Biological method for preparing heme iron not derived from porcine blood

Abstract

The present invention relates to heme iron not derived from porcine blood and a method of preparing the same, and more particularly to a method of biologically preparing heme iron not derived from porcine blood, a method of preparing a salt thereof, and an iron supplement containing the salt thus prepared as an active ingredient.

Claims

1. A method of biologically preparing a salt of heme iron of Chemical Formula 1 below, not derived from porcine blood and containing no animal component, the method comprising: A) producing heme iron by culturing Escherichia coli DH5 pLEX_HMD (Accession number: KCTC 13173BP) at 39 to 44 C. and a pH of 7 to 9 in a medium containing no animal component; B) purifying the heme iron produced in step A); and C) preparing a salt of the heme iron purified in step B) ##STR00002##

2. The method of claim 1, wherein the medium includes plant-derived peptone and a yeast extract prepared from yeast cultured in a plant-derived medium.

3. The method of claim 1, wherein the pH is adjusted using succinic acid.

4. The method of claim 1, wherein step B) is performed through filtration process using diatomaceous earth.

5. The method of claim 4, wherein the diatomaceous earth is Celite545.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the results under optimal culture temperature conditions; and

(2) FIG. 2 shows the analysis results of FT-IR (Fourier transform infrared spectroscopy) for identification of prepared heme iron.

(3) FIG. 3 shows the receipt of original deposit of Escherichia coli DH5 pLEX_HMD.

MODE FOR INVENTION

(4) Hereinafter, a better understanding of the present invention will be given through the following examples, which are merely set forth to illustrate the present invention but are not to be construed as limiting the scope of the present invention.

Example 1: Preparation of Heme Iron Production Strain

(5) With reference to the heme biosynthesis pathway in microorganisms in the present invention, a heme iron production strain was prepared through several trial-and-error steps. The heme iron production strain thus prepared was Escherichia coli DH5 pLEX_HMD, which is DH5-based Escherichia coli transformed with a plasmid designed for heme biosynthesis, particularly a plasmid containing a self-expression promoter P.sub.L, an ALA synthase gene (HemA), a NADP-dependent malic enzyme gene (MaeB), and a dicarboxylic acid transport protein gene (DctA), and the strain thus prepared was deposited at the Korean Collection for Type Cultures in the Korea Research Institute of Bioscience and Biotechnology on Dec. 21, 2016 (Accession number: KCTC 13173BP).

(6) Based on the comparing results of the heme iron production capability thereof with strains prepared using MG1655, Top10, BL21, and Rosetta gami, the production strain Escherichia coli DH5 pLEX_HMD exhibited superior heme iron production capability compared to the other Escherichia coli strains containing the same plasmid.

(7) The heme iron production capability was compared as follows. 10 ml of a LB (Luria-Bertani) medium (10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl) containing 50 g/ml ampicillin was placed in a 50 ml conical tube, and each strain was seeded therein and then cultured overnight at 37 C. and 200 rpm using a rotary shaking incubator. Next, 1 ml of the culture broth obtained after overnight culture was seeded in each of five 250 ml Erlenmeyer flasks added with 50 ml of an S medium (10 g/L peptone, 5 g/L yeast extract, 5 g/L KH.sub.2PO.sub.4, 10 g/L succinate, 2 g/L glycine, and 10 mg/L FeCl.sub.2.4H.sub.2O) containing 50 g/ml ampicillin, and was then cultured at 37 C. and 200 rpm for 4 hr. After culturing for 4 hr, 2 ml of the culture broth was seeded in a 250 ml Erlenmeyer flask added with 100 ml of an S medium containing 50 g/ml ampicillin, and was then cultured at 37 C. and 200 rpm for 48 hr, after which the resulting cells were recovered, based on which the extent of production of heme iron was compared. The results are shown below.

(8) TABLE-US-00001 TABLE 1 Comparison of heme iron production capability Strain Strain Strain Strain prepared prepared prepared prepared Production from from from from Rosetta strain MG1655 Top10 BL21 gami Recovered 13.96 13.7 10.55 13 9 cells [g/L] Recovered 6.1 2.8 4.1 4.3 3.3 heme iron [mg/L]

(9) As is apparent from the above results, the production strain Escherichia coli DH5 pLEX_HMD can be found to have superior heme iron production capability.

Example 2: Optimization of Heme Iron Production Conditions

(10) Using the production strain Escherichia coli DH5 pLEX_HMD prepared in Example 1, optimization of the production process, including optimization of the medium composition, etc., was performed. FIG. 1 illustrates the results of optimization of the culture temperature as one of conditions for optimizing the production process. The production conditions of heme iron finally established through several processes for obtaining optimal production conditions are summarized below.

(11) TABLE-US-00002 TABLE 2 Optimal heme iron production conditions Items Optimal conditions Seed culture medium 10 g/L peptone, 5 g/L yeast composition extract, 10 g/L NaCl Seed culture period 1.sup.st seed culture: overnight culture 2.sup.nd seed culture: 4 hr Main culture medium 10 g/L peptone, 5 g/L yeast composition extract, 5 g/L KH.sub.2PO.sub.4, 10 g/L succinate, 2 g/L glycine and 10 mg/L FeCl.sub.24H.sub.2O Culture temperature Seed culture: 37 C. Main culture: 42 C. Stirring rate 200 rpm pH 8 to 9 (adjusted using succinic acid) Production period 72 hr (Main culture period)

Example 3: Optimization of Heme Iron Purification Conditions

(12) In the present Example, a heme iron purification process was developed, and specific conditions thereof were optimized. The finally established purification conditions of heme iron are as follows.

(13) The culture broth of the heme iron production strain Escherichia coli DH5 pLEX_HMD was centrifuged at 3,000 g at 4 C. for 15 min, thus recovering cells thereof. The cells thus recovered were washed two times by suspending the same in PBS (Phosphate Buffered Saline) and then performing centrifugation. The finally recovered cells were naturally dried for about 30 min and then weighed. Typically, it was possible to recover 40 to 50 g of cells from 5 L of a culture broth. The recovered cells were added with cold acid-acetone and thus heme iron was extracted. Here, the cold acid-acetone that was used was prepared by mixing 998 ml of acetone at 20 C. with 2 ml of hydrochloric acid. The addition of the cold acid-acetone was conducted by a manner in which 1 L of cold acid-acetone was added to the cells recovered from 5 L of the culture broth. The extraction of heme iron using acid-acetone was performed at 4 C. for 5 days. The solution obtained through heme iron extraction for 5 days was passed through a celite-packed column to thus recover acetone containing heme iron. The acetone containing heme iron thus obtained was concentrated using a rotary evaporator. Here, concentration was performed until the volume was reduced from 1 L to 30 ml. The solution thus obtained was added with a 10-fold volume of methylene chloride, mixed thoroughly and then allowed to stand until layers were separated. After separation of the layers, the lower layer was recovered and concentrated using a rotary evaporator. Here, concentration was performed until the volume became 30 ml. A portion of the sample thus obtained was subjected to FT-IR (Fourier transform infrared spectroscopy). The analysis results thereof are shown in FIG. 2. After concentration, a NaOH aqueous solution was added in an amount of 2.1 equivalents based on the equivalents of heme iron contained in the concentrate, mixed thoroughly and then allowed to stand until layers were separated. After separation of the layers, the upper layer was recovered and freeze-dried, thereby yielding a salt of heme iron in a powder phase, as represented by Chemical Formula 1.

Example 4: Identification of Prepared Heme Iron

(14) In order to identify the prepared heme iron and the salt thereof, various analyses were performed. Specifically, FT-IR, mass spectrometry, UV-vis spectrophotometry, and ICP-OES were conducted. The analysis results of heme iron are summarized below. These results were consistent with expectations. For reference, the results of FT-IR are shown in FIG. 2.

(15) TABLE-US-00003 TABLE 3 Analysis results of heme iron Analysis method Analysis results FT-IR 1709, 1392, 1179, 1142, 938, 915, 841, 717, 608, 551 cm.sup.1 Mass (ESI) Calcd for C.sub.34H.sub.32FeN.sub.4O.sub.4: spectrometry 616.2, found: m/z 616.2 UV-vis (DMSO, nm) .sub.max 348, 386 spectrophotometry ICP-OES Calcd for Fe: 9.06%, found: 9.0%

(16) Meanwhile, the analysis results for the salt of heme iron are summarized below. These results were consistent with expectations.

(17) TABLE-US-00004 TABLE 4 Analysis results for salt of heme iron Analysis method Analysis results FT-IR 1682, 1559, 1412, 1208, 1144, 880, 834, 726, 602, 541 cm.sup.1 Mass (ESI) Calcd for C.sub.34H.sub.30FeN.sub.4Na.sub.2O.sub.4: spectrometry 660.1, found: m/z 660.2

Example 5: Evaluation for Effectiveness of Heme Iron as Iron Supplementary Source

(18) The salt of the heme iron prepared according to the present invention was dissolved in saline and administered to iron-deficiency-anemia-induced animals, whereby the effectiveness of the salt of the heme iron prepared according to the present invention on alleviating anemia was evaluated.

(19) Specifically, thirty 7-week-old Sprague-Dawley rats (female) were divided into 3 groups of 10 rats per group, among which one group was fed with normal feed in an amount of 10% of body weight daily for one month (Group 1; control), and the remaining two groups were fed with iron-deficient feed in an amount of 10% of body weight daily for one month to induce iron-deficiency anemia (Group 2 and Group 3). After one month of feeding, it was confirmed that iron-deficiency anemia was induced in the individual rat belonging to Group 2 and Group 3. Then, one of the anemia-induced groups was orally administered once a day with saline alone (Group 2), and the other anemia-induced group was orally administered once a day with saline containing heme iron (0.1 mg Fe/500 l saline) (Group 3). The administration continued for 30 days, and the occurrence of abnormal symptoms was monitored during the administration period. After 30 days of administration, blood was collected, and whether anemia was alleviated was evaluated. During 30 days of administration to Group 2 and Group 3, Group 1 was continuously fed with normal feed, and Group 2 and Group 3 were fed with iron-deficient feed. There were no abnormal symptoms in any animals during the 30 days of administration period. The analysis results of blood collection are shown below.

(20) TABLE-US-00005 TABLE 5 Analysis results of blood collection Weight of rats Blood test on 30.sup.th day Mean after Hemoglobin corpuscular Group administration content RBC volume Hematocrit No. Treatment [g] [g/dl] [10.sup.6/l] [fl] [%] Group 1 Normal feed 273.8 3.3 14.6 0.9 8.43 0.2 49.5 1.3 36.0 2.2 Group 2 Iron- 288.2 1.4 11.9 0.7 8.52 0.1 39.3 3.2 35.6 1.5 deficient feed + saline Group 3 Iron- 265.2 1.1 14.2 0.4 8.59 0.1 48.2 2.2 35.8 1.9 deficient feed + home iron

(21) As is apparent from the above results, the heme iron of the present invention can be concluded to be effective at alleviating iron-deficiency anemia and is thus efficient material as an iron supplementary source. Also, the heme iron of the present invention and the salt thereof can be confirmed to be useful not only in the treatment of iron-deficiency anemia but also in the prevention thereof.

(22) While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, those skilled in the art will appreciate that the specific description is only a preferred embodiment, and that the scope of the present invention is not limited thereto. It is therefore intended that the scope of the present invention be defined by the claims appended hereto and their equivalents.

(23) [Accession Number]

(24) Name of Depositary Authority: KCTC

(25) Accession number: KCTC 13173BP

(26) Accession date: 20161221