KR100434842B1 - The Simple manufacturing process for the heme-iron from hemoglobin - Google Patents

Abstract

The present invention relates to a method for the production of heme iron from hemoglobin, and simultaneously treated with two types of endo- and exo-type proteolytic enzymes having different proteolytic mechanisms to hemoglobin to obtain an enzymatic digestion having improved hydrolysis degree, and then the enzyme digested product is subjected to specific pH. As a method for producing heme iron by selectively sedimenting and then separating and drying the heme iron portion by adjusting to, the process is simpler than the conventional heme iron manufacturing method, and the heme iron yield is high.

Description

Simple manufacturing process for the heme-iron from hemoglobin

The present invention relates to a method for producing heme iron, which is one of the sources of iron, and more specifically, hemoglobin containing heme iron in the protein present in the blood of an animal is decomposed by enzymes, and then heme iron is used by using the difference in solubility between heme iron and peptides. It relates to a method for preparing a simple heme iron.

Iron in the living body is a kind of trace minerals, but the amount is small, but plays an important role mainly involved in oxygen transport. In Korea, the recommended daily dose of iron is 10-12 mg in children, 12-18 mg in adolescents, 12 mg in adult men, 18 mg in adult women, 26-30 mg in pregnant women, and 20 mg in lactating women. Iron in food comes in two forms, heme iron and non-heme iron, with an average of 40% of the iron content in animal foods being heme, the remaining 60% of non-heme iron, and all of the iron in vegetable foods. It exists in the form of a non-hem.

Heme iron is mostly taken in the form of hemoglobin and myoglobin, and its absorption rate is very high and is not affected by other dietary factors. However, the absorption of non-heme iron is affected by the type of food and the nutritional status of individuals. It is known to absorb only about 5 to 10% of iron. Iron deficiency, therefore, is the most common nutritional problem in the world due to inadequate intake, poor iron absorption, rapid growth and blood loss, and depletion of stored iron due to repeated pregnancy. The high risk of iron deficiency is seen in infants of 6 months to 4 years of rapid physical growth, in puberty, women of childbearing age with menstrual blood loss, and in pregnancy with increased demand for iron, especially infants and pregnant women who are prone to iron deficiency. It is known.

Ferric chloride, ferric citrate, ferric ammonium citrate, iron and sodium succinate citrate, ferrous lactate to prevent and treat iron deficiency ), Iron compounds such as ferric pyrophosphate and heme iron are used as iron reinforcing agents and pharmaceutical raw materials in food.

The rest of the iron compounds except heme iron are inorganic iron, which has a high iron content and economically inexpensive advantages, but has a low absorption rate in the living body and can cause iron poisoning when excessively ingested. The usage of is increasing.

Heme iron is a component contained in hemoglobin that is prepared by separating and purifying from animal blood. Hemoglobin (molecular weight 65,000 Daltons) is a protein consisting of four polypeptide chains containing heme groups. One mole of hemoglobin contains four moles of iron. The iron content of hemoglobin is 0.34% (= 223.2 / 65,000 × 100) when calculated according to the molecular weight, and the heme iron content is 3.8% (= 2,464 / 65,000 × 100). Therefore, in order to obtain a high concentration of heme iron, the content of the protein portion must be lowered. .

As a method for obtaining heme iron, hemoglobin is first isolated from red blood cells in blood, and carboxymethy cellulose (CMC) is added to precipitate heme into the CMC complex (Autio, et. Al., US Patent 4,518,525), Methods of using dehydrating agents such as lower alcohols or imidazol derivatives (Lindroos, US Patent 4,431,581), methods of adding amino acids of lysine and arginine (Ingberg, et. Al., US Patent 5,008,388) are known. have.

However, the above methods are designed to separate the globin protein from hemoglobin, and it is possible to separate the globin protein by adding a separate compound to the red blood cell or hemoglobin solution and to obtain heme iron as a by-product, but a separate process of recovering or removing the additive This necessitates a disadvantage in that mass production is difficult.

Another method is to decompose globin in hemoglobin by treatment with high concentration of organic acid (Liu, et. Al., J. Agric. Food Chem., 44, 2957, 1996) and protease. (Eriksson, US Pat. No. 4,411,915, Piot, et. Al., J. Chem. Technol. Biotechnol., 42, 147, 1988). The use of organic acids can contain a large amount of organic acids in the final product and adversely affect the quality.

Conventional methods using proteolytic enzymes have a low protein hydrolysis because they use only one type of endo type protease to degrade protein of hemoglobin, and hydrophobic bonds between peptides, peptides, peptides and heme groups resulting from the breakdown of hemoglobin. Aggregate is produced (Lebrun, et. Al., J. Agric. Food Chem., 46, 5017, 1998) and the molecular weight of heme iron-containing aggregates is greatly increased (Liu, et. Al., J.). Agric, Food Chem., 44, 2957, 1996) It is difficult to produce products containing heme iron at high concentrations by efficiently removing peptides.

There is a method of adding urea at a high concentration (35-50%) in order to facilitate the removal of peptides in the aggregate containing heme iron, but it is not economical because the amount of urea is added, and there is a disadvantage in that a process for removing urea is additionally required.

In addition, hemoglobin filed on September 9, 2000, the inventors of the present application to the method for producing heme iron (Application No. 10-2000-053778), hemoglobin in the endotype and exo-type proteolytic enzymes with different proteolytic mechanisms 2 There is a method to repeatedly decomposed by treatment more than once and then dried by ultrafiltration, but the process is complicated and expensive.

The present inventors have studied to overcome the disadvantages of the conventional methods for producing heme iron, and as a result, hydrolyzes only two specific peptides of different kinds of proteases, ie, specific peptide bonds of proteins, to decompose the polypeptide into oligopeptides. The solubility of heme iron and peptide in the enzymatic digestion of hemoglobin improved by hydrolyzing hemoglobin simultaneously with endo-type enzyme and exopeptidase, which non-selectively hydrolyzes the end of polypeptide by amino acid. The present invention was completed by confirming that they are different from each other by pH, and by simply adjusting the pH of the enzyme decomposition product to recover heme iron having a high iron content.

Accordingly, an object of the present invention is to provide a simple method for producing heme iron having a high iron content by simultaneously treating two or more kinds of proteolytic enzymes having different proteolytic mechanisms in hemoglobin and degrading the pH of the digestion solution.

An object of the present invention is to treat hemoglobin denatured in a basic solution at the same time to treat two or more proteolytic enzymes endo type and exo type enzymes to obtain a hydrolyzate of hemoglobin with high degree of hydrolysis and to adjust the pH of this enzyme decomposition product heme iron part The precipitates were separated and dried to obtain a dark brown powder. The yield of the dark brown powder was measured according to the pH and the content of heme iron in the powder, which were prepared in the same manner, except that the yield of the dark brown powder according to the degree of hydrolysis of hemoglobin and the heme iron in the powder were measured. By comparing the content of to achieve the optimal method of obtaining heme iron.

Hereinafter, the configuration of the present invention.

1 is a diagram showing a change in the degree of hydrolysis of hemoglobin over time.

Figure 2 is a diagram showing the solubility of heme iron and peptides in accordance with the pH in the enzymatic digest of hemoglobin.

In the present invention, hemoglobin hydrolyzate by adjusting the pH of the hemoglobin hydrolyzate is treated by simultaneously treating the proteolytic enzyme in the hemoglobin solution heated and denatured using an endotype and exo-type proteolytic enzyme suitable for hydrolysis of hemoglobin. It consists of a process required to prepare a dark brown powder containing heme iron by sedimentation and separation of the portion containing a large amount of heme iron.

Looking at the step-by-step process to process the enzyme in the hemoglobin solution to perform the hydrolysis efficiently so that the acquisition of heme iron simply and easily; In the same way, hemoglobin hydrolyzate, which is treated by endo type and exo type hydrolase simultaneously, is separated and concentrated by isoelectric point precipitation method or ultrafiltration, and then heme iron powder is prepared by hot air or spray drying. It consists of.

According to the above configuration of the present invention, the endo-type enzyme and the exo-type enzyme were simultaneously used to increase the content of heme iron, and as a result, the degree of hydrolysis could be improved, and the removal of the peptide portion in the process of separating the heme iron from the hemoglobin decomposition product was also very good. It was easy.

By treating endo-type enzyme and exo-type enzyme at the same time, the end of the polypeptide chain produced by endo-type enzyme is broken down into amino acid units, so it is easy to lower the protein content in the process of recovering heme iron and increase the heme iron content. There is this. In addition, since peptides produced by the action of endo-type enzymes are decomposed into amino acid units by the action of exo-type enzymes at the same time, in the pure peptide portion, the hydrophobicity of the peptides becomes weaker and the hydrophilicity becomes stronger due to the increase of amino and carboxyl groups, thereby increasing the solubility of the peptides. In addition, since the amino acid is liberated from the peptide by the exo-type enzyme, the peptide portion containing heme iron increases the fraction of the relatively hydrophobic iron-containing porphyrin ring, thereby decreasing the solubility of heme iron.

Therefore, since the production of aggregates generated by the hydrophobic bond between the peptide and the peptide, peptide and heme group is greatly reduced, the separation of heme iron becomes easy. This difference in solubility is proportional to the degree of hydrolysis of hemoglobin, and as the degree of hydrolysis increases, the difference in solubility between heme iron and peptide increases. Since the solubility of proteins and peptides is minimum at the pH corresponding to each isoelectric point, there is a pH condition with minimal solubility of the peptide portion containing heme iron, and it is easy to separate heme iron at specific pH conditions.

In the present invention, as a result of examining the relationship between pH and solubility of heme iron, pH 5 ~ 6 at 10-20% hydrolysis degree, pH 4-5 at 20-40% hydrolysis degree, and hydrolysis degree 40 Above pH%, the solubility of heme iron was minimized below pH 4, and as the degree of hydrolysis increased, the average isoelectric point of the peptide part including heme iron was shifted toward the acidic side. Therefore, pH conditions can be selected to efficiently separate heme iron according to the degree of hydrolysis of hemoglobin, and when the degree of hydrolysis increases, the pH of the hydrolyzate is adjusted to 3-4, minimizing the solubility of the heme iron, thereby easily producing high concentration heme iron. I can do it.

Another method for separating heme iron from hemoglobin digested by endo and exo-type proteases is chromatography, ultrafiltration, electrophoresis and the like. However, the electrophoresis method has a disadvantage in that it is difficult to process a large amount of samples, the ultrafiltration method using the chromatographic method and the membrane has the advantage of recovering heme iron in a large amount, but has a disadvantage in that the equipment cost is high. Therefore, the method of controlling the pH of the enzymatic digestion liquid as in the present invention has the advantage that the separation process is very simple, saving the equipment cost and the separation efficiency is also excellent.

As the endo type proteolytic enzymes used in the present invention, all enzymes of animal, plant, and microbial origin can be used, and since the substrate is used after denaturation of hemoglobin as a base under basic conditions, it is effective that the optimum pH of the enzyme reaction is neutral alkaline. As commercialized, alcalase, esperase, pescalase and the like are suitable for the decomposition of hemoglobin.

The exo-type degrading enzyme is economical to use Flavozyme, Peptidase FP as an enzyme of microbial origin.

The amount of each proteolytic enzyme is usually determined in the range of 0.1 to 10% (w / w) of the substrate protein in consideration of the economic time based on the enzyme reaction time and the activity of the enzyme. In addition, since the reaction conditions of the enzymes used are different, it is necessary to adjust the temperature and pH conditions to conditions in which the activity of the enzymes used can be maintained in consideration of the reaction conditions of the endo type enzyme and the exo type enzyme.

In the present invention, the degree of hydrolysis of the reaction solution is measured over time to hydrolyze hemoglobin to about 35 to 70%. At this time, the degree of hydrolysis is defined by the following formula.

In the present invention, the fraction from which the peptide and the amino acid portion are removed may be dried by hot air drying, spray drying, vacuum freeze drying, vacuum drying, or the like. The final dried product is dark brown powder with iron content of 1 to 3.2% (w / w), heme iron content of 11.1 to 35.5% (w / w), and yield of 34 to 10% (w / w) compared to hemoglobin. to be.

The present invention is to remove the protein from hemoglobin, so the degree of hydrolysis of the protein is increased, the more the protein is removed, the higher the heme iron content.

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples, but the scope of the present invention is not limited to Examples.

Example 1 Preparation of Heme Iron from Hemoglobin Treated Simultaneously with Two Proteinases

First step: hydrolysis of hemoglobin

100 g of hemoglobin (Sigma Co., Ltd.) was suspended in 1.5 L of distilled water, denatured by heating at 90 DEG C for 30 minutes, and then the temperature was adjusted to 7.5 by adding 0.1 M NaOH solution at 50 DEG C and pH to 2 L. In the same manner, three substrate solutions were prepared, the first of which was 1 g of spermase (Esperase), the second of which was 1 g of flavozyme (exoflavase), and the third of endo type enzyme 1 g of Esperase and 1 g of flavozyme, an exo-type degrading enzyme, were added simultaneously and digested for 5 hours, respectively. At this time, each substrate pH was kept constant at pH 7.5 using NaOH solution. Samples were taken over time during the hydrolysis, and the degree of hydrolysis was measured. As a result of using the endolytic enzyme and the exolytic enzyme at the same time, the degree of hydrolysis was more than doubled compared to the hydrolysis reaction using the endolytic enzyme or the exolytic enzyme alone.

Second Step: Investigation of Separation Conditions of Heme Iron from Hemoglobin Hydrolyzate

In order to investigate the separation conditions of heme iron in hemoglobin hydrolyzate, solubility of the heme iron portion and the peptide portion was examined according to pH. Take 200 ml of hemoglobin digests (45.6% hydrolysis degree) using both endo- and exo-lyases, adjust the pH to 2-9 with concentrated HCl and NaOH, and centrifuge for 15 min at 3,000 g. The total protein was determined by micro Kjeldahl method, and the content of heme iron was measured by pyridine-caustic soda method as shown in FIG. 2. The heme iron content of the supernatant in the hydrolyzate of 45.6% was determined to be the minimum in the range of pH 3-4.

Third Step: Separation and Drying of Heme Iron from Hemoglobin Hydrolyzate

After adjusting pH of 2L of hemoglobin digest (45% hydrolysis) previously prepared using endo- and exo-degradase simultaneously, centrifuge for 15 min at 3,000 g, discard supernatant and recover precipitate. Hot air was dried by the method. Analysis of the composition of the dried product 73% protein, heme iron 23%, moisture 4% yield was 16%.

<Example 2> to <Example 6> Preparation of heme iron according to the degree of hydrolysis of hemoglobin hydrolysates simultaneously treated with two kinds of protease

In the same manner as in Example 1, hemoglobin solution was prepared, and 1 g of endo-type enzyme esperase and 1 g of exo-type enzyme flavozyme were simultaneously added and digested for 5 hours while maintaining a constant pH of 7.5. At this time, take 100 ml of sample at the appropriate time differently in the degree of hydrolysis, adjust the pH according to the degree of hydrolysis, centrifuge for 15 minutes at 3,000 g, discard the supernatant, recover the precipitate, and dry by hot air in the usual way. It was.

The results of analyzing the pH conditions of the hydrolyzate and the components of the dried product are shown in Table 1.

[Table 1] Yield of heme iron according to the degree of hydrolysis of hemoglobin

Degree of hydrolysis (%) pH Protein content (%) Heme iron content (%) yield(%) 7.9 6.0 93 7 54.3 14.8 5.5 91 9 42.2 19.8 5.0 89 11 34.5 29.5 4.5 85 15 25.3 42.5 3.5 78 22 17.3

Comparative Example 1 Preparation of Heme Iron by Ultrafiltration from Hemoglobin Treated with Two Proteolytic Enzymes Simultaneously

In the same manner as in Example 1, hemoglobin solution was prepared, and 1 g of endo-type enzyme esperase and 1 g of exo-type enzyme flavozyme were simultaneously added and digested for 5 hours while maintaining a constant pH of 7.5. The low molecular weight component contained in 2 l of the final hydrolyzate (hydrolysis degree 43.3%) was removed using an ultrafiltration membrane (Millipore) made of recycled cellulose with a membrane pore size of 5,000 Daltons. The product was concentrated twice and further distilled while adding 2 L of distilled water to further remove the low molecular weight component and bring the final volume to 0.5 L. The final concentrate was dried by conventional spray drying to obtain 21 g of dark brown powder. As a result of analyzing the components of the dried product, the yield was 78%, heme iron 18%, moisture 4%, and the yield was 21%. The ultrafiltration device is required and the separation time is longer than the manufacturing method of the present invention.

<Comparative Examples 2 to 3> Preparation of Heme Iron from Hemoglobin Decomposed with One Protease

Prepare the hemoglobin solution in the same manner as in Example 1 and prepare two substrate solutions in the same manner. First, 1 g of spermase, an endo-type enzyme, and 1 g of flavozyme, an exo-type enzyme were added, and pH 7.5 The decomposition was carried out for 5 hours while keeping constant. After measuring the degree of hydrolysis of the hydrolyzate and adjusting the pH accordingly, after centrifugation at 3,000 g for 15 minutes, the supernatant was discarded and the precipitate was recovered and hot-air dried in a conventional manner.

The results of analyzing the components of the dried product are shown in Table 2. Since the degree of hydrolysis is lower than that of the present invention, the content of heme iron is low.

[Table 2] Yield of heme iron by one protease

Enzyme used Degree of hydrolysis (%) pH Protein content (%) Heme iron content (%) yield(%) Comparative Example 2 Esperase 25.2 4.5 91 9 42.2 Comparative Example 3 Flavorzyme 22.8 4.5 93 7 54.3

As described above, as described above, hemoglobin was simultaneously digested with endosomes and exo-type proteases with different proteolytic mechanisms to obtain enzyme hydrolysates with improved hydrolysis degree, and the pH of the enzyme digests was adjusted to 3-4. The sediment of heme iron is selectively precipitated and then separated and dried, which is a very useful invention for the food industry and the pharmaceutical industry because it has an excellent effect of producing dark brown powder having a high content of heme iron.

Claims (2)

delete In the manufacturing method of heme iron, Hemoglobin was treated with 0.1 to 10% by weight of endo type protease and exo type protease with different proteolytic mechanisms at the same time, respectively, to obtain enzymatic digests. After adjusting the hydrolysis degree of the enzyme to 20 ~ 45% to pH 3 ~ 5, the pH of which the solubility of heme iron is minimized, and centrifuged, Consisting of removing the supernatant and drying the precipitate to produce heme iron, Method for preparing heme iron from hemoglobin.