0 g
Cetearyl alcohol4.0 g
Polysorbate 201.0 g
carbomer0.8 g
dehydroacetate sodium0.4 g
disodium-EDTA0.3 g
antioxidant (tocopherol)50.0 mg
wateras needed
to 100.0 ml

(b) Each tube of ointment for body contains:

phosphatidylserine3.0 g
cholesterol2.0 g
of sodium hyaluronate10.0 mg
tetereluctant9.0 g
the triglyceride Caprylic/Caproic acid7.0 g
sorbitol7.0 g
Cetearyl alcohol3.5 g
Polysorbate 201.0 g
carbomer0.6 g
dehydroacetate sodium0.5 g
disodium-EDTA0.3 g
antioxidant (tocopherol)50.0 mg
water as needed to100,0 ml

1. The method of obtaining phosphatides with formula (I):

where R is a diacylglycerol, a R1is the residue of an alcohol group, which includes reaction phosphatide with formula (II):

R-O-PO(OH)-O-R2,

where R is as described above, a R2is CH2-CH2-NH2and/or CH2-CH2-N+(CH3)3,

with a primary or secondary alcohol with a chain length from C2 to C4, possibly substituted by one or more polar groups selected from the group consisting of amino group, hydroxyl group and carboxyl group in a purely aqueous medium in the presence of phospholipase D with transpeptidase activity produced by bacteria of the species Streptomyces hachijoense.

2. The method according to claim 1, characterized in that the diacylglycerol contains two fatty acids, which may be identical or different, saturated or unsaturated, and have a length of from C12 to C24.

3. The method according to claim 1 or 2, characterized in that the reaction temperature is 45±5°C.

4. The method according to any one of claims 1 to 3, characterized in that the phospholipase D purified on anion - and cation-exchange resin.

5. The method according to claim 4, characterized in that the fraction of phospholipase D elute at pH of 6.2 with a cation-exchange resin.

6. The method according to any one of claims 1 to 5, the tives such as those the bacteria Streptomyces species hachijoense represent a strain of ATSS 19769.

7. The method according to any one of claims 1 to 6, characterized in that phosphated with formula (I) is the phosphatidyl-D-serine, phosphatidylethanolamine, phosphatidylserine, phosphatedehydrogenase or phosphatidylglycerol.

8. The method according to any one of claims 1 to 7, characterized in that phosphated with formula (I) is phosphatidyl-L-serine, and phosphated with formula (II) is crude or purified soybean lecithin.

9. The method according to any one of claims 1 to 8, characterized in that phosphated with formula (I) is phosphatidyl-L-serine, and phosphated with formula (II) is egg lecithin.

10. Product phosphatidyl-L-serine obtained by the method according to any one of claims 1 to 9, having a fatty acid composition identical to the composition of soy or egg lecithin, and the peroxide number of less than 5 mEq O2/kg.

11. Pharmaceutical composition for the treatment of States of mental and physical stress, impaired attention, concentration and memory containing a pharmaceutically acceptable carrier and phosphated with formula (I)obtained by the method according to any one of claims 1 to 9.

12. The pharmaceutical composition according to claim 11, characterized in that it has a fatty acid composition that is identical to the egg or soya lecithin, and the peroxide number of less than 5 mEq O2/kg.

13. The pharmaceutical composition according to item 12, characterized in that it is the meet the form of a capsule, tablets or granules.

14. Cosmetic composition for treating disorders in the physiological functions of the skin and as an aid in the treatment of eczematous dermatitis and/or inflammatory type containing a pharmaceutically acceptable carrier and phosphated with formula (I)obtained by the method according to any one of claims 1 to 9.

15. Dietary Supplement containing media and phosphated with formula (I)obtained by the method according to any one of claims 1 to 9.

16. The food additive according to item 15, wherein phosphated with formula (I) is phosphatidyl-L-serine.

17. The food additive according to item 15 or 16, characterized in that it has a fatty acid composition that is identical to the egg or soya lecithin, and the peroxide number of less than About 5 mEq2/kg.

18. The food additive according to 17, characterized in that it has the form of capsules, tablets or granules.

19. Dietary Supplement containing media and phosphated with formula (I)obtained by the method according to any one of claims 1 to 9.

20. Dietary additive, characterized in that phosphated with formula (I) is phosphatidyl-L-serine.

21. Dietary Supplement according to claim 19 or 20, characterized in that it has a fatty acid composition that is identical to the egg or soya lecithin, and the peroxide number of less than About 5 mEq2/kg.

22. Dietary Supplement according to item 21, characterized in that it has the form of capsules, tablets or granules.

2. Cosmetic composition for topical application to the skin containing a pharmaceutically acceptable carrier and the phosphatidyl-L-serine obtained by the method according to claim 1, having a fatty acid composition that is identical to the egg or soya lecithin, and the peroxide number of less than About 5 mEq2/kg.

24. Cosmetic composition according to item 23, characterized in that it has the form of a cream or gel.

25. The use of phosphatide with formula (I)obtained by the method according to any one of claims 1 to 9, for the preparation of pharmaceutical compositions for the treatment of psychophysical stress, impaired attention, concentration and memory, usually associated with aging.

26. The use of phosphatide with formula (I)obtained by the method according to any one of claims 1 to 9, for the preparation of dietary supplements for the treatment of psychophysical stress, impaired attention, concentration and memory, usually associated with aging.

27. The use of phosphatide with formula (I)obtained by the method according to any one of claims 1 to 9, for the preparation of dietary supplements for the treatment of psychophysical stress, impaired attention, concentration and memory, usually associated with aging.

28. The use of phosphatide with formula (I)obtained by the method according to any one of claims 1 to 9, for the preparation of cosmetic compositions for the treatment of dermatitis or violations of the physiological functions of the skin.



 

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FIELD: organic chemistry, microbiology, pharmacy.

SUBSTANCE: invention relates to new biologically active compounds. Invention describes a compound of the formula (1):

wherein R1 means carbon chain with 2-30 carbon atoms that can be linear, branched, saturated and can be substituted once or twice with the following groups: -OH, =O, -(C1-C6)-alkyl or the group:

; R2 means (C1-C6)-alkyl; E means phosphorus atom (P); each X1, X2 and X3 means independently of one another -O- in its all stereochemical forms and mixtures of these forms taken in any ratio, and its physiologically acceptable salts also. Also, invention describes a method for preparing cyclipostins and the strain Streptomyces HAG 004107, DSM 13381 as a producer of cyclipostins. Invention provides preparing new compounds possessing valuable biological properties.

EFFECT: improved preparing method, valuable properties of substances.

12 cl, 9 tbl, 21 ex

The invention relates to biotechnology and characterizes the phytase, the DNA fragment encoding the phytase, an expression vector containing the DNA fragment

FIELD: biotechnology, amino acids, peptides.

SUBSTANCE: invention relates to a method for preparing a mixture consisting of amino acids and lower peptides from yeast autolyzates. Method involves autolysis of yeast, separation of cellular debris by centrifugation and purification of autolyzate in ionites resins wherein gel sulfocation-exchange resin in hydrogen form comprising 12-16% of divinylbenzene as a cross-linking agent is used as ionite. Proposed method provides preparing highly purified mixture of amino acids with the low content of peptides.

EFFECT: improved preparing method.

2 tbl, 4 ex

FIELD: biotechnology, biochemistry, amino acids.

SUBSTANCE: invention relates to a method for producing L-amino acids, for example, L-isoleucine and L-leucine prepared by culturing a microorganisms in medium and able to produce L-amino acid. This microorganism carries the avtA gene encoding enzyme alanine-valine transaminase (transaminase C) and possesses the enhanced activity of this enzyme as compared with it's the parent strain. Accumulated L-isoleucine and L-leucine are isolated from the cultural fluid. Invention provides preparing L-isoleucine and L-leucine with high effectiveness degree by diminishing the amount of amino acid as a by-side product that results to complications in the process of purification of indicated L-amino acids.

EFFECT: improved producing method of amino acids.

8 cl, 1 dwg, 2 tbl, 3 ex

The invention relates to a microbiological method of obtaining aminoacids collection aspartate and/or glutamate on PP 1-17 claims, genes of providerbased on PP 18-23 claims, gene structures by p. 24 claims, vectors for p. 25 claims, transformed cells in PP 26-31 claims, as well as to their application on PP 32-37 claims

The invention relates to biotechnology and can be used for the production of L-lysine

The invention relates to the microbiological industry and relates to a new strain of bacteria-bacteria producing L-leucine, an essential amino acid, which is used as nutritional additives to foodstuffs and animal feed, as well as a component of nutrient mixes and reagents in medical, pharmaceutical and chemical industries

The invention relates to the microbiological industry, and specifically to biotechnology, and can be used to produce mixtures of amino acids from proteins of yeast

FIELD: biotechnology, amino acids, microbiology.

SUBSTANCE: invention relates to a method for preparing L-glutamic acid by fermentation including culturing microorganisms possessing the ability for producing L-glutamic acid in medium with pH 5.0 or less wherein the total content of organic acid that inhibits the microorganism growth at this pH corresponds to the amount wherein the growth of microorganisms is not in inhibited. Also, L-glutamic acid is prepared by fermentation that includes culturing microorganism possessing the ability for producing L-glutamic acid at the first pH value wherein organic acid in medium doesn't inhibit the growth of microorganism followed by culturing microorganism at the second pH value suitable for production of L-glutamic acid by microorganism and at value lower as compared with the first value of pH. Microorganism can metabolize the carbon source in liquid medium containing L-glutamic acid at the saturation concentration and carbon source at pH 5 or less and able to accumulate L-glutamic acid in the amount exceeding the saturation concentration of L-glutamic acid. The claimed invention provides preparing L-glutamic acid with the high effectiveness degree.

EFFECT: improved preparing method.

10 cl, 13 dwg, 2 tbl, 1 ex

FIELD: organic chemistry, industrial microbiology, biochemistry.

SUBSTANCE: invention relates to a method for producing amide compound from a nitrile compound by using bacterial cells possessing nitrile hydratase activity. Bacterial cells are contacted with nitrile compound in an aqueous medium at continuous addition of a nitrile compound and without immobilization of cells by uptake that results to accumulation of amide compound in medium in the concentration 20% and above. Proposed invention provides enhancing the output of method in producing amide compound based on preparing the higher concentration of amide compound in medium and for shorter time period.

EFFECT: improved producing method.

6 cl, 4 ex

FIELD: biotechnology, biochemistry, enzymes, amino acids, microbiology.

SUBSTANCE: invention relates to a method for producing and preparing L-amino acids, such as L-tryptophan, L-phenylalanine, L-tyrosine and L-histidine that are prepared by culturing the modified microorganism of genus Escherichia with enhanced activity of 6-phosphogluconolactonase. The claimed invention provides preparing indicated amino acids with the high degree of effectiveness.

EFFECT: enhanced yield of amino acids.

15 cl, 9 dwg, 5 tbl, 14 ex

FIELD: biotechnology, biochemistry, amino acids, genetic engineering.

SUBSTANCE: invention relates to a method for producing L-threonine. Method involves partial inactivation of gene encoding enzyme threonine hydratase (tdc) in genome DNA of microorganism by using recombinant technology procedure. Strain of microorganism Escherichia coli pGm TN-PPC12 (KSMM-10236) is transformed with DNA fragment Δtdc::1oxpKan and the strain E. coli TRN212 (KSSM-1053) is prepared. This strain is cultured for preparing L-threonine. The claimed invention provides significant increasing the yield of L-threonine.

EFFECT: valuable properties of strain.

4 cl, 3 dwg, 4 tbl, 5 ex

FIELD: biotechnology, amino acids, microbiology.

SUBSTANCE: invention represents a method for preparing amino acid L-threonine by using bacterium belonging to Escherichia genus. This bacterium shows ability for production of L-threonine and modified by so manner that expression of gene chosen from the group glk, pgi, pfkA, tpiA, gapA, pgk, eno and pykA encoding glycogenolysis enzyme is enhanced. The claimed invention provides preparing L-threonine with the high degree of effectiveness.

EFFECT: improved preparing method.

7 cl, 1 dwg, 12 tbl, 9 ex

FIELD: biotechnology, amino acids, biochemistry.

SUBSTANCE: invention relates to a method for preparing L-amino acids by effect of plant phenylalanine-ammonium lyase in the presence of ammonia on derivative of acrylic acid comprising aromatic ring that comprises different substitutes and a teroatom and wherein L-amino acid represents aromatic L-amino acid. Invention provides preparing aromatic L-amino acids with high degree of effectiveness.

EFFECT: improved preparing method.

30 cl, 6 tbl, 7 ex

FIELD: biotechnology, amino acids, peptides.

SUBSTANCE: invention relates to a method for preparing a mixture consisting of amino acids and lower peptides from yeast autolyzates. Method involves autolysis of yeast, separation of cellular debris by centrifugation and purification of autolyzate in ionites resins wherein gel sulfocation-exchange resin in hydrogen form comprising 12-16% of divinylbenzene as a cross-linking agent is used as ionite. Proposed method provides preparing highly purified mixture of amino acids with the low content of peptides.

EFFECT: improved preparing method.

2 tbl, 4 ex

FIELD: biotechnology.

SUBSTANCE: L-amino acid is obtained by using bacteria belonging Escherichia genus having enhanced expression of xylose utilization gene. Claimed method includes culturing of said bacterium producer in cultural liquid containing xylose followed by isolation of L-amino acid from cultural liquid.

EFFECT: high effective method for production of l-amino acids.

18 cl, 1 dwg, 7 tbl, 6 ex

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