JP3223958B2 - Metal-bound lactoferrin and uses thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a metal-bound lactoferrin or a metal-bound lactoferrin-decomposed product in which a metal, particularly a trace element that controls the physiological action of a living body, is bound in a large amount. In addition, the present invention relates to a medicine, a food and drink, and a feed containing a large amount of a metal, particularly a trace element, containing such a metal-bound lactoferrin or a metal-bound lactoferrin hydrolyzate.

[0002]

2. Description of the Related Art Among substances that are said to be trace elements necessary for living organisms, it is said that there are 27 or more kinds of substances which have been confirmed to be essential trace elements in animal experiments. Among them, the trace elements with large required amounts, except for sodium and potassium, are 600 g of calcium, 300 g of magnesium and 10 mg of iron per day. In Japan, there is no required amount, but in the United States, trace elements with required amount
For example, 15 mg of zinc, 1.5 to 3 mg of copper, and 2 to 5 mg of manganese.

[0003] It can be said that it is desirable to ingest a sufficient amount of these trace elements from food and drink on a daily basis. However, due to factors such as eating habits, regional characteristics, and diseases, it is necessary to ingest sufficient amounts of these trace elements from food and drink. It can happen that you can't do it. In such a case, it becomes necessary to supplement these trace elements. At present, supplements containing various trace elements such as iron have been developed and marketed. However, among these trace elements, iron, zinc, copper and the like, in particular, have a peculiar metallic smell, and thus have a problem that they are not suitable for edible use as they are.
In addition, since these trace elements promote oxidation of fats and oils, there is also a problem that the flavor of foods and drinks containing fats and oils is deteriorated.

On the other hand, it is known that lactoferrin which is an iron-binding protein is present in milk. It is known that lactoferrin has an effect of suppressing bacterial growth by chelating and removing iron required for bacterial growth. It has also been proposed to use iron binding properties of lactoferrin to prepare lactoferrin to which iron is bound, and to use this as a raw material for foods and drinks for iron supplementation.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have developed an iron-linked lactoferrin with iron via carbonic acid or bicarbonate.
Lactoferrin has been proposed (JP-A-7-304798).
In addition, a zinc-lactoferrin in which zinc and lactoferrin are similarly bound was proposed (Japanese Patent Application No. 7-344831).
. Furthermore, we have been studying the binding properties between various metals and lactoferrins.As a result, it is better to react a plurality of metals with lactoferrins at the same time than to react a single metal with lactoferrins. Found that a large amount of metal can be bound to lactoferrins. In addition, they have found that lactoferrin degradation products have similar properties.

[0006] The lactoferrins to which plural kinds of metals are bonded and the decomposed products of lactoferrins to which metals are bonded do not show a metallic odor, are stable for a long time and have heat resistance. The present inventors have found that the compound does not deteriorate even when added to medicines, foods and drinks, or feeds for the purpose of supplementation, and have completed the present invention. Therefore, an object of the present invention is to provide a metal-bound lactoferrin or a metal-bound lactoferrin hydrolyzate in which a plurality of types of metals are bound in a large amount. Furthermore, another object of the present invention is to provide a medicine for supplementing minerals, a food and beverage, and a feed containing a metal-bound lactoferrin or a metal-bound lactoferrin hydrolyzate in which a plurality of types of metals are bound in a large amount.

[0007]

The present invention provides various metals,
In particular, while examining a method for easily combining trace elements with lactoferrins and lactoferrin degradation products, it is better to bind multiple types of metal to lactoferrins and lactoferrin degradation products than to bind a single metal. It has been found that a large amount of metal can be bound.

The metal-bound lactoferrin of the present invention can be prepared, for example, by adding a solution containing a plurality of metals to a solution containing lactoferrins and mixing. Further, it can be prepared by mixing a solution containing carbonate ions, bicarbonate ions, or carbonate ions and bicarbonate ions, a solution containing plural kinds of metals, and a solution containing lactoferrins.

Examples of the lactoferrin used in the present invention include lactoferrin separated from secretions of milk and the like of mammals such as humans and cows. Further, transferrin isolated from blood, organs and the like, ovotransferrin isolated from eggs and the like can be used in the same manner as lactoferrin. Many methods for preparing these lactoferrins in large quantities have already been known, and lactoferrins prepared by any method may be used. The lactoferrins do not need to be completely isolated, and may contain other components. Furthermore, it is also possible to use lactoferrins produced from microorganisms, animal cells, transgenic animals and the like by genetic manipulation.

The metal-bound lactoferrin hydrolyzate of the present invention can be prepared, for example, by adding a solution containing a plurality of metals to a solution containing a lactoferrin hydrolyzate and mixing the solution. Bicarbonate ions, or a solution containing carbonate ions and bicarbonate ions,
It can be prepared by mixing a solution containing plural kinds of metals and a solution containing lactoferrin decomposed products.
The lactoferrin hydrolyzate is obtained by decomposing lactoferrin with a protease such as trypsin, pepsin or chymotrypsin, or with an acid or alkali. These are preferably decomposed products having a molecular weight of about 1,000 or more and about 70,000 or less.

On the other hand, as the metal which can be used in the present invention, ferric chloride, zinc chloride, copper chloride, manganese chloride, nickel chloride, aluminum chloride, cobalt chloride and the like can be used. Metal sulfates and phosphates can also be used. The amount of metal to be added is preferably 3 M or more, more preferably 15 M or more, with respect to 1 M of lactoferrins, and the upper limit is 500 M or less. If the amount of one kind of metal to be added exceeds the above-mentioned upper limit, precipitation occurs during production or storage, or the metal remains in an ionic state without being completely bonded.

Further, the solution containing carbonic acid, bicarbonate, or carbonic acid and bicarbonate that can be used in the present invention includes carbonated water, ammonium bicarbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, sodium carbonate solution. ,
Calcium carbonate solution and the like, and these solutions may be mixed and used. In addition, these solutions may contain other components, for example, carbohydrates, proteins, fats, and the like.

The prepared metal-bound lactoferrin and the metal-bound lactoferrin decomposed product have no metal odor peculiar to metals, are stable for a long period of time, have heat resistance, and have an oxidizing action caused by metals. Since it has the property of being suppressed, it is useful as a mineral supplement material to be blended in medicine, food and drink, feed and the like. We have:
It has been found for the first time that by simultaneously binding a plurality of types of metals to lactoferrin and lactoferrin hydrolyzate, it is possible to bind a larger amount of metal to lactoferrin and lactoferrin hydrolyzate than to bond a single metal.

Test examples are shown below to explain the characteristics of the metal-bound lactoferrin and the metal-bound lactoferrin degradation product of the present invention.

Test Example 1 The following solutions were prepared and mixed to produce metal-bound lactoferrin, and the amount of each metal bound to lactoferrin was determined. (A solution) 1 L of a solution containing 1 M sodium bicarbonate (B1 solution) 0.2 L of a solution containing 100 mM each metal salt However, in the case of a solution containing two kinds of metals, an equal amount of each metal salt is mixed, and the metal salt is 200 mM. Was prepared. Also, 3
In the case of a solution containing a metal of the species, an equal amount of each metal salt is mixed,
A solution containing 300 mM of the metal salt was prepared. (B2 solution) 0.8 L solution containing 1 mM lactoferrin

First, a B solution obtained by mixing a B1 solution and a B2 solution was added to the A solution and stirred to prepare a solution containing lactoferrin bound to each metal. And this solution 300m
l was dialyzed against ultrapure water using a dialysis membrane with a molecular weight of 10,000 to remove unbound metal and completely desalted.
After lyophilization, the amount of each metal bound to lactoferrin was measured with an inductively coupled plasma emission spectrometer (ICP). Table 1 shows the results. As a result, metal-bound lactoferrin was obtained in a yield of 90% or more of lactoferrin.

[0016]

[Table 1] Metal salt Metal / lactoferrin (molar ratio)鉄 Iron 101 Copper 32 Manganese 9 Cobalt 7 Nickel 8 Aluminum 1 Iron + Copper 98 + 62 Iron + Manganese 92 + 31 Iron + Nickel 94 + 24 Iron + Aluminum 81 + 8 Iron + Cobalt 87 + 12 Copper + Zinc 67 + 31 Copper + Manganese 62 + 11 Iron + Copper + Zinc 96 + 60 + 29 When used, the molar ratios are described in the order of metal salts. For example, 98 + 62 in the iron + copper section is iron9
8, the meaning of copper 62. The same applies to the following tables.

The metal salts used in this test were as follows: iron was ferric chloride, copper was copper (II) chloride, zinc was zinc (II) chloride, manganese was manganese (II) chloride, cobalt was cobalt chloride, Nickel is nickel chloride and aluminum is aluminum chloride. In addition, 300 ml of an aqueous solution in which each of the metal salts used was dissolved in 300 mM was dialyzed against ultrapure water using a dialysis membrane having a molecular weight cutoff of 10,000, desalted for one week, and lyophilized to obtain a sample control. When this was similarly analyzed by ICP, it was confirmed that all the control samples had a metal amount of 0.3% or less of the amount before the dialysis, and all free metals were removed by the dialysis. From the above results, it was found that a larger amount of metal can be bound to lactoferrin when multiple kinds of metals are bound together with lactoferrin than when each metal is bound alone.

[0018]

[Reference Example 1] Trypsin (TYPE III, Sigma) against lactoferrin dissolved in 50 mM phosphate buffer (pH 7.5)
1% by weight was added and hydrolyzed at 37 ° C. for 24 hours. Then, 1% by weight of trypsin was further added and hydrolyzed at 37 ° C. for 24 hours to completely hydrolyze lactoferrin. In performing the hydrolysis reaction, the reaction solution was sterilized by a sterilization filter to suppress the growth of the bacteria, and further subjected to the reaction with one drop of toluene added dropwise. Next, the molecular weight
It was dialyzed with a dialysis membrane of 1,000 cuts and freeze-dried to obtain a lactoferrin hydrolyzate consisting mainly of peptides having a molecular weight of 55,000, 30,000 and 10,000 and other peptides having a molecular weight of 1,000 or more.

[0019]

Test Example 2 The following solutions were prepared and mixed to produce a metal-bound lactoferrin hydrolyzate, and the amount of each metal bound to the lactoferrin hydrolyzate was determined. (A solution) 1 L of a solution containing 1 M sodium bicarbonate (B1 solution) 0.2 L of a solution containing 100 mM each metal salt However, in the case of a solution containing two kinds of metals, an equal amount of each metal salt is mixed, and the metal salt is 200 mM. Was prepared. Also, 3
In the case of a solution containing a metal of the species, an equal amount of each metal salt is mixed,
A solution containing 300 mM of the metal salt was prepared. (B2 solution) 0.8 L of a solution obtained by dissolving a lactoferrin hydrolyzate in water so as to be 13.2 μM / L in terms of lactoferrin.

The lactoferrin hydrolyzate was prepared in Reference Example 1
What was obtained by was used. First, a B solution obtained by mixing a B1 solution and a B2 solution was added to the A solution and stirred to prepare a solution containing a lactoferrin hydrolyzate to which each metal was bound.
Then, the solution was dialyzed against ultrapure water at 4 ° C. for 90 hours with a dialysis membrane having a molecular weight of 1,000 and desalted completely.
After freeze-drying, the amount of each metal bound to the lactoferrin hydrolyzate was measured by ICP. Table 2 shows the results. In addition, no precipitation occurred during the process of preparing the lactoferrin hydrolyzate to which each metal was bound, but as a control, when trypsin was used instead of the lactoferrin hydrolyzate, precipitation occurred, resulting in a different behavior. showed that.

[0021]

[Table 2] Metal salt Metal / lactoferrin decomposition product (molar ratio)鉄 Iron 101 Copper 33 Iron + Copper 93 + 75 Copper + Zinc 66 + 35 Iron + Copper + Zinc 94 + 55 + 27 ─────────── ─────────────────

The metal salts used in this test were as follows: iron was ferric chloride, copper was copper (II) chloride, zinc was zinc (II) chloride, manganese was manganese (II) chloride, and cobalt was cobalt chloride. Nickel is nickel chloride and aluminum is aluminum chloride. From the above results, it was found that binding a plurality of metals simultaneously with the lactoferrin hydrolyzate allows a larger amount of metal to bind to the lactoferrin hydrolyzate than binding each metal alone.

[0023]

Test Example 3 The following solutions were prepared and mixed to produce metal-bound lactoferrin, and the heat resistance was examined. (A solution) 1L of a solution containing 1M sodium bicarbonate (B1 solution) 0.2L of a solution containing 50mM of each metal salt However, in the case of a solution containing two kinds of metals, an equal amount of each metal salt is mixed and 100mM of a metal salt Was prepared. Also, 3
In the case of a solution containing a metal of the species, an equal amount of each metal salt is mixed,
A solution containing 150 mM of the metal salt was prepared. (B2 solution) 0.8 L solution containing 1 mM lactoferrin

First, a B solution obtained by mixing a B1 solution and a B2 solution was added to the A solution, and the mixture was stirred to prepare a solution containing lactoferrin bound to each metal. In addition, B1 solution and B2
The solution B mixed with the solution was stirred to prepare a solution containing lactoferrin to which each metal was bound. The resulting aqueous solution was desalted to 0.1 mS / cm using an ultrafiltration membrane having a molecular weight of 10,000 cuts. Then, the solution containing these metal-bound lactoferrin was added to a 50 mM Tris-HCl buffer (pH 7.5) containing 0.2 M salt.
To a total metal concentration of 3.6 mM / L,
The formation of a precipitate upon heating at 10 ° C. for 10 minutes was observed. Table 3 shows the results.

[0025]

[Table 3] 有無 Presence or absence of metal salt precipitation ────────────────── Iron No Copper No Zinc No Manganese No Nickel No Aluminum No Iron + Copper No Iron + Nickel No Iron + Manganese No Iron + Aluminum No Zinc + Copper No Copper + Manganese No Iron + Copper + Zinc No ＋ ─────

The metal salts used in this test were as follows: iron was ferric chloride; copper was copper (II) chloride; zinc was zinc (II) chloride; manganese was manganese (II) chloride; nickel was nickel chloride; The aluminum is aluminum chloride. From the above results, it was found that lactoferrin to which a plurality of types of metals were bonded also had heat resistance.

[0027]

Test Example 4 In the same manner as in Test Example 1, a metal-bound lactoferrin in which 96 molecules of iron, 60 molecules of copper and 29 molecules of zinc were bound per 1 molecule of lactoferrin was prepared, and the oxidation stability to fish oil was examined. As a control, a mixture of ferric chloride, copper (II) chloride and zinc (II) chloride having the same molar ratio as that of the above-mentioned metal-bound lactoferrin was prepared, and the oxidation stability to fish oil was examined. The composition of each sample was as shown below, and the metal-bound lactoferrin or metal salt mixture was dispersed in fish oil with an ultra disperser (Yamato). Sample 1: 6,000 mg fish oil Sample 2: 6,000 mg fish oil + 20 nM metal-bound lactoferrin Sample 3: 6,000 mg fish oil + 200 nM metal-bound lactoferrin Sample 4: 6,000 mg fish oil + 2,000 n metal-bound lactoferrin
M Sample 5: Fish oil 6,000mg + metal salt mixture (iron 1.9μM, copper
Sample 6: 6,000 mg of fish oil + metal salt mixture (iron 19 μM, copper 12 μM)
M, zinc 5.8 μM) Sample 7: Fish oil 6,000 mg + metal salt mixture (iron 190 μM, copper 1
(Sample 20 has a metal amount equivalent to that of Sample 2, and Sample 6 has a metal amount equivalent to that of Sample 2.)
Is the amount of metal corresponding to sample 3, and sample 7 is the amount of metal corresponding to sample 4. The stability of each sample against autoxidation is determined by the CDM test of the standard fat and oil analysis test method.
(Standard oil and fat analysis test method Cd2.4.28.2-93)
At a measurement temperature of 80 ° C., the length of the oxidation induction period defined by the CDM test was measured and evaluated using a Rancimat apparatus (manufactured by Metrohm). The results are shown in Table 4 (the longer the oxidation induction period, the higher the oxidation stability).

[0028]

[Table 4] ──────────── Sample oxidation induction period ──────────── 1 5.2 (hours) 2 5.1 3 5.3 4 5.4 5 <0.1 6 <0.1 7 <0.1 ────────────

In the fish oil to which the metal salt mixture was added, severe deterioration was observed immediately after the start of the measurement, but in the fish oil to which the metal-bound lactoferrin was added, the oxidation induction period was not different from that of the fish oil without the metal. Therefore, it was found that by adding a metal to fish oil as metal-bound lactoferrin, deterioration of fish oil due to metal can be completely suppressed. For the sample prepared in the same manner,
, And the peroxide value (POV) was measured by sampling over time, and the same tendency as in the Rancimat test was observed.

[0030]

Test Example 5 A metal-bound lactoferrin in which 98 molecules of iron and 62 molecules of copper were bound per 1 molecule of lactoferrin was prepared in the same manner as in Test Example 1, and the oxidation stability to soybean oil was examined. As a control, a mixture of ferric chloride and copper (II) chloride having a molar ratio similar to that of the above-mentioned metal-bound lactoferrin was prepared, and the oxidation stability to soybean oil was examined. The composition of each sample is as shown below.
The metal-bound lactoferrin or metal salt mixture was dispersed in soybean oil using an ultra disperser (Yamato). Sample 1: soybean oil 6,000 mg Sample 2: soybean oil 6,000 mg + metal-bound lactoferrin 20 nM Sample 3: soybean oil 6,000 mg + metal-bound lactoferrin 200 n
M Sample 4: Soybean oil 6,000mg + metal-bound lactoferrin 2,000
0nM Sample 5: 6,000mg of soybean oil + metal salt mixture (iron 2μM, copper
Sample 6: 6,000 mg of soybean oil + metal salt mixture (iron 20 μM, copper 12
Sample 7: 6,000 mg of soybean oil + metal salt mixture (iron 200 μM, copper
Sample 5 has a metal amount equivalent to that of Sample 2 and Sample 6
Is the amount of metal corresponding to sample 3, and sample 7 is the amount of metal corresponding to sample 4. The stability of each sample against autoxidation is determined by the CDM test of the standard fat and oil analysis test method.
(Standard oil and fat analysis test method Cd2.4.28.2-93)
Rancimat device at a measurement temperature of 120 ° C (manufactured by Metrohm)
And measured and evaluated the length of the oxidative induction period as defined by the CDM test. Table 5 shows the results.

[0031]

[Table 5] 試 料 Sample oxidation induction period ──────────── 1 4.9 (hours) 2 5.2 3 5.1 4 4.8 5 <0.1 6 <0.1 7 <0.1 ────────────

In the case of soybean oil to which a metal salt mixture had been added, severe deterioration was observed immediately after the start of the measurement, but in the case of soybean oil to which metal-bound lactoferrin had been added, the oxidation induction period was not different from that of soybean oil to which no metal had been added. Therefore, it was found that by adding a metal to soybean oil as metal-bound lactoferrin, deterioration of soybean oil due to the metal can be completely suppressed. Further, the sample prepared in the same manner was subjected to an oven storage test at 40 ° C., and the POV was measured by sampling over time. The same tendency as in the Rancimat test was observed.

[0033]

BEST MODE FOR CARRYING OUT THE INVENTION The metal-bound lactoferrin of the present invention can be obtained by mixing a solution containing a plurality of types of metals and a solution containing lactoferrins.
It can be obtained by mixing bicarbonate, or a solution containing carbonic acid and bicarbonate, a solution containing a plurality of types of metals, and a solution containing lactoferrins. The metal-bound lactoferrin hydrolyzate of the present invention can be obtained by mixing a solution containing a plurality of types of metals and a solution containing a lactoferrin hydrolyzate, or a solution containing carbonic acid, bicarbonate, or a solution containing carbonic acid and bicarbonate. , A solution containing a plurality of metals and a solution containing lactoferrin decomposed products. Since the metal-bound lactoferrin and the metal-bound lactoferrin hydrolyzate of the present invention are stable to heat,
When producing these powders in large quantities, it is preferable to perform spray drying.

When the metal-bound lactoferrin or the metal-bound lactoferrin hydrolyzate of the present invention is blended in food or drink or feed, there is no particular limitation on the blending amount and the like, and a physically possible amount can be blended. The metal-bound lactoferrin or the metal-bound lactoferrin hydrolyzate of the present invention does not show any deterioration of the food even if it is blended in an amount of 50 to 300 g per 1 kg of the oil-containing food. There is no problem even if it is directly added to the composition. Therefore, soybean oil,
Rapeseed oil, cottonseed oil, sesame oil, safflower oil, olive oil,
It can also be directly blended with vegetable oils such as corn oil, fish oils, animal oils such as milk fat and lard, and fat-soluble vitamins such as vitamin A, vitamin E and vitamin D, which are easily oxidized. When manufacturing a food or drink or feed containing the metal-bound lactoferrin or the metal-bound lactoferrin hydrolyzate of the present invention, heat sterilization or sterilization can be performed. When used as a medicine, metal-bound lactoferrin or a metal-bound lactoferrin hydrolyzate itself may be used in the form of a powder or a solution. Mixed with nutrient supplements, powders, granules,
Used as tablets, capsules or liquids. In addition, since the metal itself has excess disease, it is necessary to determine an appropriate compounding amount from a nutritional point of view so as to avoid an overdose situation.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example 1 10 L of 200 μM bovine lactoferrin (manufactured by DMV) solution (solution A), 10 mM ferric chloride, 10 mM copper (II) chloride and
After preparing 10 L of a solution (B solution) containing 10 mM zinc (II) chloride, the B solution was added to the A solution, followed by stirring and mixing. And
This mixed solution is further added to a 1 M aqueous sodium hydrogen carbonate solution (20 L).
, And desalted and concentrated by an ultrafiltration membrane having a molecular weight of 50,000, and spray-dried to produce 172 g of a metal-bound lactoferrin in which iron, copper and zinc are bonded. The obtained metal-bound lactoferrin is 50 μl of iron per 1 μM of lactoferrin.
M, copper 50 μM and zinc 50 μM were bound. For this metal-bound lactoferrin, the total metal concentration is 5 mg / 100 ml
When a sensory evaluation test was carried out after dilution, none of 10 panelists felt metallic taste.

[0036]

Example 2 1 L of a solution (A solution) containing 1.2 M sodium bicarbonate and 10 μM bovine lactoferrin (DMV) and 1 L of a solution (B solution) containing 1.0 mM ferric chloride and 0.5 mM copper (II) chloride
Was prepared, and the B solution was added to the A solution, followed by stirring and mixing. Then, this mixed solution was desalted and concentrated using an ultrafiltration membrane having a molecular weight of 5,000 cut to prepare a solution containing metal-bound lactoferrin in which iron and copper were bound. The obtained metal-bound lactoferrin has 99 μl of iron per 1 μM of lactoferrin.
M and 49 μM of copper were bound. With respect to the solution containing the metal-bound lactoferrin, 50 mM Tris-HCl buffer (pH 8.
Diluted at 0). Then, seal in a test tube with a screw hole and 90
After heating at room temperature for 10 minutes and cooling naturally to room temperature,
When stored for months and the presence or absence of a precipitate was visually determined, no precipitate was observed. When a sensory evaluation test was conducted, none of the 10 panelists felt metallic taste.

[0037]

Example 3 1 L of a solution (solution A) containing 1.2 M sodium bicarbonate and 10 μM bovine lactoferrin (manufactured by DMV) and 1 L of a solution (solution B) containing 5.0 mM ferric chloride and 1.0 mM copper (II) chloride
Was prepared, and the B solution was added to the A solution, followed by stirring and mixing. Then, this mixed solution was desalted and concentrated using an ultrafiltration membrane having a molecular weight of 5,000 cut to prepare a solution containing metal-bound lactoferrin in which iron and copper were bound. For the solution containing this metal-bound lactoferrin, the total metal concentration is 10 mg / 100
50 mM Tris-HCl buffer (p
H 8.0). Then, it was sealed in a test tube with a screw hole, heated at 90 ° C. for 10 minutes, allowed to cool naturally to room temperature, and stored at room temperature for one month to determine the presence or absence of a precipitate with naked eyes.
No precipitation was observed. When a sensory evaluation test was conducted, none of the 10 panelists felt metallic taste. The solution containing the metal-bound lactoferrin was dialyzed against ultrapure water using a dialysis membrane with a molecular weight cutoff of 10,000, completely desalted, freeze-dried, and the amount of each metal bound to lactoferrin was measured by ICP. As a result, it was found that 382 molecules of iron and 70 molecules of copper were bound per lactoferrin molecule.

[0038]

Example 4 0.2 L of a solution (A solution) containing 1.0 mM copper sulfate and 0.2 mM zinc (II) chloride and 0.8 L of a solution (B solution) containing 50 μM transferrin (Apo type, manufactured by Wako Pure Chemical Industries) were prepared. After that, the B solution was added to the A solution, followed by stirring and mixing. Then, the mixed solution was desalted and concentrated using an ultrafiltration membrane having a molecular weight of 5,000 to prepare a solution containing copper- and zinc-bound metal-bound transferrin. In the obtained metal-bound lactoferrin, 70 μM of iron and 18 μM of zinc were bound per 1 μM of lactoferrin. The solution containing the metal-bound transferrin was diluted with an imidazole buffer (pH 6.8) containing 0.2 M sodium chloride so that the total metal concentration was 10 mg / 100 ml. And sealed in a test tube with a screw hole
After heating for 10 minutes and naturally cooling to room temperature, it was stored at 37 ° C. for one month, and the presence or absence of a precipitate was judged by the naked eye. As a result, no precipitate was observed.

[0039]

Example 5 A solution (A solution) obtained by adjusting 1 L of a solution containing 0.5 M sodium carbonate and 0.7 M potassium bicarbonate to pH 8.3 with acetic acid, and a solution containing 1.0 mM ferric chloride and 0.5 mM aluminum chloride (B1 solution) ) Ovotransferrin (type) with pepsin (Sigma) in 0.2L, 0.01N hydrochloric acid solution
IV, Sigma) hydrolyzed at 37 ° C for 1 hour, 800 mg of ovotransferrin hydrolyzate (10 µm in terms of transferrin)
After preparing 0.8 L of a solution (corresponding to M) (solution B2), B1 solution and B2 solution were first mixed to prepare solution B, and then solution B was added to solution A, followed by stirring and mixing. Then, the mixed solution was desalted with a dialysis membrane having a molecular weight of 1,000 to prepare a solution containing a metal-bound transferrin hydrolyzate in which iron and aluminum were bound. For the solution containing this metal-bound transferrin degradation product, the total metal concentration
The mixture was diluted with a 50 mM phosphate buffer (pH 7.0) to a concentration of 30 mg / 200 ml. Then, the tube was sealed in a test tube with a screw hole, heated at 90 ° C. for 10 minutes, allowed to cool to room temperature, and then stored at 37 ° C. for 1 month, and the presence or absence of a precipitate was visually judged. No precipitate was observed at all. Was. The solution containing the metal-bound transferrin hydrolyzate was dialyzed against ultrapure water using a dialysis membrane with a molecular weight cutoff of 10,000, completely desalted, freeze-dried, and mixed with the ovotransferrin hydrolyzate. The amount I
Ovotransferrin degradation product 1 was measured by CP.
It was found that 0.05 mg of iron and 0.002 mg of aluminum were bound per mg.

[0040]

EXAMPLE 6 A 20 L solution containing the metal-bound lactoferrin obtained by binding iron and copper obtained in Example 2 was mixed with an iron concentration of 5 mg / 2.
The mixture was homogenized by adding it to raw milk at a concentration of 00 ml and a copper concentration of 3 mg / 200 ml, sterilized at 150 ° C. for 2 seconds, immediately cooled to 4 ° C., and aseptically filled into a 250 ml paper container. And 37
After storage at 3 ° C. for 3 months, the mixture was centrifuged to examine the presence of a precipitate, but no precipitate was observed. The number of E. coli and the number of general bacteria were both 0. Furthermore, when a sensory evaluation test was performed using raw milk sterilized and stored under the same conditions as a control, none of the 10 panelists felt abnormalities in metal taste, taste, aroma, and the like.

[0041]

Example 7 A bovine lactoferrin hydrolyzate was prepared according to the method shown in Reference Example 1. On the other hand, 1L of a solution containing 1.3M sodium bicarbonate (solution A), 0.9mM ferric chloride and 0.6m
After preparing 0.2 L of a solution containing M manganese chloride (B1 solution) and 0.8 L of a solution containing the above-described bovine lactoferrin hydrolyzate equivalent to 10 μM in terms of lactoferrin (B2 solution),
The solution B is prepared by mixing the solution 1 and the solution B2.
Solution B was added to solution A, stirred and mixed to prepare a solution containing a metal-bound lactoferrin hydrolyzate in which iron and manganese were combined. The solution containing the metal-bound lactoferrin hydrolyzate was sealed in a test tube with a screw hole and heated at 90 ° C. for 10 minutes.
After naturally cooling to room temperature, the mixture was stored at 37 ° C. for 1 month, and the presence or absence of a precipitate was visually determined. As a result, no precipitate was observed. When a sensory evaluation test was conducted, none of the 10 panelists felt metallic taste.

[0042]

Example 8 To a solution of 500 g of bovine lactoferrin, 250 g of ferric chloride hexahydrate and 50 g of copper sulfate dissolved in 50 L of water, 25 g of sodium bicarbonate was added with stirring and mixed. Then, the mixed solution was desalted and concentrated using an ultrafiltration membrane having a molecular weight of 5,000 cut, and spray-dried to produce 410 g of metal-bound lactoferrin in which iron and copper were bonded. Pure lard 20 obtained by heating and dissolving 410 g of this metal-bound lactoferrin to 55 ° C.
In addition, the mixture was vigorously stirred with a homomixer to perform a dispersion treatment to produce lard reinforced with iron and copper.

[0043]

Example 9 A freeze-dried powder of metal-bound lactoferrin combined with iron and copper obtained in the same manner as in Example 2 was used to produce a 150 mg capsule containing vitamin E having the following composition by a punching method. Mixing ratio (%) Capsule solution Gelatin 45 Glycerin 22 Water 33 Raw material composition Vitamin E 35 (200 IU) Metal-bound lactoferrin 20 Wheat germ oil 40 Emulsifier 5 This capsule is used as a health food. In addition,
This capsule was stored at 37 ° C. for 3 months and subjected to a sensory evaluation test. None of the panelists felt off-flavors and metallic taste of the fats and oils.

[0044]

Example 10 Using the freeze-dried powder of metal-bound lactoferrin bound to iron and copper obtained in the same manner as in Example 2, margarine having the following composition was produced. Blending ratio (%) Hardened soybean oil (melting point: 42 ° C) 10 White soybean oil 40 Palm oil 25 Glycerin fatty acid ester (iodine value 60) 0.5 Skim milk powder 1 Metal-bound lactoferrin 0.5 Lactic acid 0.1 Water 22.9

As a comparative example, ferric chloride hexahydrate and copper chloride (11) were used in place of metal-bound lactoferrin.
-Using dihydrate , margarine having the following composition was produced. Blending ratio (%) Hardened soybean oil (melting point: 42 ° C) 10 White soybean oil 40 Palm oil 25 Glycerin fatty acid ester (iodine value 60) 0.5 Skim milk powder 1 Ferric chloride hexahydrate 0.2 Copper chloride (11) dihydrate 0.15 lactic acid 0.1 water 23.05

Further, margarine having the following composition was prepared as a control. Blending ratio (%) Hardened soybean oil (melting point 42 ° C) 10 White soybean oil 40 Palm oil 25 Glycerin fatty acid ester (iodine value 60) 0.5 Skim milk powder 1 Lactic acid 0.1 Water 23.4 And these margarines, immediately after production and 15%
The POV after storage at 6 ° C. for 6 months was measured. Table 6 shows the results.
Shown in

[0047]

[Table 6] From the above results, it was found that the use of metal-bound lactoferrin completely suppressed the degradation of margarine by metal.

[0048]

Example 11: 890 g of whey protein concentrate (WPC) and lactose
After dissolving 5.2 kg in 60 kg of hot water, 7.5 kg of milk and 400 g of casein are added, and water-soluble vitamins (vitamin B
_1, vitamin B _2, vitamin B _6, vitamin B _12, vitamin C, pantothenic acid, niacin, folic acid, biotin,
75 g of choline and inositol) and 75 g of minerals other than iron, copper and zinc (calcium carbonate, potassium chloride and magnesium sulfate) were added and dissolved. Next, to this solution, 10.3 g of metal-bound lactoferrin obtained by binding iron, copper and zinc obtained in the same manner as in Example 1 was added and dissolved. And fat-soluble vitamins (Vitamin A, Vitamin D, Vitamin E, Vitamin K, β-carotene) are dissolved and mixed. Add 2.5 kg of prepared fat, mix, sterilize, concentrate and spray dry to prepare infants. 10 kg of milk powder was produced. When a sensory evaluation test was performed on this infant formula, none of the 10 panelists felt metallic taste. In addition, the infant formula was found to be a product of stable quality with no increase in POV.
In addition, this infant formula has 6 mg of iron and 100 mg of copper per 100 g.
It contained 31 µg and 2.6 mg of zinc, and satisfied the various recommended values and recommended values, including the standards for special nutritional foods and FAO / WHO.

[0049]

Example 12 Iron- and copper-bound metal-bound lactoferrin obtained in the same manner as in Example 2 2 kg, soybean meal 120 k
g, 140 kg of skim milk powder, 40 kg of soybean oil, 20 kg of corn oil, 280 kg of palm oil, 148 kg of corn starch, 90 kg of flour, 20 kg of bran, 20 kg of vitamin mixture, 30 kg of cellulose, 30 kg of cellulose, 20 kg of mineral mixture excluding iron and copper, for dog breeding 1,000 kg of feed was produced.

[0050]

The metal-bound lactoferrin and the metal-bound lactoferrin hydrolyzate of the present invention can contain a large amount of metal as compared to a metal-bound lactoferrin or a metal-bound lactoferrin-decomposed product of one kind of metal. Despite the fact that a large amount of seed metals are bound, the taste of the metal is not expressed, and the oxidizing action caused by the metal is also suppressed.
It can be blended in foods and drinks, feeds and the like.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61P 3/02 A61K 37/14 (72) Inventor Kazumasa Hamashi 2-11-4 Minamiotsuka, Kawagoe-shi, Saitama Minamidai Heights 401 (72) Inventor Akira Tomizawa 5-3-33 Toyooka, Iruma City, Saitama Prefecture Arden 710 (72) Inventor Hitoshi Aikawa 12-89 Goryocho, Higashimatsuyama City, Saitama Prefecture Park Town 5-503, Gori 5-503 (72) Inventor Ken Takahashi Saitama 25-19 Matsubacho, Tokorozawa-shi Maison Matsuba 302 (56) References Milchwissenschaft, Vol. 45, no. 4 (1990) pp. 225-229 (58) Fields investigated (Int. Cl. ⁷ , DB name) C07K 14/79 BIOSIS (DIALOG) CA (STN) JICST file (JOIS) JAFIC file (JOIS) MEDLINE (STN) ) WPI (DIALOG)

Claims (7)

(57) [Claims] 1. A metal-bound lactoferrin or a metal-bound lactoferrin hydrolyzate in which a plurality of metals are bonded to lactoferrin or a hydrolyzate thereof. 2. A metal-bound lactoferrin or a metal-bound lactoferrin-decomposed product in which 3 to 500 molecules of each of a plurality of types of metals are bonded to one molecule of lactoferrin or its decomposed product. 3. The metal-bound lactoferrin or the metal-bound lactoferrin degradation product according to claim 1, which is obtained by mixing a solution containing a plurality of types of metals and a solution containing lactoferrins or a degradation product thereof. 4. The method according to claim 1, which is obtained by mixing a solution containing carbonic acid, bicarbonate, or a mixture of carbonic acid and bicarbonate, a solution containing plural kinds of metals, and a solution containing lactoferrins or a decomposition product thereof. Or a metal-bound lactoferrin hydrolyzate. 5. The metal-bound lactoferrin or the metal-bound lactoferrin degradation product according to any one of claims 1 to 4, wherein the plurality of types of metals are trace elements that control physiological functions of a living body. 6. The metal-bound lactoferrin or metal-bound according to claim 5, wherein the plurality of trace elements are two or more minerals selected from iron, copper, zinc, manganese, cobalt, nickel, and aluminum. Lactoferrin degradation product. 7. A medicine, food, drink or feed for supplementing minerals, which comprises the metal-bound lactoferrin or the metal-bound lactoferrin hydrolyzate according to claim 1.