RU2286685C1 - Method for production of iron-containing biologically active food supplement - Google Patents

Abstract

FIELD: production of iron-containing biologically active food supplement useful in prophylaxis of iron-deficit condition in human.

SUBSTANCE: claimed method includes Andara broughtoni haemolymph frosting, defrosting, centrifugation, filtering on membrane with pore size of 0.2 mum. Further obtained product is concentrated and washed from contaminants with water in ultrafiltration apparatus with membrane, passing globular proteins with molar mass of 300 kDa or less to produce oxyhemoglobin. Then proteolytic enzyme is added to oxyhaemoglobin in oxyhaemoglobin/enzyme ratio of 100:1-2. Mixture is incubated for 4-5 h at 29-30°C. Obtained haemoprotein is washed with distilled water to remove low molecular hydrolysis products and concentrated in ultrafiltration apparatus with membrane of 30 kDa. Claimed food supplement contains bivalent iron in amount of 3-5 % based on dry material mass that is higher by 15-20 times in contrast to supplement obtained from animal blood.

EFFECT: bioactive food supplement with sufficiently increased bivalent iron content.

2 cl, 1 dwg, 1 tbl, 3 ex

Description

The invention relates to the field of production of biologically active food additives that can be used to prevent iron deficiency conditions in humans.

According to the World Health Organization, iron deficiency anemia (IDA) affects 2 billion people in the world. The latent iron deficiency (pre-stage IDA) among residents of Europe and Russia is 30%. IDA occurs with a decrease in the amount of hemoglobin in blood compared with the physiological norm due to insufficient intake of iron from food.

Cattle blood is known to be a valuable source of iron-containing proteins for the pharmaceutical industry.

A known method of purification of hemoglobin from a crude blood solution by ion exchange chromatography [RU 2145873 C1, 02.27.2000]. This method of obtaining hemoglobin is time-consuming and quite complicated.

There is a method for isolating hemoglobin, used as a dietary supplement, from the blood of farm animals by producing erythrocyte hemolysate by diluting the erythrocyte mass with distilled water and freezing, followed by concentration of the hemolysate by ultrafiltration, sterilized filtration and lyophilization [RU 2080865 C1, 06/10/1997]. This method is multi-stage, requires significant costs and does not allow to produce hemoglobin that does not contain pathogenic viral particles, without additional purification steps.

A dietary supplement, which is a powder composition, including purified cattle blood hemoglobin, contains heme iron in an amount of 0.3 ± 0.05% by weight of the hemoglobin contained in the additive and is used in the manufacture of sweets [RU 2161888 C1, 20.01. 2001].

Currently, a search is underway for alternative sources of hemoproteins, phylogenetically remote from mammals and not susceptible to human-dangerous viral diseases.

There is a method of complex processing of anadara ″ Anadara endtoni ″, in which hemolymph is used as a raw material for the production of biologically active food additives. Hemolymph, selected during cutting from anadara, is frozen or dried, while in order to prevent coagulation of proteins, it is mixed with ethyl alcohol in a ratio of 2: 1 and dried by sublimation. The dried powder is used as a biologically active food supplement or as a raw material for the isolation of dietary supplements - heparin, which prevents blood coagulation and blood clots [RU 2231272 C2, 06/27/2004].

This method involves the use of an organic solvent to stabilize extractive substances and freeze-drying an alcohol solution, which does not allow one of the active components of hemolymph to be isolated - a heme-containing protein in a chromatographically pure form.

The objective of the invention is the development of a method for producing iron-containing biologically active food additives using an unconventional source of this substance.

For this, the heme-containing product is isolated from the hemolymph of the commercial mollusk Anadara headtoni, which remains in the process of industrial processing and contains a large number of respiratory pigments.

Hemolymph is frozen (to carry out hemolysis of cellular structures and the convenience of transportation, storage), defrosted, centrifuged, filtered on a membrane with a pore size of 0.2 μm, concentrated and washed from foreign impurities with water on an ultrafiltration unit with a membrane passing 300 globular proteins with a molecular weight of 300 kDa and below to obtain oxyhemoglobin. According to atomic absorption spectral analysis, the preparation contains 0.2% iron.

Then, a proteolytic enzyme is added to oxyhemoglobin in the ratio oxyhemoglobin: enzyme 100: 1-2, the mixture is incubated for 4-5 hours at a temperature of 29-39 ° C. The resulting hemoprotein is washed with distilled water from low molecular weight hydrolysis products and concentrated on an ultrafiltration unit with a 30 kDa membrane. Get biologically active food supplement of hematoprotein nature, containing ferrous iron in the amount of 3-5% by weight of dry matter.

As a proteolytic enzyme, a complex of king crab proteinases, or papain, or pepsin, is used.

In order to increase the shelf life, the target product is freeze-dried.

There is evidence of better absorption of ferrous iron in the presence of ascorbic acid [Annals N.Y. Academy of Sciences Vol. 355, p. 32-44. (1980)]. Therefore, when applied to the target product, it is advisable to add ascorbic acid (0.05 mg / ml).

The technical result provided by the invention is to obtain an iron-containing biologically active food supplement, in which the iron content is 15-20 times higher compared to the iron content in the additive obtained from the blood of farm animals.

Using as a source of heminic iron, the A. mollusk shellfish, phylogenetically removed from mammals and not susceptible to human viral diseases, allows producing an iron-containing biologically active food supplement that does not contain pathogenic viral particles, without additional purification steps as opposed to obtaining one from the blood farm animals.

The technical result also consists in expanding the raw material base for obtaining iron-containing biologically active food additives, as well as in expanding the arsenal of iron-containing biologically active food additives.

The inventive method is based on modern membrane technologies, does not require significant material costs, since the raw material used is a by-product of the industrial processing of commercial mollusk A. bytontoni.

The drawing shows the absorption spectrum of oxyhemoglobin obtained from hemolymph A. transferredtoni.

The implementation of the invention.

Example 1

1.0 L of anadara hemolymph is frozen, defrosted, centrifuged until the cellular material is completely removed, sterilized by filtration on a membrane with a pore size of 0.2 μm, concentrated by the ultrafiltration method on an installation with a membrane that transmits globular proteins with a molecular weight of 300 kDa and below. Concentrated oxyhemoglobin is washed from impurities with distilled water under the same ultrafiltration conditions. Kamchatka crab proteinase complex V = 5.0 ml (45.0 mg, activity 1.3 u / mg) is added to the obtained product V = 0.2 L (3.0 g dry matter), incubated for 4 hours and at a temperature of 37 ° C, pH 8. Next, the hematoprotein obtained by enzymatic hydrolysis is washed with distilled water from low molecular weight hydrolysis products and concentrated on an ultrafiltration unit with a 30 kDa membrane. The target product (V = 50.0 ml, 108.0 mg) contains iron in an amount of 5.0% based on dry matter.

Example 2

The method is carried out as described in example 1, but papain is used as an enzyme. The target product (V = 50.0 ml, 190.0 mg) contains iron in an amount of 3.0% based on dry matter.

Example 3

The method is carried out as described in example 1, but pepsin is used as an enzyme. The target product (V = 50.0 ml, 136.0 mg) contains iron in an amount of 4.2% based on dry matter.

Organoleptic, physico-chemical and microbiological indicators of hemoprotein from anadara are presented in the table.

Table
Organoleptic, physicochemical and microbiological indicators of anadara hemoprotein No. p / p The name of indicators Indicator values one Appearance Amorphous powder 2 Color Maroon 3 Smell Peculiar to fresh seafood four Mass fraction of moisture,%, no more one 5 Mass fraction of ash in terms of absolutely dry matter,%, no more 6 6 Mass fraction of crude protein in terms of absolutely dry matter,%, not less
97 7 Hemoprotein content,%, not less than 96 8 Mass fraction of lipids in terms of absolutely dry matter,%, no more
2 9 Iron content, g / kg, not less thirty 10 Lead content, mg / kg, no more Not allowed eleven Cadmium content, mg / kg, no more Not allowed 12 Mercury content, mg / kg, not more than Not allowed 13 Magnesium content, mg / kg, no more twenty fourteen Metallomagnetic impurity, mg / kg, no more Not allowed fifteen Solubility, 1 g in 10 ml of distilled water, min, not less than 10 16 Total bacterial contamination of 1 g of cell product, not more than 4 × 10 ⁵ 17 E. coli bacteria Not allowed eighteen Salmonella bacteria Not allowed 19 Toxicity Not allowed

Claims (3)

1. The method of obtaining iron-containing biologically active food additives, characterized in that the Anadara hemolymph (Anadara endtoni) is frozen, defrosted, centrifuged, filtered on a membrane with a pore size of 0.2 μm, concentrated and washed from impurities with water on an ultrafiltration unit with a membrane, transmitting globular proteins with a molecular mass of 300 kDa and below to obtain oxyhemoglobin, then a proteolytic enzyme in the ratio oxyhemoglobin: enzyme 100: 1-2 is added to oxyhemoglobin, the mixture is incubated in 4-5 hours at a temperature of 29-39 ° C, then the resulting hemoprotein is washed with distilled water from low molecular weight hydrolysis products and concentrated on an ultrafiltration unit with a 30 kDa membrane. 2. The method according to claim 1, characterized in that, as a proteolytic enzyme, a complex of king crab proteinases, or papain, or pepsin is used. 3. The method according to claim 1, characterized in that the target product is freeze-dried.