BG64077B1 - Method for the preparation of metal chelates by protein hydrolysates - Google Patents

Abstract

The chelates are used in veterinary and human medicines for the prevention and treatment of iron-deficiency anaemias, as well as for the supply of the microelements required for the organisms. By the method the metals are supplied in the form of metallic proteins or metallic peptide chelates, as a result of which their absorption by the gastric mucose membrane is facilitated as they are carried by the peptide or protein chelate bound by them. The invention relates to a protein hydrolysate of soya bean protein concentrates and isolates and protein hydrolysate and isolates of other origin having protein content over 70 g kg-1, produced by enzymic grinding with alkaline proteinase for 2 to 4 h and temperate range from 40 to 80 deg.C. The proteins and the peptide chelates with metals are produced by mixing of the so produced hydrolysates with hot solutions of the metallic salts in alkaline medium. 4 claims

Description

Technical field

The invention relates to a method for the preparation of metal chelates with protein hydrolysis, intended for the prevention and treatment of iron deficiency anemias and conditions, as well as for supplying the necessary trace elements for the body in veterinary and human medicine.

Iron (Fe), copper (Cu), cobalt (Co), zinc (Zn), manganese (Mn), magnesium (Mg), molybdenum (Mo) and some other metals are vital for the development of the human and animal organism. Iron is required for oxygen transfer (formation of hemoglobin) and for some other metabolic functions. Copper is involved in the action of important enzyme systems as well as in the formation of hemoglobin. Cobalt is an integral part of vitamin B _{| 2} and is required in the formation of blood elements. Zinc is required for the activation of important enzyme systems and in cell division. Manganese takes part in the growth processes of the body. Magnesium is involved in vasodilation and nervous system activity. Molybdenum is associated with the activity of some enzyme systems.

Iron and the other metals mentioned may occur as salts in the lower and higher valence states (lower and higher oxidized form). In such a condition, their absorption and passage through the gastric mucosa is difficult because it is positively charged and repels positively charged metal cations. Therefore, oral orally accepted metals as salts are only absorbed in the body by 4 - 5%, and the remaining amount is thrown out through the colon, most often with diarrheal effects. Binding of metals in organic salts (acetates, lactates, citrates, etc.) or as chelates with EDTA (ethylenediaminetetraacetic acid) also did not produce the desired results: in the first case, the metal is sufficiently ionized, and in the second, the metal is tightly bound in the chelate. connection to EDTA.

BACKGROUND OF THE INVENTION

In veterinary medicine, iron-containing preparations are now mainly used as anti-anemic iron-containing preparations, such as injectable solutions and preparations containing iron sulfate and other salts, as food additives. In the scientific literature, there are two preparations based on iron-protein complexes for oral administration: Organofer and Ferogen. For both preparations, there is a considerable amount of research that proves their exceptional effectiveness as anti-anemic preparations, with no data on the methods for their preparation [1, 2, 3]. Oral preparations based on ferric chloride, ferrous sulphate, ferric fumarate, ferrous aspartate and ferrous polyisomaltose are known as human medicine preparations [4,5].

In the patent literature, there is a known method for producing iron protein and proteins of the other said metals based on amino acids, di- and tripeptides, which are obtained by enzymatic digestion with proteolytic enzymes for six days in the presence of an antibacterial agent [6]. Under these conditions, proteins are obtained whose anti-anemic action is demonstrated. However, the method of producing the proteins is distinguished by its duration (six days) and low efficiency for industrial application. Selected enzymes, papain, bacterial and fungal proteinase, could not retain their activity in an alkaline environment for such a length of time.

SUMMARY OF THE INVENTION

The technical nature of our proposal consists in the choice of the enzyme, the sequence of steps, the time of enzymatic digestion and the reaction conditions between the protein hydrolyzate and the metal ions. Alkaline proteinase (subtilisin) is selected because of its high activity and low specificity (almost no preference for peptide bond rupture). We achieve high concentration (40%) of protein hydrolyzate by reversing the sequence of mixing the enzyme with the substrate. Instead of adding the enzyme solution (as always) to the substrate solution, we prepare the enzyme solution and gradually add the protein substrate. In this case, the protein substrate is immediately exposed to high concentrations of the enzyme, and due to the high initial digestion rate, the reaction mixture is flowable at the beginning of the reaction and is easily stirred. Under these enzymatic digestion conditions, the reaction lasts only about 2 hours, wherein the reaction mixture is a mixture of peptides with a molecular weight below 1000 Da (9 - 13%), with a molecular weight of 1000 to 5000 Da (5-15%), with molecular weight from 5000 to 10000 Da (50-60%) and with a molecular weight above 10000 (10-15%). This molecular weight distribution of peptides is demonstrated by high performance liquid chromatography (HPLC, HPLC). The formation of a chelate bond between the metal and the peptides are proof characteristic bands in the infrared spectrum at a wavelength λ = 880 cm ^'1 (the field of "fingerprinting"). Of great importance for the formation of chelates is the maintenance of pH above the neutral point, but not in the high alkaline region, as is done in the sources mentioned in the literature, but at a pH close to the neutral point. The biological activity of the metallo chelate proteins thus obtained is evidenced by experiments with animals - sows and newborn piglets [7,8] conducted at the Central Veterinary Medical Institute in Sofia. In all cases, the high absorption of the metal in the body, its passage into breast milk and transmission in the little piglets, is demonstrated. This provides favorable conditions for the production of medicines, both in veterinary and human medicine. The iron protein and the proteins of the other metals mentioned above have a protein content of not less than 42 g kg ' ¹ and a metal content of not less than 8 g kg-1. The color of the protein depends on the metal: for iron - brownish, for copper - blue, for cobalt - dark violet, for zinc - white to creamy white, for manganese - light beige, for magnesium - white and for molybdenum - light blue.

Examples of carrying out the invention

The method is demonstrated by the following embodiments

Example 1. Prepare a 0.1 solution of sodium hydroxide solution by dissolving 1 kg of sodium hydroxide in 250 l of water, add 40 g of calcium chloride to the solution, dissolve 4 kg 5 enzyme alkaline proteinase (subtilisin) with activity of 50 000 E / g and the temperature rises to 55-60 ° C. With continuous stirring for 20 minutes, 100 kg of soy protein isolate (or 100 kg of casein or 10 115 kg of soy protein concentrate or other protein product with a protein content exceeding 75 g kg are added ¹ ). With continuous stirring, the temperature was maintained in the range of 55 to bSS. 20 min after the addition of the last 15 portions of protein concentrate, 2 1 16% sodium hydroxide solution was added and after another 20 min another 2 1 16% sodium hydroxide solution was added. Stirring at the indicated temperature was continued for a further 60 min, after which 4 1 32% sodium hydroxide solution was added to the reaction mixture.

Without stopping stirring, 100 kg of ferrous sulfate (FeSO ₄ .7H ₂ O), previously dissolved in 50 l of water at 50 to 60 ° C and neutralized with 3.5 1 32% sodium hydroxide solution, were added. The reaction mixture was stirred for a further 30 min and then subjected to drying in a preferred manner (ventilation dryer, vacuum dryer30. Spray dryer or lyophilizer).

EXAMPLE 2 Protein hydrolyzate and the addition of ferrous sulfate were carried out as in Example 1, but before drying, the reaction mixture was filtered through filtration tissue 35 or centrifuged and the insoluble material was dried.

EXAMPLE 3 Protein hydrolyzate was prepared as in Example 1, but instead of a solution of ferrous sulfate to the protein hydrolyzate, 40 solutions of the following salts were added: 100 kg of copper sulfate (CuSO ₄ .5H ₂ O) or 40 kg of zinc chloride (ZnCl ₂ ), or 50 kg cobalt sulfate (CoSO ₄ .7H ₂ O), or 70 kg of manganese chloride (MnCip, or 70 kg of magnesium sulfate (MgSO _4), 45 or 50 kg molybdenum chloride. further, the reaction mixture was processed as in Examples 1 and 2.

Claims

Claims (8)

1. A method for the preparation of metallic proteinates, in particular metal-peptide chelates, characterized in that a portion of soy protein isolate or concentrate or any other protein concentrate is added to an alkaline solution of the enzyme alkaline proteinase (subtilisin). or an isolate with a protein content of not less than 70 g kg ' ¹ and enzymatic hydrolysis is carried out for 1 to 4 hours, during which time 8 N (320 g I ¹ ) sodium hydroxide solution is added. to keep the pH between 7.5 and 9.5, followed by the addition of a solution of 10 metal to the enzyme hydrolyzate. and a salt for the preparation of a metal proteinate. A method for the preparation of metal proteins (metal peptide chelates) according to claim 1, characterized in that the enzyme is from 1 to 8% to the protein substrate and 15 its alkaline solution is prepared in 0.05 N to 0.5 N sodium solution base or from 0.2 to 2.0 g 1 * and the temperature at which the enzymatic hydrolysis is carried out is from 35 ° C to 80 ^p C. 3. A process for the preparation of metallic proteinates (metal-peptide chelates) according to claim 1, characterized in that the soy protein isolate or concentrate or any other protein concentrate or isolate having a protein content of not less than 70 g kg ¹ was added 25 in an amount of 300 to 450 g I ¹ to the alkaline solution of the alkaline proteinase enzyme. Process for the preparation of metal proteins (metal peptide chelates) according to claims 1 and 2, characterized in that the 30 metal salt is one of the water soluble salts of iron (Fe ²⁺ ) or copper (Cu ²⁺⁾ ), or cobalt (Co ²⁺ ), or (Zn ²⁺ ), or manganese (Mn ²⁺ ), or magnesium (Mg ² *), or molybdenum (Mo ² *), which is added as a hot saturated solution to the enzyme hydrolyzate, stirred for 5 to 15 minutes, then dried. Literature 1. YamamotoH et al. (1982), Acomparative study of iron proteinate and ferrous fumarate on pig performance In: Chelated mineral nutrition in Plants, Minerals and Man (Ashmead D ed.) Springfields: Charles C Thomas. 2. Ashmead W. Beck B, Hopson H (1977), A new prophylactic approach to piglet mortality reduction, Modem Veterinary Practices, June, pp 334-337. 3. Fosta · J (1980), In-feed iron increases numbers weaned, Pig Farming, September, pp 330-331. 4. Mashkovsky, MD, (1988), “Medicines” (Pharmacotherapy Manual for Doctors), vol. 1 and vol. 2, “Medicine”, Moscow. 5. Arnaudov, DA, Arnaudova, GA, (1984), Medicinal therapy ”,“ Medicine and physical culture ”, Sofia. 6. Ashmead N.N. (1980), Compositions containing metal chelates., US 1 575 577. 7. Angelov, L., Dilov, P., Dalev, Π. (1994), Opportunities for Overcoming Hypotrophies in Newborn Pigs, Jubilee Scientific Session, Agricultural Academy, 2-3. XI. 1995, Abstracts, p.49. 8. Angelov, L. Encheva, Y. Dilov, P. Boyadzhieva, A. (1994), “Testing of iron-containing preparations in calves”, SCIENTIFIC SCIENCES - Sofia, Volume III, pp. 248-251.