CS261398B1 - Process for preparing matrix proteins and peptides - Google Patents

Abstract

The solution concerns a method for preparing matrix proteins and peptides, where the starting biological material containing significant proportions of blood, for example placenta, is extracted after preliminary mechanical treatment in an acidic environment at pH 2 to 4 and at a temperature of 42 to 125 °C. This causes the blood proteins to precipitate into a solid coagulum and the respective matrix oroteins or peptides to pass into a solution, which is subsequently separated from the solid residue. The aqueous component of the extract can be removed completely or only partially. The product is expected to be used in cosmetics, food industry, medicine, biology and biotechnology.

Description

The subject of the invention is a method for preparing matrix proteins and peptides.

□A number of procedures suitable for the isolation of collagen proteins from various connective tissues - extracellular matrix - have been described. These procedures are mainly based on extraction techniques, where the starting material, i.e. suitable biological material such as skin, tendon, bone, is exposed to the action of appropriate extraction agents. These are usually aqueous solutions of organic or inorganic acids or salts, urea, etc., which, by their nature, are used in a wide range of hydrogen ion concentrations; pH 2 to 12.

In some cases, these extraction techniques are combined with so-called limited digestion, in which the starting biological material is first exposed to the action of common proteolytic enzymes, e.g. pepsin in an acidic environment. Such a procedure is particularly suitable for mature tissues with a high content of cross-links, e.g. articular cartilage, blood vessels, trachea, etc. Here, the action of proteases cleaves off small peptides at the ends of collagen molecules, where the aforementioned cross-links are mainly concentrated. In this way, relatively intact collagen chains can be released from the extracted tissue and then transferred to solution in greater yield.

- 2,261,398

In the temperature range of +4 to 40°C, the matrix proteins can be isolated more or less intact by the above procedures. In contrast, extraction carried out at temperatures around 60°C and above usually leads to irreversible denaturation of the protein material and is accompanied by cleavage of peptide bonds in the main chain of the protein. This results in a significant reduction in the molecular weight of the resulting extraction product, i.e. peptides of various sizes are formed. Gelatins for technical or food applications are commonly prepared in this way.

However, there are tissues with extensive vascularization and rich in blood proteins and blood cells, such as placental tissue, which makes the isolation of pure matrix proteins or peptides very difficult. The usual washing of the raw material with flowing cold water is very lengthy and expensive. This produces large volumes of wastewater and poses a problem of its purification. In addition, bacterial contamination easily occurs here, which degrades the final product. Blood proteins present in the tissue are very susceptible to microbial decomposition and are an ideal substrate for numerous microorganisms. The use of inhibitors, such as sodium azide or organomercury compounds, is effective in suppressing the growth of microorganisms, but very problematic due to the need to remove these toxic substances from the product.

The above difficulties mean that the available biological material is not yet used and its valuable matrix components are wasted.

It has now been discovered and practically verified that by using a suitable combination of an acidic environment and heat, blood proteins can be almost quantitatively coagulated together with blood cells so that they do not pass into the extract of matrix proteins or peptides. These can then be

- 3,261,398 easily separated from the solid coagulum and remnants of the starting tissue, e.g. by centrifugation.

The essence of the method for preparing matrix proteins and peptides according to the present invention is based on the empirically established knowledge that blood and matrix proteins behave completely differently in an acidic environment when exposed to heat.

Under these conditions, blood proteins are denatured and thus pass into an insoluble form - a blood clot. At the same time, most of the blood cells are also entrapped in the resulting clot. Here, some of the known coagulation factors, which have the nature of enzymes, are apparently also used, at least partially. As is known, they are more active at slightly elevated temperatures, e.g. 37°C up to temperatures around 56°C, and at higher temperatures they are already denatured and inactivated.

Matrix proteins are also denatured under the above conditions, but the denaturation products are highly soluble in an acidic environment, which practically allows their extraction from the relevant biological material.

The temperature required for the effective extraction of matrix proteins or peptides can be chosen within relatively wide limits. However, the lower limit should be at least 42°C and the upper limit of the temperature is practically limited to about 125°C, i.e. when using an autoclave. In general, the rule applies here that the higher the extraction temperature, the deeper the degradation changes take place in the given biological material. Protein chains thus produce larger or smaller peptides, the size or mass distribution of which depends mostly on the chosen temperature regime.

Another factor that is significantly applied in processes

- 4 261 398 extraction, is the time during which the given environment acts on the processed raw material. It has been empirically verified that this time has only a small effect on the extraction yield if it takes place at a milder temperature regime, i.e. up to 95°C at most. However, at temperatures around 100°C and higher, when deeper degradation changes occur in the starting material, the time of contact of the raw material with the given environment should be as short as possible. Otherwise, the product is burdened with unsuitable and also differently colored substances, which are created here by the decomposition of some components of the blood clot, such as heme compounds.

It follows from the above that the choice of temperature regime and extraction time are decisive factors for the quality of the final product. The definition of an acidic environment can be defined by the pH value here in the range of 2 to 4. Suitable inorganic or organic acids can be used to acidify the environment, e.g. hydrochloric, formic, acetic, propionic, malonic, lactic, citric, etc. The latter substances are well known from isolation techniques for collagen proteins. These substances are added to the processed raw material suitably diluted with water, with the ratio of the aqueous phase to the initial amount of raw material preferably chosen as 1:1 to 2:1; a larger amount of the aqueous phase then represents higher costs for energy necessary for its subsequent removal.

Sometimes it is advantageous to use volatile acids, e.g. hydrochloric or acetic, which can be easily removed during further processing of the extract. This is the case when drying an aqueous extract of matrix proteins or peptides by freeze sublimation or lyophilization, or by evaporation in a vacuum under heat - on a vacuum evaporator. However, it is not always necessary to remove all the water and then only a partial concentration of the extract is sufficient. In such and similar

- 5 261 398 cases, the presence of acids or other suitable preservatives is rather desirable. These substances prevent microbial attack and product deterioration. In addition to the aforementioned preservatives, other ingredients can be added to the aqueous extract of matrix proteins or peptides, depending on the intended use of the product.

Protection against microbial contamination also applies to the starting material, which must be ensured from the very beginning, i.e. when collecting the biological material. For example, in the case of bovine placenta, preservation is carried out by freezing it to at least -10°C, preferably -18 to -20°C. The raw material is stored in this way until its further processing; frozen it can last for many months without deterioration.

The preparation of matrix proteins or peptides, according to the method of the present invention, is characterized by the following steps:

(a) cutting the frozen raw material into smaller pieces, (b) grinding the raw material in a meat grinder in the cold, (c) mixing the raw material with an aqueous acid solution, (d) swelling the raw material in the cold and homogenizing it, (e) thermal extraction in an acidic environment, (f) separating the extract from the solid components of the material, (g) possible concentration of the extract and/or addition of ingredients, (h) possible drying of the extract to a solid product.

The method according to the invention can be used to prepare matrix proteins or peptides in relatively pure form and in high yield. According to the selected physical and physicochemical conditions, the above substances can be prepared in a wide range of molecular weights. These substances can find significant application in various fields, e.g. cosmetics, food industry, medicine, biology and biotechnology.

- 6,261,398

The examples given below serve to illustrate the invention and do not in any way limit the number of possible combinations, substance ratios and conditions for the preparation of matrix proteins and peptides.

Example 1

Beef placenta, stored at -Í8°C, was mechanically cut into pieces approximately in the shape of 3x3x5 cm fries, which were then ground on a knife meat grinder with a steel sieve with openings 0 2 mm. This yielded about 5 kg of ground raw material, which was immediately mixed with three liters of an aqueous solution of acetic acid with a concentration of 1%-volume. The total volume was then adjusted to ten liters by adding cold water. The material was left to swell in the cold at +4°C for 15 hours; the mixture was occasionally stirred with a wooden spoon.

The swollen material was then briefly mixed and 200 ml samples were taken from it; these were placed in 1/4 liter glass infusion bottles, which were equipped with a rubber stopper and a metal screw cap.

The actual thermal extraction in an acidic environment (pH 3.85) was carried out by heating the containers at a temperature of 42°C, or 60°C, or 90°C, or 100°C - all for 2, or 4, or 6 hours; or at 120°C for 1, or 2, or 3 hours. Heating to 42°C was carried out in a water thermostat; at 60 to 100°C in a hot air circulation dryer; at 120°C in a sterilization autoclave.

After cooling the containers to room temperature, their contents were transferred to centrifuge tubes and centrifuged at 10,000 rpm for 30 minutes at 20°C; the material gels easily in the cold.

- 7,261,398

The clear supernatants were then separated from the solid residue by pouring into additional 1/2 liter infusion bottles.

In them, after freezing to approximately -60°C in a bath of alcohol and solid carbon dioxide, their lyophilization was carried out. The solid material residues were dried using a hot-air dryer at a temperature of 105°C until constant weight. A sample of the original ground material was taken for control, for which the dry matter was determined by the same method, here 8.3%-wt.

The following table shows the extraction conditions and the corresponding yields; weight of dry extract + residue » 100%.

Terms and Conditions extraction: Profit extraction: Product Features: temperature time extract the rest approximate Mw, event. (°C) (hours) mass) product coloring 42 2 15.8 83.2 • Λ <\5 proteins, 10 42 4 18.1 81.9 ditto 42 6 18.6 81.4 ditto 60 2 27.4 72.6 peptides, 10 ³ ” ⁴ 60 4 28.3 71.7 ditto 60 6 29.9 70.1 ditto 90 2 52.7 47.3 peptides, 10 ³ “ ⁴ 90 4 54.3 45.7 ditto 90 6 56.5 43.5 ditto 100 2 67.8 32.2 peptides, 10 ^{2 4} 100 4 69.1 30.9 ditto 100 6 70.2 29.8 ditto, color, prod. 120 1 69.7 30.3 , peptides, 10^ 120 2 72.1 27.9 ditto, color prod. 120 3 75.2 24.8 ditto, color, prod.

- 8 Example 2

261,398

Frozen bovine placenta was mechanically processed according to the procedure given in Example 1; 5.3 kg of ground raw material was obtained. To this was added 5.5 liters of cold water with stirring, together with 55 ml of concentrated hydrochloric acid. The mixture was left in a refrigerated box at +4°C for 24 hours; the acid-swollen mass had a pH of 2.18.

Thermal extraction of the material placed in the enameled vessel was carried out by heating it in a boiling water bath. After about 3/4 hour, a temperature of 95 to 96°C was reached in the stirred material and this heating was continued for another hour. The mixture was then strained while hot through two layers of gauze, with approximately 7.5 liters of the liquid portion being collected and the remainder disposed of. The extract was purified by centrifugation under the previously stated conditions. Part of the product, i.e. 2.5 liters, was dried by lyophilization and the remaining solution was stored in the cold at +4°C; dry matter 2.4%-wt.

Example 3

About L

Matrix peptides with an approximate Mw of 1CT, prepared by lyophilization of acidic placenta extract as described in Example 2, were experimentally used in the formulation of a lanolin-based skin cream. Preliminary application tests showed beneficial nutritional effects of the cream for dry and older skin.

Example 4

The liquid matrix peptide product, prepared according to Example 2, was neutralized by the addition of ΙΜ-NaOH and further used to prepare culture plates for rabbit endothelial cells isolated from the aorta. The adherence and growth of these cells were stimulated by tissue-specific peptides from placental basement membranes.

Claims (3)

SUBJECT OF THE INVENTION 261 398 A method for the preparation of matrix proteins and peptides, characterized in that the starting biological material containing substantial proportions of blood, such as the placenta, is extracted, after a mechanical pretreatment, in an acidic pH of 2 to 4 and at a temperature of 42 to 125 ° C. the blood proteins precipitate into the solid coagula and the respective matrix proteins or peptides pass from the extracted tissue into a solution which is subsequently separated from the solid residue, for example by centrifugation. 2. Process according to claim 1, characterized in that a suitable water-dilute inorganic or organic acid, e.g. hydrochloric or acetic acid, is used to acidify the medium, the proportion of the aqueous phase to the starting material being preferably 1: 1 to 2: 1 parts by volume. 3. The method according to claim 1, wherein the aqueous component of the resulting matrix protein or peptide extract is partially or completely removed, e.g., by vacuum evaporation or lyophilization.