CS269834B1 - Phospholipoproteins and method of their production - Google Patents

Abstract

Phospholipoproteins are new complexes containing acid phospholipids and peptides, or proteins in a weight ratio of 1 to 50: 99 to 50. Peptides must contain at least two amino acid residues, at least one of which it consists of a basic amino acid. Getting by mixing acid phospholipids with the peptides or proteins with vigorous cooling at 10 to 100 ° C. Phospholipoproteins find use as new biological factors. in medicine, cosmetics, food industry and agriculture.

Description

. CS 269834 81

The present invention relates to phospholipoproteins comprising acidic phospholipids complexed by peptides or proteins and a process for their production.

Phospholipoproteins are new biological factors that can be prepared using phospholipids obtained by known isolation and synthesis procedures. Especially suitable for industrial preparation are mixtures of phosphatidic acids and bis / diacylglycerol / phosphoric acid, their lysoderivatives, which are prepared by phosphorylation of a mixture of partial acylglycerols, e.g. Author's Certificate No. 173 220, MS. autorského certifikč. 203 421, MS. Copyright Certificate No. 256 691 and MS. Copyright Certificate No. 259 440.

The complexes that result from the interaction of proteins with phospholipids occur in all life objects, where they perform a number of functions, the most important of which is the function of the structural / bio-membrane (and the transport / transport of substances through the membranes or lipoproteins). In biological membranes, complexes form at the lipid bilayer phase boundary with integral and peripheral proteins. In terms of the structure of the complexes formed with integral proteins, the structures where the binding of both components is mainly mediated by hydrophobic interactions between the hydrocarbon chains of the acyl groups of the phospholipids and the lipophilic portions of amino acids in the polypeptide molecules such as aromatic nuclei in tryptophan, tyrosine and phenylalanine and aliphatic , optionally branched chain leucine, isoleucine and valine. Peripheral proteins that are located on the membrane surface are bound to polar membranes and mostly hydrogen bonds. Even these bonds are relatively weak, since peripheral proteins can be easily released from the surface, for example, by dilute solutions of neutral salts. The number of molecules of phospholipids bound to a protein molecule depends both on the size of the molecule of the integral protein and on the composition and structure of its surface which is in contact with the lipid bilayer. The protein-bound phospholipids are constantly exchanged at a rate comparable to the lateral diffusion rate of these molecules in the membrane space and their composition significantly affects the functional complex. Lipoproteins, which are the most prominent transport form of blood plasma lipids,, like biomembranes, are formed by the nucleus (containing triacylglycerol and cholesterol esters) and the mantle formed by the phospholipid layer, on which special protein molecules, called apoproteins, are bound. Here, too, binding occurs primarily through hydrophobic interactions between the hydrophobic chains of phospholipids and the hydrophobic side chains of apolipoproteins (whose structure is said to be amphiphilic). The hydrophilic side chains of apoproteins protrude into the aqueous phase and allow the transport of lipid blood plasma. The binding of all components in the lipoprotein particles is relatively loose, since, when moving lipoproteins in blood plasma, individual molecules of apoproteins, or lipids, are easily exchanged for others. The existence of the above described phospholipoprotein biomembrane and lipoprotein complexes has been predominantly derived from the physical properties of active systems. To our knowledge, due to their low stability, such complexes have not yet been isolated. Lipid-peptide-protein interactions are significantly involved in some other physiological processes, for example, in myelination of nerve fibers where lysophosphatidylcholine is bound to the basic myelin protein, as well as in the formation of anti -ectectactic lipoproteins, which resides in internal alveoli and the formation of ionophores, lipid complexes with lower peptides that allow the transport of ions through membranes and the like. In recent years, attention has also been paid to the importance of the interaction of lipids and proteins in foods, where a significant role in digestion and resorption of nutrients from the gastrointestinal tract is implicated in complex formation. In this connection, interactions of neutral / amphoteric / phospholipids / fo-spatidylcholine, lysophosphatidylcholine / with some food proteins and their fractions, especially with milk and soy proteins, were mainly studied. These works also showed that the hydrophobic interactions between the acyl chain phospholipids and the hydrophobic groups of the protein are predominantly applied to the complex formation and that the polar portion of the phospholipids remains relatively free. In other words, the isolation of these complexes for the practical use of Jakobiological factors in medicine and nutrition would be difficult to implement due to their small chemical CS 269834 B1 and physical stability. The fact is that the complexes of this type have never appeared on the market.

According to the invention, phospholipoproteins characterized in that they contain acidic phospholipids and peptides or proteins in a weight ratio of 1 to 50: 99 to 50 are suitable for industrial production and for applications as biological factors in medicine, food industry and animal nutrition. phospholipids may be natural or synthetic glycerol phospholipids or sphingophospholipids containing at least one free acid phosphate or carboxyl group capable of interacting with basic groups of peptides or proteins. These include, but are not limited to, phosphatidic acid, bis / diacylglycerol / phosphoric acid, plasmanic acid, plasminic acid, and phosphatidylglycerol phosphoric acid; Suitable peptides are both low molecular weight and high molecular weight peptides containing at least two amino acid residues / dipeptides, at least one of which is a basic amino acid such as arginine, lysine, histidine or ornithine. They can be prepared either by isolation from natural materials or by suitable methods of chemical synthesis and biosynthesis. The protein component in the phospholipoproteins can be any proteinaceous, microbial or animal origin or synthetically / purely chemically or biochemically / native or denatured that carries basic groups capable of binding phosphate or carboxyl anions in its side chains. These may include, but are not limited to, protein concentrates from soybean, rape, potato, algae, leaf tree plant, yeast, milk, bone, skin, blood and blood plasma, egg white and yolk, or mixtures thereof.

The phospholipoproteins of the invention can be prepared by intensively mixing both components at temperatures of 10 to 100 ° C. Phospholipids, which are liquid at a given temperature, can be mixed with powdered peptides and proteins, and the reaction is accelerated by the addition of a small amount of water. The solid phospholipids can be pre-dissolved in suitable organic solvents, for example aliphatic hydrocarbons, in chlorinated and fluorinated hydrocarbons, in diethyl ether and higher molecular ethers, in lower alcohols, etc. Excess solvent is removed by evaporation after the reaction. The reaction between acidic phospholipids and peptides or proteins can also be accomplished by dispersing phospholipids in suitable protein solutions and evaporating the water in a suitable apparatus at elevated temperature, optionally reduced pressure.

The phospholipids in the phospholipoproteins prepared according to the invention are bound not only by common hydrophobic interactions and hydrogen bonds, but also by their phosphate and free carboxyl groups and by stronger electrostatic bonds to basic groups of lysine, histidine, arginine in the side chains of peptides and proteins. The formation of the already-bonded ion pair without proton transfer or ion bonding results, among other things, in a distinct change in the pH of the reacting system.

By binding phospholipids to peptides or proteins, their solubility in water and organic solvents will change significantly. Most phospholipoproteins, in contrast to the initial phospholipids, are well soluble or dispersible in water and behave, in proportion to the phospholipid and peptide or protein, as a typical surfactant, forming an oil-in-water or water-in-oil emulsion with hydrophobic oils. . The presence of a high molecular weight peptide or protein component ensures good stability of the emulsion or dispersion formed. Example 1 85 kg of soy concentrate powder containing 72 wt. The protein was mixed in a sealed homogenizer connected to a cooled condenser with a dispersion of 15 kg of phosphatid and bis / diacylglycerol / phosphoric acid acidic ammonium salts in 100 kg of p-hexane. Acid salts were prepared by phosphorylating diacylglycerols from sunflower oil and partial neutralizing with gaseous ammonia and containing 40 wt. % phosphatidic acids, 15 wt. bis / diacylglycerol / phosphoric acids, 6 wt. their lysoderivatives, 1% fatty acids, 2% wt. % diacylglycerols and 36 wt. triacylglycerols. After addition of 2 kg of water, the reaction mixture was stirred at 40 to 55 ° C for 2 hours under reduced pressure. The phospholipoprotein obtained was made into a sieve in a screen mill and was used as a nutritional factor in dietetic preparations for athletes. It contained a complex of phospholipids and proteins in a weight ratio of 25: 75. Example 2 1 kg of lyophilized human seralbumin reacted with a dispersion of 10 kg of synthetic 1-hexadecyl-2-lyso-sn-glycerol-3-phosphoric acid (1-hexadecyl-2-lyso-plasman) acid / 200 ml of diethyl ether and 20 ml of water. After stirring for 30 minutes, diethyl ether was distilled off under reduced pressure at 30 ° C and the so obtained phospholipoprotein powder was thin-layered in a vacuum oven for 2 hours at 30 ° C. The composition of the complex of phospholipids and proteins in a weight ratio of 1: 99 was tested as a modifier for malignant transformations and cell growth. Example 3 10 kg of serum milk protein ultrafiltrate containing 65 wt. The proteins were dispersed in a dispersion of 7 kg of phosphatidylcholine fraction of soybean lecithin in 30 liters of hexane and after 20 minutes of stirring the solvent was evaporated at 40 ° C under reduced pressure. 16.5 kg of a phospholipid-containing protein complex was obtained in a 50: 50 weight ratio. Example 4 90 kg of potato protein / 85 wt. of protein / was mixed with 10 kg of a mixture of phosphatide and bis / diacylglycerol / phosphoric acids obtained by phosphorylation of low-oleic diacylglycerols from rapeseed oil in a tempered 80 ° C homogenizer. The phospholipld mixture contained 60 wt. % phosphatidic acid, 10 wt. bis / diacylglycerol / phosphoric acid, 15 wt. % of their lysoderivatives, 4 wt. fatty acids and 11 wt. triacylglycerols. After stirring for 2 hours, when the initially acidic mixture was neutral, the phospholipoproteins obtained were screened in a sieve mill and used as nucleating factors in the production of dietary pastries and as emulsifiers in emulsified fats. The weight ratio between phospholipids and proteins in this complex was 16: 84. Example 5 In 1 kg of liquid egg whites containing 10.5 wt. 10 g of soya lecithin, obtained by chromatography of lecithin on an alumina column, were dispersed in the protein. % phosphatidylinosit, 12 wt. % phosphatidyl serine, 10 wt. % phosphatidylethanolamine, 5 wt. % phytoglycolipids, 4 wt. phosphatidylcholesterol, 1 wt. % lysophosphatidylcholesterol, 3 wt. phosphatidic acid and 1 wt. fatty acids. After 10 minutes of stirring at 25 ° C, the resulting emulsion was sprayed in a stream of hot air in a spray dryer. The final phospholipo-protein powders were used as a nutritional factor and emulsifier in enteral nutrition. They contained phospholipids and proteins in a 9: 91 weight ratio

To 500 mg of an aqueous solution (4%) of bradykine nonapeptide was mixed at 40 ° C with 100 mg of a 1% dispersion of 1,2-diolevl-sn-glycerol-3-phosphate / phosphatidic acid prepared by the reaction of 1,2-sn-dioleylglycerol with oxychloride phosphorous. After 5 minutes, stir

Claims (3)

The original acidic reaction of the mixture changed to neutral. The obtained phospholipoprotein solution was used as a biologically active substance in the study of experimental inflammations. The weight ratio between the acid phospholipid and the peptide was 5: 95. Phospholipoproteins are new substances that will require considerable research in medicine, the food industry, agriculture, or other fields. According to the results obtained so far, they can be used for the preparation of dispersion enteral dietary mixtures for application in medicine, which are introduced as a full-value supplement of parenteral nutrition. In dermatology, albumin and collagen complexes are likely to be used as additives to creams and lotions, and as pharmaceuticals for the treatment of wounds and burns. In human nutrition, they can be applied as additives with a high nutritional value to new types of dietetics and mixtures with a corrosive effect, especially for athletes, convalescents, cancer patients as well as for physiologically aging populations. Furthermore, as non-toxic emulsifiers and stabilizers for special food emulsions and as fat dispersants in meat products and the like. OBJECT OF THE INVENTION Phospholipoproteins composed of acidic natural or synthetic glycerophospholipids and proteins of vegetable, microbial or animal origin, characterized in that they contain acidic phospholipids and peptides or proteins in a weight ratio of 1 to 50: 99 to 50. 2. The phospholipoproteins of claim 1, wherein the peptides comprise at least two amino acid residues, at least one of which is a basic amino acid such as arginine, lisine, histidine, ornithine. 3. A process for producing phospholipoproteins according to claim 1, wherein acid phospholipids or solutions thereof in organic solvents or their dispersion in water with powdered peptides or proteins are mixed with vigorous stirring at a temperature of 10 to 100 ° C. with aqueous solutions of peptides or proteins, and after completion of the reaction excess water is removed by evaporation or azeotropic distillation. 5