KR102429286B1 - The biological production method of heme-iron which is not derived from porcine blood - Google Patents

Abstract

The present invention relates to heme iron not derived from pig blood and a method for producing the same, and more particularly, to a method for producing heme iron not derived from pig blood through a biological production process, a method for preparing a salt thereof, and a salt prepared in this way It relates to an iron supplement containing as an active ingredient.

Description

The biological production method of heme-iron which is not derived from porcine blood

The present invention relates to heme iron free from animal-derived components and a method for producing the same, and more particularly, to a method for producing heme iron not derived from pig blood through a biological process, characterized in that it does not contain animal-derived components. It relates to a method for preparing a salt thereof, and an iron preparation comprising the salt prepared in this way as an active ingredient.

Iron (Fe, iron) is a trace element that plays an essential role in oxygen transport in the body, and is an important element in hemoglobin, myoglobin, citchrome, iron/sulfur proteins and biomolecular structures. The total amount of iron in the human body is about 3-4 g, of which 60-65% is in the form of binding to hemoglobin in circulating red blood cells, and the remaining 30-35% exists as stored iron (ferritin). In addition, it exists in the form of tissue iron and serum iron (transferrin), and a small amount of iron is also present in myoglobin in muscle.

Since iron is not synthesized in the body, it must be absorbed entirely through ingestion, and it exists in two forms: heme iron and non-heme iron. Heme iron refers to an iron complex having a portion having the same structure as the heme of hemoglobin in the body, and non-heme iron refers to an iron complex having no component having the same structure as the heme of hemoglobin. Both can be used as iron supplements (iron supply agents), and it is known that the bioavailability of heme iron is significantly higher than that of non-heme iron. Also, the absorption of heme iron into the body is not affected by other dietary factors. In addition, heme iron has the advantage that it does not cause various side effects (constipation, gastrointestinal disorders, etc.) reported with non-heme iron.

In general, heme iron is produced from slaughter blood, such as pig blood. Heme iron is prepared from slaughtered blood by first separating hemoglobin from slaughtered blood and separating heme iron from the isolated hemoglobin. When heme iron is separated from hemoglobin, alcohol and imidazole derivatives are used (Lindroos, US Patent 4,431,581), amino acids are added (Ingberg, et. al., US Patent 5,008,388), and high-temperature decomposition using a high concentration of organic acid Methods (Liu, et. al., J. Agric . Food Chem . , 44, 2957, 1996), a method using proteolytic enzymes, etc. are used.

Heme iron produced in this way has other problems that non-heme iron does not have, such as a risk of infection by an animal-derived infectious agent, a problem of contamination of livestock growth hormone, and a problem of residual antibiotics. In addition, since heme iron production in this way is produced from the blood of slaughtered animals such as pigs, which is forbidden in Islam, the heme iron produced in this way does not conform to halal, which is a problem for Muslims to use as iron supplements. there was.

Therefore, there is a need to develop a method for producing heme iron not derived from pig blood.

Accordingly, an object of the present invention is to solve the problems of the prior art and the technical problems that have been requested from the past.

It is an object of the present invention to provide a biological manufacturing process capable of producing heme iron not derived from pig blood.

Another object of the present invention is to provide a heme iron producing strain Escherichia coli DH5α pLEX_HMD (Accession No. KCTC 13173BP) that can be used in the biological production process of heme iron not derived from pig blood.

Another object of the present invention is to provide a method for preparing a salt of heme iron prepared through a biological manufacturing process of heme iron not derived from pig blood.

Another object of the present invention is to provide a pharmaceutical that does not contain animal ingredients that can be used for the prevention of iron deficiency anemia, which contains a salt of heme iron prepared through the biological manufacturing process of heme iron not derived from pig blood. It is to provide an enemy composition.

Another object of the present invention is a pharmaceutical that does not contain animal components that can be used for the treatment of iron deficiency anemia, which contains a salt of heme iron prepared through the biological manufacturing process of heme iron not derived from pig blood. It is to provide an enemy composition.

In order to solve the above problems, the present inventors have made various efforts, as a result of various efforts, a biological manufacturing process of heme iron, characterized in that it is not derived from pig blood, a method for preparing a salt of heme iron prepared in this way, and in addition, The present invention was completed by developing a pharmaceutical composition that does not contain an animal component by using a salt of heme iron and confirming that the composition can be effectively used for the treatment of iron deficiency anemia.

In the biological manufacturing process of heme iron not derived from pig blood, Escherichia as a production strain coli DH5α pLEX_HMD (Accession No. KCTC 13173BP) is used. Escherichia coli DH5α pLEX_HMD was deposited at the Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center on December 21, 2016 (accession number KCTC 13173BP).

In the biological manufacturing process of heme iron not derived from pig blood, a culture temperature slightly higher than the culture temperature adopted in the general E. coli culture process is adopted. As a culture temperature suitable for the implementation of the present invention, preferably 39-44 ° C. may be applied, more preferably a culture temperature of 41-43 ° C. may be applied, and most preferably a culture temperature of 42 ° C. may be applied. can be applied. Applying such a rather high culture temperature enables maximum heme iron production using the production strain of the present invention.

In the biological manufacturing process of heme iron not derived from pig blood, it is characterized by using an animal-derived component-free medium. To this end, in the present invention, plant-derived peptone and yeast extract prepared from yeast cultured in plant-derived medium conditions are used as medium components.

In the biological production process of heme iron not derived from pig blood, the pH of the culture process is maintained between 7-9, preferably, the culture pH is maintained at 8-9. At this time, pH adjustment is performed using succinic acid. The use of succinic acid for pH control can provide the advantage that succinic acid is a material used as a substrate in the biosynthesis of heme iron, which is advantageous for high-efficiency heme iron production.

The extraction of heme iron produced from the cultured cell body in the biological production process of heme iron not derived from pig blood is achieved through the implementation of the primary extraction process and the secondary extraction process. In the present invention, the organic solvent is used as the primary extraction process. It is characterized by using a celite filtration method using Application of this method facilitates the recovery of heme iron, and also increases the yield of heme iron. And as the secondary extraction process, a solvent extraction method was used. In this case, the solvents that can be used include ethyl acetate, butyl acetate, methylene chloride, 1,2-dichloroethane, chloroform, carbon tetrachloride, methyl ethyl ketone, etc. The use of methylene chloride or chloroform is most preferred.

In the method for preparing a salt of heme iron prepared through the biological manufacturing process of heme iron not derived from pig blood, the salt of heme iron to be prepared is a material having the structure of the following [Formula 1].

In the method for producing a salt of heme iron prepared through the biological manufacturing process of heme iron not derived from pig blood, the chlorination reaction for preparing the salt is a method of reacting at room temperature by adding an aqueous solution of NaOH or KOH, etc. can be achieved, it is more preferable to use an aqueous NaOH solution.

As used herein, "heme iron" refers to an iron complex having a portion having the same structure as heme of hemoglobin in the body, and "non-heme iron" refers to an iron complex having no component having the same structure as heme of hemoglobin.

Pharmaceutically acceptable carriers included in the composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.

The composition of the present invention is prepared in unit dosage form by being formulated using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person of ordinary skill in the art to which the present invention pertains, or It can also be manufactured by pouring into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

The present invention provides a biological manufacturing process capable of producing heme iron not derived from pig blood. Heme iron prepared in this way can provide various characteristic advantages of heme iron as compared to non-heme iron, and in addition, it can solve various problems of heme iron produced from pig blood. In particular, since the use of pig blood is fundamentally eliminated, heme iron produced in this way can be used to manufacture halal iron products. On the other hand, since the heme iron manufacturing process of the present invention is based on a biological manufacturing process, it also provides an advantage that the manufacturing process is mild.

1 is a result showing the culture temperature optimization result.
2 is a result of analyzing the prepared heme iron using infrared spectroscopy (FT-IR; Fourier transform infrared spectroscopy) to confirm the produced heme iron.

Hereinafter, the present invention will be described in more detail based on Examples, but these Examples are merely illustrative of the present invention and the scope of the present invention is not limited to these Examples.

Example One: heme iron production strain produce

The present inventors produced a heme iron-producing strain through several trials and errors with reference to the heme biosynthesis pathway in microorganisms. The prepared heme iron-producing strain is a plasmid containing the self-expressing promoter PL, ALA synthetase gene (HemA ₎ , NADP-dependent malic enzyme gene ( MaeB ), and dicarboxylic acid transporter protein gene ( DctA ) , which are plasmids designed for heme biosynthesis. DH5α-based Escherichia transformed with coli DH5α pLEX_HMD, and this strain was deposited at the Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center on December 21, 2016 (accession number KCTC 13173BP).

As a result of comparing the production of heme iron with the strains produced using MG1655, Top10, BL21, and Rosetta gami, the produced strain Escherichia coli DH5α pLEX_HMD had superior heme iron production capacity compared to other Escherichia coli having the same plasmid.

Heme iron production capacity was compared as follows. 10 ml of LB (Luria-Bertani) medium (Peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) containing ampicillin at a concentration of 50 μg/ml was added to a 50 ml conical tube. And, after inoculating each strain here, overnight culture was performed at 37°C and 200 rpm in a rotary shaker incubator. Then, 50 ml of S medium (peptone 10 g/L, yeast extract 5 g/L, KH ₂ PO ₄ 5 g/L, succinate 10 g/L, glycine) containing ampicillin at a concentration of 50 μg/ml 2 g/L, and FeCl ₂ 4H ₂ O 10 mg/L) were inoculated with 1 ml each of the overnight culture solution in 5 Erlenmeyer flasks of 250 ml. After inoculation, incubation was performed at 37° C. and 200 rpm for 4 hours. After culturing for 4 hours, 2 ml of the culture solution was inoculated into a 250 ml Erlenmeyer flask to which 100 ml of S medium containing ampicillin at a concentration of 50 μg/ml was added. This was again carried out in a manner in which the cell body was recovered after incubation at 37° C. at 200 rpm for 48 hours, and the degree of heme iron production was compared therefrom. The result was as follows.

Comparison of heme iron production capacity production strain Strains produced from MG1655 Strains from Top 10 Strains produced from BL21 Strains produced from Rosetta gami Recovered cell mass [g/L] 13.96 13.7 10.55 13 9 Amount of heme iron recovered [mg/L] 6.1 2.8 4.1 4.3 3.3

The production strain Escherichia produced from the above results coli DH5α pLEX_HMD was confirmed to have excellent heme iron production ability.

Example 2: heme iron Optimization of production conditions

Production strain Escherichia prepared in Example 1 coli DH5α pLEX_HMD was used to optimize the production process, such as optimizing the medium composition. As one of the production process optimization, the culture temperature optimization result is presented as an example as shown in FIG. 1 . The following is a summary of the production conditions for heme iron that were finally established through the optimization process of several production conditions.

Optimal production conditions for heme iron Item optimum conditions Composition of the culture medium Peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L Seed culture period Primary seed culture: overnight culture
Second seed culture: 4 hours Main culture medium composition Peptone 10 g/L, yeast extract 5 g/L, KH ₂ PO ₄ 5 g/L, succinate 10 g/L, glycine 2 g/L and FeCl ₂ 4H ₂ O 10 mg/L incubation temperature Seed culture: 37°C
Main culture: 42℃ agitation speed 200 rpm pH 8-9 (adjusted with succinic acid) Production period (main culture period) 72 hours

Example 3: heme iron Optimization of purification conditions

In this example, a heme iron purification process was developed and detailed conditions were optimized. The purification conditions for the finally constructed heme iron are presented as follows.

Heme iron producing strain Escherichia coli DH5α pLEX_HMD was centrifuged at 3,000 g at 4°C for 15 minutes to recover the cells. For the recovered cells, the cells were suspended in PBS (Phosphate Buffered Saline) and washed twice by centrifugation. The finally recovered cells were dried naturally for about 30 minutes, and then the weight was measured. In general, it was possible to recover 40-50 g of cell bodies from 5 L of culture solution. Heme iron was extracted by adding low-temperature acid-acetone to the recovered cells. The low-temperature acid-acetone used at this time was prepared by adding 2 ml of hydrochloric acid to 998 ml of acetone at -20°C. Addition of low-temperature acid-acetone was carried out by adding 1 L of low-temperature acid-acetone per cell recovered from 5 L culture solution. The extraction of heme iron using acid-acetone was performed in a manner performed at 4° C. for 5 days. Acetone containing heme iron was recovered by passing the solution obtained by performing heme iron extraction for 5 days through a column filled with celite. Acetone containing heme iron thus obtained was concentrated using a rotary evaporator. Concentration was carried out until 1 L became 30 ml. To the obtained solution, 10 times the volume of methylene chloride was added, mixed well, and left until the layers were separated. After the layers were separated, the lower layer was recovered and concentrated using a rotary evaporator. Concentration was carried out until it became 30 ml. Some of the samples thus obtained were analyzed by Fourier transform infrared spectroscopy (FT-IR), and the analysis results are shown in FIG. 2 . After concentration, an aqueous solution of 2.1 equivalents of NaOH was added to the equivalents of heme iron contained in the concentrate. After the NaOH aqueous solution was added, it was mixed well, and after mixing, it was allowed to stand until the layers were separated. When the layers were separated, the upper layer was recovered and lyophilized to prepare a salt of heme iron, a substance represented by [Formula 1] in powder form.

Example 4: Manufactured heme iron Confirm

Various analyzes were performed to confirm the prepared heme iron and salts thereof. Specifically, the analysis was performed using infrared spectroscopy, mass spectrometry, UV-vis spectrophotometry, and ICP-OES analysis. The analysis results for heme iron were summarized as follows, and these results were consistent with the expected results. Infrared spectroscopy results are presented for reference in FIG. 2 .

Analysis results for heme iron method Analysis infrared spectroscopy 1709, 1392, 1179, 1142, 938, 915, 841, 717, 608, 551 cm ^-1 mass spectrometry (ESI) Calcd for C ₃₄ H ₃₂ FeN ₄ O ₄ : 616.2, found: m/z 616.2 Ultraviolet-Visible Spectroscopy (DMSO, nm) λ _max 348, 386 ICP-OES method Calcd for Fe: 9.06%, found: 9.0%

On the other hand, a summary of the analysis results for the salt of heme iron is as follows, which also coincided with the expected results.

Results of analysis for heme iron salt method Analysis infrared spectroscopy 1682, 1559, 1412, 1208, 1144, 880, 834, 726, 602, 541 cm ^-1 mass spectrometry (ESI) Calcd for C ₃₄ H ₃₀ FeN ₄ Na ₂ O ₄ : 660.1, found: m/z 660.2

Example 5: As a source of iron heme iron validation

The effectiveness of the salt of heme iron prepared according to the present invention was investigated by dissolving the salt of heme iron prepared according to the present invention in physiological saline and administering it to an animal induced by iron deficiency anemia to examine whether anemia was improved.

Specifically, it is as follows. Thirty 7-week-old Spraque-Dawley rats (female) were divided into 3 groups of 10 per group, and then one group was fed a regular diet at 10% of body weight daily for 1 month (Group 1; Control), and the remaining Both groups were fed iron-deficient diet at a rate of 10% of body weight daily for 1 month to induce iron-deficiency anemia (Group 2 and Group 3). After confirming that iron deficiency anemia was induced in subjects belonging to Groups 2 and 3 after 1 month of the feed, only physiological saline was orally administered to one anemia induction group once a day (Group 2), and another To the anemia induction group, physiological saline containing heme iron (0.1 mg Fe/500 μl physiological saline) was orally administered once a day (Group 3). Administration was continued for 30 days, and abnormal symptoms were observed during the administration period. After 30 days of administration, blood was collected to examine whether anemia was improved. For 30 days when group 2 and group 3 were administered, group 1 was fed the normal feed as it was, and group 2 and group 3 were fed the iron-deficient feed. There were no specific symptoms in all animals during the 30-day dosing period. The blood sampling analysis results are presented as follows.

Blood draw analysis result group name process Weight of rats at 30 days after initiation of administration
[g] blood test hemoglobin content
[g/㎗] RBC
[×10 ⁶ /μl] Average
hematocrit
[fl] in whole blood
hematocrit
[%] group 1 general feed 273.8±3.3 14.6±0.9 8.43±0.2 49.5±1.3 36.0±2.2 group 2 Iron-deficient feed + saline 288.2±1.4 11.9±0.7 8.52±0.1 39.3±3.2 35.6±1.5 group 3 iron deficient feed + heme iron 265.2±1.1 14.2±0.4 8.59±0.1 48.2±2.2 35.8±1.9

As can be seen from the above results, it was confirmed that the heme iron of the present invention was effective in improving iron deficiency anemia, and from this, it was confirmed that it was an effective material as an iron source. This can be said to show that the heme iron and salts thereof of the present invention can be utilized not only for the treatment of iron deficiency anemia, but also for the purpose of prevention.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

[accession number]

Name of depository institution: KCTC

Accession number: KCTC 13173BP

Deposit date: 20161221

Claims (7)

A) using Escherichia coli DH5α pLEX_HMD (Accession No. KCTC 13173BP) to produce heme iron by culturing in a culture medium without animal components at a culture temperature of 42° C. and pH 7-9;
B) purifying the heme iron produced in step A); and
C) comprising the step of preparing a salt of heme iron purified in step B),
The animal component-free medium contains plant-derived peptone and yeast extract prepared from yeast cultured in plant-derived medium conditions and an iron source FeCl ₂ 4H ₂ O,
The purification step consists of a two-step purification process consisting of a primary extraction process consisting of organic solvent extraction and Celite filtration and a secondary extraction process of organic solvent extraction,
Wherein the pH is adjusted using succinic acid, a method for producing a salt of heme iron of Formula 1 that is not derived from pig blood and has no animal components through a biological manufacturing process.

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