JPH0776524A - Antianemic agent for animal - Google Patents

Abstract

PURPOSE:To obtain an antianemic agent for animals, capable of safe administration to an animal such as a piglet without a side effect and provide a method for preventing and improving animal anemia by using this agent. CONSTITUTION:An antianemic agent for animals, characteristically containing a composite material composed of dextrancarboxylic acid and an iron salt prepared according to the microbial method and a method for preventing and improving animal anemia by administrating it. A high-concentration iron is contained while securing the safety and, further, a low-viscous preparation can be obtained. This agent, therefore, can be safely administrated to an animal without causing a colored spot or a pain. This agent is low toxic and has an excellent preventive and therapeutic effect on anemia.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an anti-anemic agent for animals. More specifically, the present invention relates to iron supplements and anti-anemic agents for iron deficiency anemia in animals, especially piglets.

[0002]

2. Description of the Related Art Piglets have a small amount of iron stored due to their small weight at birth, and their growth rate is very fast. Therefore, breast milk with a low iron content cannot provide sufficient iron. It is well known that iron deficiency anemia occurs within a short time after birth. Especially in recent years, as breeding pigs are often kept under closed house system, iron intake from soil becomes difficult, and iron deficiency anemia occurs in piglets, especially in young pigs. The frequency of is increasing. On the other hand, ferrous fumarate, dextran iron, etc. have been conventionally administered. Dextran is a lactic acid bacterium belonging to the Leuconostoc mesenteroides (Leuconos
is a general term for high molecular weight glucans mainly composed of α1 → 6 bonds generated from sucrose by toc mesenteroides). Dextran iron, which is produced from dextran and iron hydroxide, is a prophylactically and therapeutically effective product, which is used mainly by parenteral administration such as intramuscular injection to supplement iron in animals, especially piglets, and iron deficiency. It has been used to ameliorate the symptoms of sex anemia.

In particular, a dextran iron preparation comprising a complex of dextran and ferric hydroxide and a preparation of a complex of dextran heptonic acid and ferric hydroxide obtained by a carbonization reaction from dextran are pig iron deficient. Prevention of sexual anemia,
It is used as a therapeutic agent. On the other hand, various chemical modification methods of dextran have been tried, but it is difficult to obtain a product with a high reaction rate in a regioselective manner in a commonly used chemical reaction, and a large number of side reaction products are given. In this way, many polysaccharides including dextran
As it is composed of homologues of various polymers with different molecular weights,
In chemical modification, there are many cases in which various side reactions occur and the product becomes complicated and the actual substance cannot be clarified. ["Biotransformations in Preparative Organic Chemistry"]
in Preparative Organic Chemistry HG Davis et a
l, Academic Press)))

Particularly, it has been attempted to chemically oxidize dextran with sodium hypochlorite, sodium bromate, chlorine, bromine, iodine or the like (see, for example, JP-A-61-233001). ), The structure of the grade is not necessarily clarified or is not fully supported. Conventionally known anti-anemia preparations have problems that it is difficult to formulate iron in a stable high concentration and low viscosity, and that colored spots occur at the intramuscular injection site. It is considered that these problems are due to the purity of the modified dextran itself, which is derived from dextran by a chemical synthetic means, and in particular, the inclusion of side reaction products accompanying the chemical reaction. An object of the present invention is to provide a safer anti-anemic agent for animals, which is more effective than the conventionally used preventive / therapeutic agents for anemia and has less toxicity and fewer side effects, and a method for preventing / treating anemia of animals. It is to be.

[0005]

[Means for Solving the Problems] In order to achieve the above-mentioned object, the present inventors have searched for compounds having various anti-anemic effects and have conducted extensive studies. As a result, dextran obtained in high purity by a microbiological method. A complex of carboxylic acid and iron salt
It was found that the anemia of animals such as piglets is improved, and further studies were conducted to complete the present invention. That is, the present invention comprises (1) an animal anti-anemic agent characterized by containing a complex of dextran carboxylic acid and an iron salt obtained by a microbiological method, (2) a hydroxymethyl group and / or hemiacetal Corresponding by reacting a microorganism belonging to the genus Pseudogluconobacter having the ability to oxidize a hydroxymethyl group and / or a hydroxyl group-containing hemiacetal part of dextran having a hydroxyl group to a carboxyl group, to dextran having such a group The animal anti-anemic agent according to (1) above, which uses dextran carboxylic acid obtained by producing and accumulating carboxylic acid and collecting the carboxylic acid, and (3) the microorganism is Pseudogluconobacter saccharoketgenes. ) Described animal anti-anemic agents,
(4) The microorganism is Pseudogluconobacter saccharoketgenes FERM BP-1128, FERM BP-
1129, FERM BP-1130, FERM BP
-1131, FERM BP-1132 or FERM
The animal anti-anemic agent according to (2) above, which is BP-1133, (5) The animal anti-anemic agent according to (1) above, wherein the iron salt is iron hydroxide or iron oxide, and (6) the iron salt is water. Oxidation second
The animal anti-anemic agent according to (5) above, which is iron, (7) the animal anti-anemic agent according to (2), wherein the processed product of the microorganism is a culture solution of the microorganism, and (8) dextran carboxylic acid has the formula ( I
I), (III) or (IV):

[0006]

[Chemical 2]

[0007] [In the formula, n represents 1 to 50] The anti-anemic agent for animals described in (1) above, (9) The anti-anemic agent for animals described in (8) above, wherein n is 1 to 15. (1) The anemia agent, (10) the complex, which is obtained by reacting dextran carboxylic acid with ferric hydroxide sol, the animal anti-anemic agent (11), and the injection agent (1). The animal anti-anemia agent described in (1), the animal anti-anemic agent described in (1) above which is an intramuscular injection, (13) the animal anti-anemia described in (1) used in piglets, (14). (1) an animal anti-anemic agent applied to iron deficiency anemia, (15) an effective anti-anemic effect of a complex of dextran carboxylic acid and ferric hydroxide obtained by a microbiological method Prevention of anemia in animals, characterized in that an amount is administered to an animal in need of prevention or treatment of anemia Other treatment methods, and (16) a process for the (15), wherein the animal piglets.

Conventionally, the oxidation of a compound such as a saccharide having a hydroxymethyl group- and / or hydroxyl group-containing hemiacetal moiety into a corresponding carboxylic acid by using a microorganism has a substrate specificity of the microorganism to be used. It was applied only to a very limited number. In addition, the chemical oxidation of the hydroxymethyl group and the hydroxyl group-containing hemiacetal part is
As described above, since there are many side reactions and complicated purification steps are unavoidable, a method for producing an oxidant of dextran or a derivative thereof by a highly selective and efficient oxidation reaction and the purity thus obtained High dextran carboxylic acids have been sought. According to the present invention, a highly pure dextran carboxylic acid can be obtained in high yield and with high selectivity by oxidizing the hydroxymethyl group- and / or hemiacetal hydroxyl group-containing hemiacetal part of dextran using a microorganism.

The microorganisms belonging to the genus Pseudogluconobacter that can be used to obtain dextran carboxylic acid microbiologically in the present invention have the ability to oxidize the hydroxymethyl group- and / or hydroxyl group-containing hemiacetal moiety of sugars to carboxyl groups. Any microorganism can be used as long as it is a microorganism belonging to the genus Pseudogluconobacter. . Especially, Pseudogluconobacter saccharoketgenes is preferable. More specifically, for example, European Patent Publication (EP-A) No. 22
The following strains described in No. 1,707 are listed as typical examples. Pseudo-Gluconobacter Saccharoketgenes K59
1s strain: FERM BP-1130, IFO 1446
4 Pseudogluconobacter Saccharoketgenes 12-
5 strains: FERM BP-1129, IFO 14465 Pseudogluconobacter saccharoketgenes TH
Strain 14-86: FERM BP-1128, IFO 1
4466 Pseudogluconobacter Saccharoketgenes 12-
15 strains: FERM BP-1132, IFO 1448
2 Pseudogluconobacter Saccharoketgenes 12-
4 strains: FERM BP-1131, IFO 14483 Pseudogluconobacter saccharoketgenes 22-
3 strains: FERM BP-1133, IFO 1148
4.

In the method of the present invention, the bacterial cells of the microorganism belonging to the genus Pseudogluconobacter may be allowed to act,
Alternatively, the processed product may be used. As the treated product, for example, a culture solution of these microorganisms can be used. Further, enzymes produced by these microorganisms may be used. However, in the case of the microorganism of the genus Pseudogluconobacter of the present invention, the enzyme is usually accumulated in the cells. Usually, it is convenient to use the bacterial cells themselves and bring them into contact with and act on the starting saccharides to produce carboxylic acids or salts thereof. Especially, it is preferable to use resting cells. The cells or the culture solution thereof can be produced, for example, according to the method described in JP-A-64-85088 or a method similar thereto.
That is, seed culture was performed from a slant, then main culture was performed to obtain a culture solution, and if necessary, this culture solution was centrifuged, the precipitate was collected, and then washed with a saline solution several times to obtain a culture solution. The resulting precipitate can be subjected to cell reaction. Further, the microorganism is a nutrient source that can be utilized by the strain under aerobic conditions, that is, carbon source (carbohydrate such as glucose, sucrose, starch or organic matter such as peptone, yeast extract), nitrogen source (ammonium salts, urea or Inorganic and organic nitrogen compounds such as corn steep liquor and peptone), inorganic salts (salts such as potassium, sodium, calcium, magnesium, iron, manganese, cobalt, copper, phosphoric acid and thiosulfate), and C as a micronutrient
It can be cultured in a liquid medium containing vitamins / coenzymes such as oA, pantothenic acid, biotin, thiamine, riboflavin, FMN (flavin mononucleoside) or amino acids such as L-cysteine and L-glutamic acid or natural products containing them. The resulting culture solution can be used in the method of the present invention. Culturing can be performed at pH 4 to 9, preferably pH 6 to 8.

The culturing time varies depending on the microorganisms used, the composition of the medium, etc., but is preferably 10-100.
It's time. A suitable temperature range for performing the culture is 10 to
40 degreeC, Preferably it is 25-35 degreeC. By adding a rare earth element to the medium at the time of culturing, the target substance can be produced more efficiently. The rare earth elements added to the medium include scandium (Sc), yttrium (Y), lanthanum (La), and cerium (C).
e), praseodymium (Pr), neodymium (Nd), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (D)
y), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb) and lutetium (Lu). These rare earth elements may be added as metal powder or metal pieces, and they are also used as compounds such as chlorides, carbonates, sulfates, nitrates, oxides or oxalates. They may be used alone or two or more kinds of rare earth elements such as cerium carbonate and lanthanum chloride may be used at the same time. Further, a crude product obtained in the process of separating and refining various elements can also be used. The amount of rare earth element added to the medium may be selected within a range that does not inhibit the growth of the microorganism used,
Generally, the range of 0.000001 to 0.1% (W / V), preferably 0.0001 to 0.05% (W / V) is effective. As a method of addition to the medium, it may be added to the medium in advance, but it may be added intermittently during the culture or continuously.

When dextran carboxylic acid is produced and accumulated in a culture solution, if pseudogluconobacter is cultivated in the coexistence of another bacterium as described in EP-A-221,707, pseudogluconobacter will be obtained. The amount of product accumulated can be increased as compared with the case where the genus bacterium is cultured alone. Such mixed culture, that is, culture in which Pseudogluconobacter bacteria are coexistent with mixed bacteria of different species can be appropriately performed according to the disclosure of EP-A-221,707. As such a mixed bacterium, Bacillus megaterium bacterium is particularly preferable.

In producing dextran carboxylic acid according to the present invention, the saccharide to be subjected to the reaction is dissolved or suspended in water or a solvent miscible with water, for example, methanol, acetone, polyethylene glycol or the like to contact with a microorganism. You may. The amount of solvent used may be selected within a range that does not delay the reaction, and the substrate concentration is usually 0.1 to 20% (W / V), preferably 1 to 5% (W
The range of / V) is effective. A suitable temperature range for carrying out the oxidation reaction by the microorganism of the present invention is 10 to 40 ° C, preferably 25 to 35 ° C. The reaction is preferably carried out under aerobic conditions. For example, while aerating air at 0.1 to 5 liters / minute, if necessary, 50 to 2000
It is also possible to stir by rotation. The reaction time is 5 minutes to 3 days, usually 1 hour to 24 hours, depending on the nature of the primary hydroxyl group substituted in the saccharide to be used in the reaction. The pH of this reaction is preferably adjusted. It is generally effective to carry out the treatment in the range of pH 4 to 9, preferably pH 6 to 8. As the base used for pH adjustment, any base may be used as long as it does not inhibit the reaction. For example, inorganic salts such as sodium hydroxide, potassium hydroxide, calcium carbonate, magnesium hydroxide and ferrous hydroxide, and organic salts such as sodium morpholinoethane sulfonate and calcium morpholinoethane sulfonate can be used.

Further, by adding an anion exchange resin if desired, it is not necessary to add the above-mentioned neutralizing agent such as an alkali metal salt for adjusting the pH, and the reaction can be selectively controlled. it can. The method of adding the anion exchange resin is particularly preferable in the case of selectively reacting to obtain one equivalent of the oxidant. The anion exchange resin used here may be any anion exchange resin as long as it adsorbs the produced carboxylic acid. Among them, styrene type and acrylic type anion exchange resins are preferable. Specifically, for example, Amberlite (trade name, Organo Co.) IRA-400, IRA-401, IRA-4
02, IRA-410, IRA-900, IRA-91
0, IRA-35, IRA-68, IRA-94S, etc., Diaion (trade name, Mitsubishi Kasei) SA-10A,
SA-20A, PA-306, PA-308, PA-4
06, WA-10, WA-11, WA-20, WA-3
0 and the like.

When the sugar added as a substrate (that is, dextran having a hydroxymethyl group and / or a hemiacetal hydroxyl group) disappears in the reaction solution, stirring is stopped and the reaction solution and anion exchange resin are separated, The anion exchange resin is eluted by adding a different eluent to obtain the desired product. Examples of the eluent include an aqueous solution of sodium chloride, an alkali metal salt, or an acidic aqueous solution of hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, or the like. The sugar carboxylic acid thus eluted and accumulated can be collected by known means (eg HP
It can also be collected and purified by a method such as separation or purification with an adsorptive resin such as -20 or SP205) or a method similar thereto.

Further, in order to remove inorganic ions from the obtained dextran carboxylic acid, it can be subjected to a treatment of passing it through an ion exchange resin, or a desalting treatment by dialysis, electrodialysis or ultrafiltration. Examples of such dextran carboxylic acid include the following formulas (II), (III),
A typical example is a compound represented by (IV).

[0017]

[Chemical 3]

[In the formula, n represents 1 to 50. Preferably n is 1-15. The dextran used in the present invention is actually a mixture of polymers having different molecular weights, but is preferably represented by the following formula (I).

[0019]

[Chemical 4]

[In the formula, n has the same meaning as described above. The substrate subjected to the microbiological oxidation in the present invention may be a derivative of dextran, or may include it. Examples of such dextran derivatives include dextranyl gluconic acid, dextranyl glucuronic acid represented by the above formula (II) or (III), and mixtures thereof. It is also possible to subject these compounds to oxidation by the above-mentioned microorganisms, leading to other oxidants such as the compounds of the above formula (IV). The resulting dextran carboxylic acid is easily complexed (including complexed) with iron salts to produce a non-toxic, stable, injectable liquid complex containing 5-30% or more iron. can do. Examples of the iron salt include ferrous hydroxide, ferric hydroxide, iron oxide and the like. Usually, ferric hydroxide is more preferably used.

The complex of dextran carboxylic acid and iron salt can be generally produced by a known method for producing a complex of dextran and iron salt or in accordance with it. For example,
The complex can also be produced by reacting dextran carboxylic acid with a sol of an iron salt such as ferric hydroxide. Preferably, dextran carboxylic acid and the sol are
At H4.0 to 10, heated temperature condition, for example, 100
A colloidal solution or suspension is obtained by heat treatment for 30 minutes in an autoclave or the like at ℃ to 120 ℃. As a method for producing the ferric hydroxide sol, an alkali metal salt, especially a sodium carbonate solution, can be used as the ferric chloride solution according to a conventional method. However, when these are used, there is a drawback that the addition rate must be extremely low. As a result of various studies, it was found that neutralization with an alkali hydrogencarbonate solution, especially a sodium hydrogencarbonate solution, enables the addition speed to be higher than that of the conventional method. By this method, iron can be formed by processes such as salt formation, chelate formation and hydration.
A complex of dextran carboxylic acid and iron salt is formed.
As described below, the elemental iron content of the colloidal suspension iron salt complex in the aqueous phase is about 50-300 mg / solution or suspension.
ml, and if desired, a high iron content iron salt complex can be prepared from a low iron content product by operations such as evaporation and concentration. The high iron content iron salt complex thus obtained is itself extremely stable for a long period of time, and in particular, has the property that its colloidal state is stably maintained in the aqueous phase.

The characteristics of the complex used in the present invention were examined. As a result, a complex of a low-molecular-weight dextran carboxylic acid and an iron salt, for example, formula (II), (III) or (IV) [wherein n
It has been found that a complex of a low-molecular-weight dextran carboxylic acid represented by [1 to 15] and an iron salt is preferable.
Among these, a low-molecular-weight dextran gluconate iron salt complex is particularly preferable, and a stable formulation of low iron content and high iron content can be produced by using the complex. In fact, the iron content of conventional dextran iron preparations is at most about 200 mg / ml, whereas the use of the complex of the low molecular weight dextran gluconic acid and iron salt of the present invention provides a high iron content of 300 mg / ml. It is possible to manufacture a formulation. The iron-rich preparation can be advantageously used because it can reduce the administration volume, improve the injection administration work, and shorten the administration time. Further, it is possible to provide an anti-anemia agent which can cope with the enlargement of young pigs due to breeding improvement.

The complex of dextran carboxylic acid and iron salt thus obtained may be diluted with distilled water for injection, physiological saline or the like and parenterally administered to animals as an injection. In addition, according to a known pharmaceutical manufacturing method, if necessary, a pharmaceutically acceptable diluent or excipient is used, and large pills,
It can be orally administered as tablets, powders, granules, capsules, emulsions, solutions, premixes, syrups and the like. In addition, it can be used as a single agent directly or after being dispersed in a carrier and mixed with feed, drinking water and the like.

Therefore, the preventive / therapeutic agent for anemia of the present invention is prepared by, for example, diluting the complex of dextran carboxylic acid and iron salt with a solid or liquid carrier or stabilizing it by coating or the like, for example, powder, powder, granules. Agents, tablets, large pills, liquids, emulsions, pastes, capsules, premixes,
It is produced as an injection or the like, or by adding it to a feed, a beverage or the like directly or by dispersing it in a carrier. Examples of solid carriers include lactose, sucrose, starch, wheat flour, corn flour, bran, soybean meal, defatted rice bran, rapeseed meal, tofu meal, fibrin, yeast cells, fish meal, peanut squeezed meal, shell powder, and carbonate. The liquid carrier includes, for example, water, physiological saline, physiologically harmless organic solvent and the like. Other appropriate auxiliary agents such as emulsifiers, dispersants, suspension agents, wetting agents, thickening agents, gelling agents, and solubilizing agents may be added in appropriate amounts. It can also be in the form of a premix. further,
Antiseptic, disinfectant, anthelmintic, antioxidant, colorant, fragrance,
Antibacterial agents, antibiotics, enzyme preparations, lactic acid bacteria preparations, antipyretics, analgesics, antiphlogistics and the like may be mixed, and other prophylactic / therapeutic agents for anemia may be mixed and used together. Further, various vitamins, minerals, amino acids and the like may be mixed.

The amount of the complex in the anti-anemic agent of the present invention is usually 0.1 to 100% by weight, preferably 1 to 10% by weight, based on the total amount of the preparation. The complex is preferably administered as an injection such as intramuscular injection, subcutaneous injection and intraperitoneal injection. Intramuscular injection is particularly convenient. For these injections, it is usually preferable to use a complex of dextran carboxylic acid and iron salt diluted with distilled water for injection, physiological saline or the like.

The anti-anemic agent of the present invention is used for the purpose of preventing and / or treating anemia caused by various diseases including iron deficiency anemia, for livestock production of pigs, cows, horses, dogs, cats, etc., livestock or pets. Administered to animals. In particular, it is preferably applied to piglets and the like that are prone to anemia. The dose depends on the administration subject, symptom, and administration route, but for example, an injection usually has an iron content of 5 to 500 mg / head, preferably 100 to 3
The range is 00 mg / head. If necessary, the number of times may be increased depending on the symptoms. In addition, solid dosage forms such as tablets may be administered directly or by mixing with feed or the like so as to correspond to the above dose. In particular, the anti-anemia agent of the present invention is useful for preventing or treating anemia of piglets by intramuscular injection. For example, the present anti-anemic agent shows a uniform dispersion and good absorption into living tissues when injected intramuscularly into the hips of young pigs. In addition, the anti-anemic agent of the present invention has low toxicity and is safe without showing any remarkable abnormalities such as side effects. For example, when administered to piglets as shown in the following examples, no side effects or abnormalities were observed for 4 weeks. As shown in the following examples, in the administration experiment of the anti-anemic agent of the present invention of hematological test items to young pigs, particularly hemoglobin amount (HGB), hematocrit value (HCT), mean red blood cell volume (MCV), Compared to the control group for many parameters such as weight gain,
Significantly improved.

[0027]

EXAMPLES The present invention will be described more specifically with reference to the following Reference Examples and Examples, but the present invention is not limited thereto. Reference Example 1 Pseudogluconobacter Saccharoketgenes (Pseu
dogluconobacter saccharoketogenes) TH14-86
Preparation of bacterial solution; Pseudogluconobacter saccharoketgenes TH14-86 (FERM BP-1128) strain slant was added to 20 ml of the medium shown in the following Table 1 by 1 platinum loop, and this was added to a 200 ml smooth flask at 30 ° C, 1 Incubate under agitation using a rotary shaker for a day. Then, 2
Take 1 ml of the above culture medium per 0 ml of medium,
Shaking culture was carried out for 2 days to give a seed culture. On the other hand, Bacillus megaterium (IFO 1
2108) slant per 20 ml of the composition shown in Table 2 below, and added to 1 white ear, 200 ml smooth flask.
Shake culture is performed at 0 ° C. for 2 days.

[0028]

[Table 1] Medium components of Pseudogluconobacter saccharoketgenes TH14-86 % (W / V) lactose 1 yeast extract 1 (Prodivel) (NH ₄ ) ₂ SO ₄ 0.3 corn steep Liquor 3 CaCO ₃ 2 (Super) pH before addition of CaCO ₃ = 7.0

[0029]

[Table 2]

Next, the following main culture was carried out. Pseudogluconobacter Saccharoketgenes TH14-8
100 ml of the seed (seed) culture medium of 6 strains and 1.5 ml of Bacillus megaterium seed medium were added to 900 ml of the main culture medium shown in Table 3 below, and then about 20 ° C. at 30 ° C.
The culture was carried out with shaking for a period of time to obtain a culture solution of Pseudogluconobacter saccharoketgenes TH14-86.

[0031]

[Table 3] Main culture medium components% (W / V) sucrose 0.05 (single sterilization) Corn steep liquor 2 (single sterilization) (NH ₄ ) ₂ SO ₄ 0.3 (single sterilization) FeSO ₄ · 7H ₂ O 0.1 (alone sterile) vitamin B ₂ 1 mg / l (before sterilization pH = 7.0) sorbose 10 (alone sterile) LaCl ₃ 0.01 (alone sterile) CaCO ₃ 4 (Sterilization by itself) (Super)

To 1.5 liters of the Pseudogluconobacter saccharoketgenes culture solution thus obtained, 30 g of dextran-4 (molecular weight: 4000 to 6000; manufactured by Extra Synthese (France)) was added to 5 by Biot. It was put in a liter jar fan mentor, and then 1.7 liter of sterilized water was added to prepare a reaction solution. 32 this
Air was aerated at 1.6 liters / minute while stirring at 600 ° C./600 rpm, and the pH was adjusted to 6.3 with 0.5% NaOH solution.
Was added automatically as the reaction progressed. The reaction was stopped in 3 hours, and then 12.0 liters of the reaction solution was added to 8
The cells were separated at 000 rpm with a cooling centrifuge to remove the cells, and 1.98 liters of a supernatant was obtained. The supernatant was filtered through a cellulose acetate membrane filter (φ0.2 to μm) to sterilize again. The obtained filtrate was passed through an Amberlite IRA-68 (OH type) column (50 ml),
It was washed with a small amount of water (100 ml) to obtain 2 liters of passing liquid. This passing solution was passed through an HP-20 column (900 ml). Then, elute with 2 liters of water to give 7
Discard the 00 ml, then collect 3.3 liters of the eluate, add 13.8 ml of concentrated hydrochloric acid to the mixture to make it acidic, and add this to a cellulose acetate membrane filter (φ0.2).
μm), and the filtrate is Sepha beads (trade name) SP2
05 (manufactured by Mitsubishi Kasei Co., Ltd.) Then,
After washing with 700 ml of 0.05M hydrochloric acid and 2 liters of water,
Elution was performed with 2 liters of a 20% aqueous methanol solution to obtain a fraction of the desired product. This was concentrated under reduced pressure to obtain 14 g of dextranyl-glucuronic acid white powder. HPLC of this product (Conditions: Asahi Pack GS320 (φ7.6mm × 50m
m; manufactured by Asahi Kasei), mobile phase: water 1 ml / min, detection; RI
(Waters 410) and 200 nm (Tosoh UV-
8020) Rt of sample 0.5% aqueous solution 20 μl injection)
A single peak was shown at 8.48. (Rx of dextran-4 as a raw material was 10.83). To confirm the structure of this product, an enzymatic digestion treatment experiment was performed. 100 μl of a 1.5% aqueous solution of this product was added with 100 μl of a glucoamylase (manufactured by Wako Pure Chemical Industries, Ltd.) solution (4 mg / ml), and the mixture was incubated at 30 ° C. for 1 day and the enzyme-treated solution was examined by HPLC. As a control experiment, dextran-4 was similarly treated. As a result, it was found that this product did not undergo glucoamylase digestion. On the other hand, Dextran-4
Subsequent to glucoamylase digestion, the substrate disappeared and glucose was regenerated. From this, this product has the above formula (II)
(However, in the formula, n is 8 on average) It was confirmed to be dextranyl glucuronic acid.

Reference Example 2 Pseudogluconobacter Saccharoketgenes K59
The 1s (FERM BP-1130) strain was cultivated for 3 days at pH 7.5 under shaking at 30 ° C. and 230 rpm using a peptone yeast (PY) medium composed of 1% Bactopeptone and 1% yeast extract. . Then, the culture was transferred to the main culture, 0.1% calcium chloride was added to the PY medium, and the culture was carried out for 48 hours while shaking at 30 ° C. and 230 rpm. Then, the obtained culture broth was centrifuged at 10,000 rpm for 10 minutes to collect the cells, and the cells were washed with 500 ml of water to obtain 7.59 g of cells per liter of the culture broth. Then, Dextran-4 (molecular weight 4000-60
00; 30 g of Extra Synthese (France) was dissolved in 1 liter of water, and 56 g of wet bacterial cells was suspended in 1 liter of water, and the mixture was stirred at 32 ° C. and 800 rpm for 60 minutes with air for 1 minute. The mixture was aerated, and the reaction was carried out for 6.5 hours while controlling the pH to 6.3 with a 0.1% NaOH solution. The reaction solution was centrifuged, 2 liters of the supernatant was collected, filtered and sterilized with a membrane filter, and then HP-20 column (900
ml) and eluted with 2000 ml of water. Concentrated hydrochloric acid was added to the flow-through to make the final concentration 0.05M, acidified, and filtered with a membrane filter.
Conduct the solution through a -205 column (100 ml) and initially use 0.05M.
After washing with hydrochloric acid (1000 ml) and then with water (2000 ml), the fractions eluted with 20% methanol (2000 ml) were collected, concentrated and freeze-dried to obtain 14 g of white powder dextranyl gluconic acid. HPLC conditions of this product [Asahi Pack GS320 (φ7.6 mm × 50 mm: manufactured by Asahi Kasei Corp.), mobile phase: water 1 ml / min, RI (Waters 41)
0) and 200 nm (Tosoh UV-8020), 20 μl injection of 0.5% aqueous solution of the sample] showed a single peak at Rt 8.70. The Rt of dextran-4 used as a starting material was 10.83. When this product was digested with glucoamylase under the conditions described above, it was easily digested, and glucose and glucosylgluconic acid could be detected. Therefore, it was confirmed that the present compound is dextranyl gluconic acid represented by the above formula (III) (wherein, n is 8 on average).

Reference Example 3 Pseudogluconobacter saccharoketgenes was cultured in the same manner as in Reference Example 2 to obtain 50
30 g of dextranyl glucuronic acid (prepared by the method described in Reference Example 1) was added to a solution prepared by dissolving 1 liter of water, and air was aerated for 1 minute at 60 ml while stirring at 32 ° C. and 800 rpm, PH = 6 with 0.1% NaOH solution.
While controlling to 3, the reaction was carried out for 21 hours. The reaction solution was centrifuged, 2 liters of the supernatant was collected, sterilized by filtration with a membrane filter, passed through an HP-20 column (900 ml), and eluted with 1.5 liters of water to collect the target fractions. After concentrating and freeze-drying, 15 g of white powder of glucuronyl dextranyl gluconic acid sodium salt was obtained.

Reference Example 4 50 ml of a 50% aqueous solution of ferric chloride hexahydrate (FeCl ₃ 6H ₂ O) was heated to 30 ° C., and then 24% Na ₂ CO ₃ was added.
50 ml of the solution are added dropwise at a rate of 0.4 ml for 1 minute with good stirring. Then, 50 ml of a 10% dextranyl glucuronic acid solution obtained in Reference Example 1 was added dropwise at a rate of 3 ml / min, and a 16% Na ₂ CO ₃ solution was added dropwise at a rate of 0.4 ml / min to obtain a pH of 4. Adjusted to 3. Next, 200 ml of ethanol
, Stir vigorously to form a slurry, and centrifuge (500
0 rpm), collect the precipitate, then dissolve in 40 ml of water, 6
The mixture was thoroughly stirred with 0 ml of ethanol and centrifuged to remove soluble substances. 20 ml of water was added to disperse the precipitate well, ethanol was removed well with an evaporator under reduced pressure, and 0.2 ml was added.
10% NaOH solution was added with good stirring at a speed of 1 / min to adjust the pH to 5-6, heated at 100 ° C. for 20 minutes, and then phenol was added to 4 mg / ml to obtain the desired dextranil. 22 ml of a complex of glucuronic acid and ferric hydroxide sol solution was obtained.

Reference Example 5 100 g of ferric chloride hexahydrate was dissolved in 100 ml of water, and this solution was added to 1 with Branson G-220.
Sonicate for time to dissolve well. Then add water to make 200 ml and transfer the solution to a 500 ml beaker. Then, 200 ml of a 24% Na ₂ CO ₃ aqueous solution was added with good stirring at 30 ° C. at a rate of 0.8 ml / min to prepare a pale tan ferric hydroxide sol. 100 ml of the ferric hydroxide sol thus prepared was taken, transferred to a 300 ml beaker, and 50 ml of 10% dextranyl gluconic acid solution was gradually added thereto while stirring well at 30 ° C. Then,
A 16% NaCO ₃ solution was added very slowly, 0.1-0.2.
The pH was adjusted to 4.3 by adding at a rate of ml / min. Next, 200 ml of ethanol was added with stirring, the resulting precipitate was centrifuged, separated from the supernatant, the precipitate was well suspended in 80 ml of water, and 120 ml of ethanol was added to reprecipitate, and the precipitate was centrifuged. Separated and separated. This operation was repeated once more, and the obtained precipitate was suspended in 20 ml of water and stirred well. 10% NaOH solution 0.1-0.2 m
It was added at a rate of 1 / min and the pH was added to 6.0. This solution was autoclaved at 120 ° C for 10 minutes,
Phenol as a preservative (to a final concentration of 1%)
Was added, and then the mixture was concentrated with an evaporator to obtain a desired complex of dextranyl gluconic acid and ferric hydroxide sol. The product formed a stable ferric hydroxide sol.
The property was analyzed and it was as follows. Total iron salt concentration: 200 mg / ml, viscosity: 32 cP, conductivity: 4
It was 7 mS / cm.

Example 1 Test material: As an anti-anemic agent, a complex of dextranyl glucuronic acid obtained in Reference Example 4 and ferric hydroxide sol (12
9 mg Fe / ml content, hereinafter abbreviated as glucuronic acid / iron) and a complex of dextranyl gluconic acid obtained in Reference Example 5 and ferric hydroxide sol (containing 206 ml Fe / ml,
Hereinafter, abbreviated as gluconic acid / iron) was used. Test animals: Landrace young pigs were used. Test method: At the age of 6 days, 9 animals per litter were divided into 3 groups [n = 3
(3 dogs in each group)], and 200 mg / head of iron sample was intramuscularly administered to the left hip. At the start of the test and every week thereafter, body weight measurement and blood collection were performed to determine the amount of hemoglobin (HG
B), hematocrit value (HCT), mean red blood cell volume (MCV) and body weight parameters were measured and compared with the untreated control group. The results are shown in Table 4.

[0038]

[Table 4]

As shown in Table 4, each anemia parameter of blood significantly increased as compared with the control group, and the progress of anemia was obviously suppressed by the administration of the anti-anemic agent of the present invention. In addition, serum iron and total iron binding capacity at the third week after administration were measured, and unsaturated iron binding capacity (total iron binding capacity-serum iron) and iron saturation (= serum iron /
The total iron binding capacity x 100) was calculated and compared with the control group. The results are shown in Table 5.

[0040]

[Table 5] Samples Serum iron Total iron binding capacity Unsaturated iron binding capacity Iron saturation (μg / dl) (μg / dl) (μg / dl) (%) Control 60.5 755.8
695.3 8.1 Glucuronic acid / iron 101.5 646.4
544.8 15.7 Gluconic acid / Iron 152.8 588.1 435.3 27.8

As shown in Table 5, when the complex of the present invention was administered, the serum iron concentration was remarkably higher than that of the control group, the increase in the total iron binding capacity was suppressed, and the iron saturation was clearly improved. Is recognized. Further, it can be seen that the anti-anemia complex of the present invention is efficiently absorbed and utilized from the tissue at the administration site.
From the above results, the anti-anemia agent of the present invention is effective for the prevention / treatment of anemia in animals.

Reference Example 6 A washed bacterial cell of Pseudogluconobacter saccharoketgenes K591s obtained in the same manner as in Reference Example 2 was obtained. Then, Dextran-4 (molecular weight 4000-6
000; Extra Synthesis (French) 30g water 1
It was dissolved in liter, and then the solution was added to 56 g of wet microbial cells suspended in 1 liter of water, and air was aerated with 60 ml for 1 minute while stirring at 32 ° C. and 800 rpm, and 0.1% NaOH was added.
The reaction was carried out for 5 hours while controlling the pH to 6.3 with an aqueous solution. This reaction solution was analyzed by HPLC (analysis conditions were the same as those described in Reference Example 2) to find that the composition was 62% of dextranyl gluconic acid with Rt 8.70 and 38% of unoxidized dextran with Rt 10.90. It turned out to be Then, the reaction solution was centrifuged, 2 liters of the supernatant was collected, and filtered through a membrane filter to remove bacteria. Then, the sterilization solution is I
R-120B (H ⁺ ) (100 ml) was passed through, followed by water (20 ml).
The eluate was collected, concentrated under reduced pressure and freeze-dried to obtain 27 g of a white powder. This product was confirmed by HPLC to be a mixture of dextranyl gluconic acid and dextran, and the composition ratio thereof was 6: 4.

Then, ferric hydroxide sol was prepared by the method described in Reference Example 5 and 100 ml of the sol was taken and put into a dialysis tube, Spectra / Pore (SPECTRA / PO).
R) 2 (fraction molecular weight 12,000 to 14,000) [SPECTRUM MEDICAL INDUSTRIES INC. (SPECTRUM MEDICAL I
NDUSTRIES, Inc., USA] and dialyzed overnight. After the dialysis, iron hydroxide sol solution, the above white powder
A solution of 5 g (6: 4 mixture of dextranyl gluconic acid and dextran) in 50 ml of water was gradually added at 30 ° C. with good stirring. Then a 16% Na ₂ CO ₃ solution was added very slowly to adjust the pH to 4.3. Three
After stirring at 0 ° C for 1 hour, autoclave at 100 ° C for 30 minutes to completely dissolve it, and add 1N-NaOH solution to pH.
Adjusted to 6.41. Then, the solution was concentrated with an evaporator, and phenol (to a final concentration of 0.5%) was added as a preservative to obtain a desired complex of dextranyl gluconic acid and iron hydroxide sol. This product formed a stable iron hydroxide sol. The property was analyzed and it was as follows. Total iron salt concentration: 197 mg / ml, viscosity: 62 cP, conductivity:
50 mS / cm.

Example 2 Anti-anemia of the complex of Reference Example 6 was obtained by using a dextran iron 200 mg Fe / ml-containing preparation (trade name: Anemex, Fujita Pharmaceutical Co., Ltd.) as a control drug and using Landrace young pigs. The activities were compared and examined. The complex and the control drug (elemental iron: 200 mg / head) were intramuscularly administered to the buttocks of two 3-day-old pigs. Two weeks after the administration, hemoglobin amount (HGB), hematocrit value (HCT), mean red blood cell volume (MCV) and weight gain were measured. The results are shown in Table 6 as an index with the control drug being 100.

[0045]

[Table 6] Item Control drug administration group Invention product administration group HGB 100 109 HCT 100 110 MCV 100 106 Weight gain 100 115

From the above results, it was found that when the complex of the present invention was administered, a superior improvement was observed in all the tested anemia parameters as compared with the case where the control drug was administered.

Reference Example 7 Pseudogluconobacter Saccharoketgenes K59
One platinum loop of the 1s strain was inoculated into a test tube (16 mm x 160 mm) containing 5 ml of the complete medium shown in Table 7, and shake-cultured at 30 ° C for 2 days.

[0048]

[Table 7] Complete medium components g / liter D-sorbitol 25 peptone 10 yeast extract 10 pH = 6.5 (in solid medium, add 20 g / liter of agar)

1 ml of this culture broth was transferred to a test tube containing 5 ml of the same medium and cultivated with shaking for 5 hours. 5 ml of the obtained culture solution
Were collected aseptically by centrifugation (6,500 rpm) at 5 ° C for 15 minutes, and then 0.05M Tris-maleic acid buffer solution (1
0 ml, pH 6.5) and centrifuge again (6,500 rp)
m) I did. This was repeated twice. 5 ml of the above-mentioned buffer solution containing 0.5 mg / ml of N-methyl-N'-nitro-N-nitrosoguanidine (nitrosoguanidine) was obtained.
And was shaken at 30 ° C. for 1 hour. This treatment liquid is 5 ℃
The cells were collected by centrifugation at 6,500 rpm for 15 minutes.
In the same manner as above, Tris-maleic acid buffer solution (10 m
The cells were washed twice with l) to obtain nitrosoguanidine-treated cells. This is diluted appropriately with 0.85% saline and added to 15 ml.
Spread on a plate (9 cm in diameter) containing the complete medium (solid) of 3
The cells were cultured at 0 ° C for 5 days to form colonies. The colonies on the complete medium were replicated on the minimal medium (solid) plate shown in Table 8 and cultured at 30 ° C for 3 days.

[0050]

[Table 8]

The bacterial solution cultured as described above was appropriately diluted and spread on a selective medium (solid) plate shown in Table 9 to form about 50 colonies per plate.

[0052]

[Table 9]

After culturing at 30 ° C. for 12 days, strains in which no calcium carbonate dissolution circle was observed around the colonies were selected. Under the same culture medium and culture conditions as in Reference Example 1, a bacterial solution of the above strain was prepared to obtain washed bacterial cells. Then, Dextran
(Molecular weight 1500, Bio Chemika)
(Switzerland) 30 g was dissolved in 1 liter of water, then 40 g of wet bacterial cells was suspended in 1 liter of water, and the mixture was added at 32 ° C.
While stirring at 800 rpm at 80 rpm, air was aerated for 60 minutes at 60 ml, and the pH was adjusted to 6.3 with 0.1% NaOH solution.
It was allowed to react for 21 hours. When this reaction solution was analyzed by HPLC (the analysis conditions are described in Reference Example 2), it was found that the composition was 96% of dextranyl gluconic acid with Rt 9.69 and 4% of unoxidized dextran with Rt 15.12. found.

Then, the reaction solution was centrifuged, the supernatant was separated, filtered and sterilized with a membrane filter, and then the sterilized solution was passed through an IR-120B (H ⁺ ) column (100 ml) and washed with water.
Elute with (200 ml). The eluates were collected, concentrated under reduced pressure and freeze-dried to obtain 29 g of white powder. This product from HPLC
The composition ratio of the mixture of dextranyl gluconic acid and dextran was 90:10. Then, an iron hydroxide sol was prepared by the method described in Reference Example 5, 100 ml of which was collected, and used for dialysis tube, Spectra / Pore (SPEC).
TRA / POR [Spectrum Medical Industries, Inc. (SPECTRU
M MEDICAL INDUSTRIES Inc)
Company, USA] and dialyzed overnight. Hydroxylation second after dialysis
To the iron sol solution, 5 g of the above-mentioned white powder (a 90:10 mixture of dextranyl gluconic acid and dextran) was added to 5 parts of water.
The solution, dissolved in 0 ml, was added slowly at 30 ° C. with good stirring. Then, a 16% Na ₂ CO ₃ aqueous solution was slowly added to adjust the pH to 4.3. Then, the mixture was stirred at 30 ° C. for 1 hour and then autoclaved at 100 ° C. for 30 minutes to completely dissolve it. Then, with 1N-NaOH solution, the pH is 6.4.
After adjusting to 0, concentrate with an evaporator, add phenol as a preservative (so that the final concentration is 0.5%),
The desired complex was obtained. This product was a stable ferric hydroxide sol. The property was analyzed and it was as follows. Total iron salt concentration: 238 mg / ml, viscosity: 21.9 cP, conductivity: 47 mS / cm.

Example 3 The anti-anemic activity of the complex of Reference Example 7 was examined by using a 5-day-old young landrace pig. The complex was intramuscularly administered to the buttocks of two pigs (200 mg / elemental iron / head). Two weeks after administration, hemoglobin level (HG
B), hematocrit value (HCT), mean red blood cell volume (MC
V) and weight gain were measured. The results are shown in Table 10 as an index with the untreated control group being 100.

[0056]

[Table 10] Item Untreated control group Product of the present invention administration group HGB 100 309 HCT 100 259 MCV 100 143 Weight gain 100 133

From the above results, it can be seen that administration of the complex of the present invention significantly improved the anemia symptom as compared with the control group.

Reference Example 8 Ferric hydroxide sol was prepared by the method described in Reference Example 5, 100 ml of the sol was taken, transferred to a dialysis tube, Spectra / POR, and dialyzed overnight. . A solution prepared by dissolving 5 g of dextranyl gluconic acid (an oxidant of dextran having an average molecular weight of 1500) in 50 ml of water was added to the ferric hydroxide sol solution after dialysis with good stirring at 30 ° C. Then a 16% Na ₂ CO ₃ solution was added very slowly to adjust the pH to 4.3. After stirring at 30 ° C for 1 hour, the mixture was autoclaved at 100 ° C for 30 minutes to completely dissolve it, and then adjusted to pH 6.40, and then dialyzed tube, Spectra / Pore (SPECTRA / PO) again.
R) and dialyzed overnight. The dialyzed solution was concentrated by an evaporator, and phenol (so that the final concentration was 0.5%) was added as a preservative to prepare a desired stable iron hydroxide sol preparation. The properties of this product were analyzed and were as follows. Total iron salt concentration: 331 mg / ml, viscosity: 38.0 cP, conductivity: 6.6 mS / cm.

Reference Example 9 500 g of ferric chloride hexahydrate is dissolved in 1000 ml of water, and this solution is sonicated with Pasolina USC-1 type for 30 minutes to sufficiently dissolve it. This solution was transferred to a 5000 ml beaker, 1600 ml of a 10% NaHCO ₃ aqueous solution was added thereto with good stirring at 25 ° C. at a rate of 30 ml / min, and then heated at 70 ° C. for 1 hour. 10% NaHC in this solution
While stirring approximately 3200 ml of an O ₃ aqueous solution at 25 ° C.,
The pH was adjusted to 4.5 by adding at a rate of 30 ml / min. This solution was used as a lab module ACP1010 (molecular weight cut-off:
(13000) for about 20 minutes for ultrafiltration to obtain 2000 ml of ferric hydroxide sol after filtration. 30 water in this sol
00 ml was added, and ultrafiltration was performed under the same conditions as above. This operation was repeated twice more to desalted ferric hydroxide sol 1
600 ml were obtained (iron content found: 66.6 mg / ml). To 130 ml of this sol (8658 mg as iron), 8130 mg of dextranyl gluconic acid (average molecular weight 1500) prepared by the same method as in Reference Example 7 was added and stirred to dissolve.
About 5 ml of 10% NaHCO ₃ aqueous solution was added to adjust the pH to 4.5, and then the mixture was heated at 100 ° C. for 30 minutes to obtain a blackish brown dextranyl gluconic acid / iron sol. This sol was concentrated to a measured iron concentration of 305 mg / ml on a water bath at 50 ° C using a rotary evaporator, phenol (final concentration 0.5%) was added as a preservative, and then water was added to obtain a target iron concentration of 25
A 0 mg / ml solution was obtained. This solution was autoclaved at 121 ° C. for 20 minutes to prepare a desired stable ferric hydroxide sol preparation. As a control formulation to compare the viscosity separately,
Dextran (average molecular weight 1500, used as a raw material of dextranyl gluconic acid (average molecular weight 1500),
Made by Bio Chemika (Switzerland)
A ferric hydroxide sol preparation of dextran was prepared in the same manner as in this Reference Example. The properties of these preparations were analyzed and were as follows. Iron hydroxide sol preparation using dextranyl gluconic acid: Total iron salt concentration: 259 mg / ml, viscosity: 37.6 cP Iron hydroxide sol preparation using dextran: Total iron salt concentration: 252 mg / ml, viscosity: 112 cP

[0060]

INDUSTRIAL APPLICABILITY According to the present invention, a novel animal anti-anemic agent containing a complex of dextran carboxylic acid and iron salt, and a method for preventing / treating anemia of animals using the same are provided. INDUSTRIAL APPLICABILITY The animal anti-anemic agent of the present invention remarkably improves the anemia state of animals such as pigs, and is extremely low in toxicity and safe.
Particularly, according to the present invention, it is possible to manufacture a preparation containing iron in a stable and high concentration and having a low viscosity. Therefore, even when the anti-anemic agent of the present invention is used as an injection, it can be safely administered to animals including juvenile pigs and the like without causing coloring spots and pain.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area C12R 1:38)

Claims (16)

[Claims] 1. An anti-anemia agent for animals, which comprises a complex of dextran carboxylic acid and an iron salt obtained by a microbiological method. 2. A microorganism belonging to the genus Pseudogluconobacter having the ability to oxidize the hydroxymethyl group- and / or hydroxyl group-containing hemiacetal portion of dextran having a hydroxymethyl group and / or hemiacetal hydroxyl group, or a treated product thereof. The dextran carboxylic acid obtained by reacting dextran carboxylic acid with a dextran having such a group to produce and accumulate the corresponding dextran carboxylic acid, and collecting the dextran carboxylic acid. 3. The anti-anemic agent for animals according to claim 2, wherein the microorganism is Pseudogluconobacter saccharoketgenes. 4. The microorganism is Pseudogluconobacter saccharoketgenes FERM BP-1128, FERM.
BP-1129, FERM BP-1130, FER
The anti-anemic agent for animals according to claim 2, which is M BP-1131, FERM BP-1132 or FERM BP-1133. 5. The animal anti-anemic agent according to claim 1, wherein the iron salt is iron hydroxide or iron oxide. 6. The animal anti-anemic agent according to claim 5, wherein the iron salt is ferric hydroxide. 7. The animal anti-anemic agent according to claim 2, wherein the treated product of the microorganism is a culture solution of the microorganism. 8. A dextran carboxylic acid represented by the formula (II) or (II
I) or (IV): [In the formula, n represents 1 to 50] The anti-anemic agent for animals according to claim 1. 9. The animal anti-anemic agent according to claim 8, wherein n is 1 to 15. 10. The anti-anemic agent for animals according to claim 1, wherein the complex is obtained by reacting dextran carboxylic acid with ferric hydroxide sol. 11. The anti-anemic agent for animals according to claim 1, which is an injection. 12. The animal anti-anemic agent according to claim 1, which is an intramuscular injection. 13. The anti-anemic agent for animals according to claim 1, which is used for piglets. 14. The animal anti-anemic agent according to claim 1, which is applied to iron deficiency anemia. 15. An effective amount of a complex of dextran carboxylic acid and ferric hydroxide obtained by a microbiological method, which exerts an anti-anemic effect, is administered to an animal in need of prevention or treatment of anemia. A method for preventing or treating anemia of animals. 16. The method according to claim 15, wherein the animal is a piglet.