JPH0717875A - Method for producing iron-lactoferrin powder - Google Patents

Abstract

PURPOSE:To obtain an iron-lactoferrin powder which can stably hold a large amount of atomic iron exceeding the amount capable of being chelated with a milk-derived lactoferrin which has iron ion-binding function. CONSTITUTION:This pave iron-lactoferrin powder is obtained by a process comprising an addition of an iron salt to a lactoferrin solution, raising of pH value with the addition of an alkali, removal of isolated iron from the solution and evaporation of the solution. Iron-lactoferrin powder which can hold iron of >=2.1mg per 1g of lactoferrin in a stable state is obtained by this process.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing iron-lactoferrin powder, which is capable of binding a large amount of iron to milk-derived lactoferrin, which originally has the ability to bind iron ions.

[0002]

2. Description of the Related Art Milk-derived lactoferrin is known as a protein that binds two irons in one molecule like serum transferrin or ovalbumin.

[0003] In particular, milk-derived lactoferrin is isolated as a red protein because it binds iron ions and exhibits a pink or red color. Due to this property of iron binding, bacteriostatic action against iron-requiring Escherichia coli and other pathogenic bacteria has been the main research subject, but recently, research as an iron agent aiming at improvement of iron deficiency anemia has also been actively conducted. It is being advanced.

[0004] In comparison between human-derived lactoferrin and transferrin, both bind iron of 2 atoms per molecule, but the binding strength is about 300 times stronger than that of transferrin ( Biochim. B
iophys. Acta 257, 314-323, 1972), suggesting that the iron-binding mechanism of lactoferrin is different from that of other iron-binding proteins.

As a conventional method for binding iron to lactoferrin, J. Mazrier et al. (Biochim. Biophys, Act
a 629,399-408,1980), Masson et al. (Europian J. Bioche
m, 6,579-584, 1968) and Kawakami et al. (JP-A-63-22525).

However, conventionally, the iron-binding ability of lactoferrin is 2 iron atoms per molecule of lactoferrin, which is 1.4 iron per 1 g of lactoferrin in terms of weight.
It becomes mg. The daily iron requirement in humans is said to be 10 mg, and even if iron is supplemented from other foods, it is necessary to use a large amount of expensive lactoferrin to utilize it as an iron agent.

Kawakami et al. (JP-A-61-145200)
States that when the iron-binding ability of bovine lactoferrin separated with a monoclonal antibody was measured, 2.4-3.7 mg of iron was bound per 1 g of lactoferrin, but this value is used only as an index of the denaturation degree. It is the numerical value.

[0008] Further, Tomita et al. (JP-A-4-141067)
Lactoferrin shows a method of maintaining iron in a non-free state in a solution. This is an extension of the method of Kawakami et al. (JP-A-63-22525) described above. Is a phenomenon that occurs only under very limited acidic conditions.

This is a phenomenon observed only when the "non-free state" is defined as "a state in which iron does not precipitate", and the iron bound to lactoferrin and the lactoferrin are not bound. This is a state in which free iron coexists without precipitation.

[0010]

As described above, there has been no technique related to the production of lactoferrin powder in which iron of 3 atoms or more per molecule of lactoferrin is retained, but the present invention solves this problem. Is an issue.

[0011]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the inventors have found that after adding an iron salt to a lactoferrin solution, the pH of the solution is increased to 2. It was found that 1 mg or more of iron was retained in a stable state, and the present invention was completed.

That is, the present invention provides lactoferrin 1
This is a method for producing a lactoferrin powder in which 2.1 mg or more of iron per g is held in a stable state.

[0013]

The present invention makes it possible to maintain an unstable iron salt, which becomes insoluble in the neutral range, in a stable state without producing any precipitate, and does not have astringent taste due to iron. A stable iron-lactoferrin powder as an ironing agent, which comprises lactoferrin 1
It contains 2.1 mg or more of iron per g.

[0014]

EXAMPLE The lactoferrin used in the present invention may be separated by a commonly known method or may be commercially available. Further, as the iron salt used in the present invention, there is no problem in food hygiene, iron chloride,
Iron sulfate or iron citrate can be used.

The manufacturing method of the present invention specifically comprises the following steps. That is, (1) an iron salt is added to a lactoferrin solution in a solution state or in a solid state (molar ratio 1: 100 or less) and mixed. (2) Since the solution in this state is in an acidic state, sodium hydroxide or the like is added, and the pH is gradually raised while stirring.

(3) Since free iron ions having an astringent taste are also present in this solution, the free iron ions are removed by gel filtration or ultrafiltration. (4) After that, sterilization treatment is performed by a microfilter or heat treatment, and then freeze-dried or spray-dried to obtain iron-lactoferrin powder that retains a large amount of iron.

Next, the production method of the present invention will be described in more detail with reference to some examples, but the present invention is not limited to these examples. Example 1: Whey was obtained from 2 liters of raw milk by a conventional method, and 50 mM acetate buffer solution (pH
Diafiltration in 7.2), then 100
It was concentrated to 0 ml. This was applied to a heparin sepharose column equilibrated with the same buffer, the non-adsorbed fraction was sufficiently washed out, and then eluted with a 0-1M NaCl gradient. The eluate was dialyzed against deionized water and then lyophilized to give about 200 mg of lactoferrin.

10 mg of this lactoferrin,
Dissolve in 50 mM ammonium oxalate solution (pH 8.0), add ferric chloride dissolved in the same buffer solution to a concentration of 1 mg / ml, and add 0.1 NHCL or NaO.
Adjust the pH of the solution with H to 4.5, 5.5, 7.0, 7.5,
After adjusting to 8.0 and 9.0, lactoferrin was adsorbed on a heparin sepharose column, free iron was removed, and lactoferrin was eluted with 1 M sodium chloride and recovered.

The protein in each solution was dye-bonded and iron was Ni.
It was measured with Fe-C test Wako (Wako Pure Chemical Industries) using troso-PSAP method. The results are shown in Table 1, and it was confirmed that a large amount of iron was stabilized in all cases compared with ordinary bound iron.

[Table 1]

Example 2: 10 g of commercially available lactoferrin was added to 1
Dissolve in 1 liter of deionized water and add ferric chloride 1.
25 g was dissolved. The pH of the solution was adjusted to 12 with 1N NaOH, and then diafiltration was performed with deionized water using an ultrafiltration membrane having a molecular weight cut off of 50,000. When the pH of the solution returned to neutral, the solution was filtered through a 0.43 μm membrane filter and freeze-dried to obtain iron-stabilized lactoferrin. When the iron content of this product was measured in the same manner as in Example 1, it was 13 mg per 1 g of lactoferrin, and no astringent taste peculiar to iron was felt in the obtained powder.

Example 3: To 1 ml of a solution containing lactoferrin at a concentration of 20 mg / ml, an equal amount of ferrous sulfate solution was added so that the amount of iron would be 0.1 mg / ml, and they were mixed well and then Sephadex G- Lactoferrin and free iron were separated by gel filtration with 50 (1 × 15 cm), and the protein and iron contents of the lactoferrin fraction were measured as in Example 1. The same gel filtration operation was performed after adjusting the pH to 12 with 0.1N NaOH, and the test was also conducted. The results at that time are shown in Table 2.

[Table 2]

As is clear from Table 2, when the pH adjusted by the method of the present invention is compared with the unadjusted one which is the comparative example, the pH adjusted one has a retained iron amount of about. The value was 10 times higher, indicating that pH adjustment is essential and efficient in order to retain a large amount of iron. Regarding the taste of the solution, the astringent taste of iron was not felt.

[0023]

The iron-containing lactoferrin currently on the market has no astringent taste of iron and is considered to be a good ironing agent, but its iron content is very low at 0.14% or less, and it is suitable for use as a food material. Is problematic in terms of cost, and in the case of lactoferrin containing a large amount of free iron ions by the method of Tomita et al. (JP-A-4-141067) described above, the astringent taste peculiar to iron and a large amount of inorganic When salt is used as an iron supplement, problems such as gastric disorders are involved, but the iron-lactoferrin powder of the present invention makes it possible to provide a good iron preparation in a form in which all of these adverse effects are eliminated. Is.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Hiromi Taniguchi 2-11-4 Higashi 2-chome, Okegawa City, Saitama Prefecture (72) Toshiyuki Okamoto 2-1044-2 Amanumacho, Omiya City, Saitama Prefecture

Claims (3)

[Claims] 1. A method for producing a lactoferrin powder which holds 2.1 mg or more of iron per 1 g of lactoferrin in a stable state, which comprises adding an iron salt to a lactoferrin solution and then adding an alkali to increase the pH of the solution. . 2. The method for producing lactoferrin powder according to claim 1, wherein free iron in the pH-adjusted iron-lactoferrin solution is removed and then dried. 3. The method for producing a lactoferrin powder according to claim 2, wherein iron in the iron-lactoferrin powder is not detected as free iron in a neutral region in a solution state.