JPH07330625A - Growth promoter - Google Patents

Abstract

PURPOSE:To provide a safe and effective growth promoter, containing a kind of acyl based antibiotics as an active ingredient and having improving actions on the feed utilization rate for animals. CONSTITUTION:This growth promoter contains a compound of plusbacins of the formula [X is L-HyPro or L-Pro; R is CH3 or CH(CH3)2; (n) is 9-12] or its salt as an active ingredient. The daily dosage of this growth promoter is 0.1-5mg/kg, especially preferably 0.5-2.5mg/kg. In the case of using the compound of the formula by mixing thereof with a feed, its concentration is within the range of 0.5-100ppm, especially preferably 5-20ppm. The compound of the formula has antibiotic activities against harmful intestinal bacteria in animals, reduces the consumption of glucose and proteins in the intestine, suppresses the production of lactic acid, increases the production of volatile lower fatty acids which are an effective energy source, especially the production ratio of propionic acid, and on the other hand hardly affects the intestinal bacterial flora. This compound is obtained by culturing the bacteria having the activity of producing plusbacin, e.g. Pseudomonas sp. PB-6250 (FERM BP-2938) in a medium.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a growth promoter, and more particularly to an animal growth promoter containing an acyloctapeptide compound as an active ingredient.

[0002]

It has been pointed out that the addition of streptomycin to the feed of livestock such as chickens, pigs and cows can promote the growth of these livestock [Moore PR et al. .), J. Biol. Chem. 16: 43
7 (1946)]. Since then, various antibiotics that can be used for such purposes have been identified. It is believed that these antibiotics increase the utilization rate of the feed ingested by the animal by the animal and, as a result, exert a growth promoting effect.
That is, the feed orally ingested by an animal is a protein into an amino acid, a carbohydrate into glucose or a volatile lower fatty acid (hereinafter, referred to as VFA) by the action of various enzymes and microorganisms existing in the digestive tract of the animal, In addition, fat is decomposed into glycerin and fatty acids, and among these decomposition products, glucose,
Most of amino acids and glycerin are absorbed in the small intestine, and VFA is absorbed in the large intestine. However, not all the nutrients ingested by animals are absorbed into the body, but a large number of intestinal bacteria in the digestive tract deprive some of the nutrients, resulting in low feed utilization rates by animals. . In addition to nutrients consumed by intestinal bacteria, the lactic acid produced by these bacteria is known to stimulate peristaltic movements of the large intestine, which reduces absorption time, which also reduces feed utilization. To do. Moreover, if an animal becomes infected with a bacterial disease, naturally, the reduction of feed utilization will be promoted.

As described above, harmful intestinal bacteria in animals have various adverse effects on feed utilization rate.
For example, Clostridium (Clostridium), Bacteroides (Bacteroides), Staphylococci (St
aphylococcus ), Streptococcus
s ) and other Gram-positive bacteria are known. Among them, Clostridium perfringens Pell (C. Perfringens)
The antibacterial activity against the strain is considered to be one of the indicators for in vitro screening of antibiotics having a growth promoting effect [Ann. NY].
Sci. 78: 321-327, 1959; Poultry Science 62: 1633.
~ 1638 (1983): Poultry Science 63: 2036 ~ 2042 (19
84)].

By the way, of the nutrients ingested, some of the carbohydrates are decomposed by microorganisms in the large intestine, particularly in the proximal colon, and are metabolized into a large amount of organic acids such as VFA and lactic acid. Among these metabolites, formic acid is used. It has been pointed out that VFAs such as acetic acid, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, isocaproic acid and caproic acid are important energy sources for livestock such as pigs and ruminants (cattle etc.). [Kas
s, ML (1980), Utilization of dietary fiber from
alfalfa by growing swine, II. Volatile fatty acid
concentrations in and disappearance from the gastr
ointestinal tract., J. Animal Sci. 50: 192-197; and Takashi Sakata (1994) Message from Enterobacteriaceae "Chemistry and Biology" 32: 23-31]. Of these VFAs, acetic acid, propionic acid and butyric acid are known to have high utility values in vivo. That is, 95% or more of the produced VFAs are absorbed from the surface of the large intestine epithelium by diffusion, and, for example, medium pigs (body weight about 4
8 Kg), it is reported that the amount of sustaining energy reaches 6.9 to 12% (Kass, Sakata, supra). These V
Regarding the relative utilization efficiency of FA, for example, acetic acid and butyric acid are also utilized in pigs, but propionic acid is used more efficiently, and when the utilization of propionic acid is too low, keto-cis [a large amount of ketone is used]. Body (acetoacetic acid, β
-Hydroxybutyric acid, acetone) is accumulated in the body]
al., (1971), Digestive Physiology and Nutrition of
Ruminants. Vol. 2: 622-625].

From the above, it is considered that an antibiotic capable of increasing the feed utilization rate of animals and promoting the growth needs to exhibit at least all or some of the following actions. (1) The above-mentioned intestinal harmful bacteria that are the causative bacteria of reduced feed efficiency, for example, the antibacterial action against Gram-positive bacteria such as Clostridium and Bacteroides ; (2) Glucose and protein by intestinal bacteria (3) Inhibition of lactic acid production by intestinal bacteria; and / or (4) Increasing effect of VFA production, which is an energy source of animals, and in particular increasing propionic acid production ratio. . In addition to these effects, antibiotics routinely administered as growth promoters need to be ecologically safe. The above-mentioned actions are interrelated with each other, and an antibiotic having such an action exhibits an antibacterial action against harmful bacteria in the intestine, suppresses the production of lactic acid, and at the same time, converts V from carbohydrates.
Efficiently produces FA, and in particular a higher proportion of propionic acid, thereby increasing carbohydrate utilization and reducing the incidence of ketosis, improving overall feed efficiency and improving animal growth. Expected to be feasible.

[0006] Conventionally, the lactone-containing peptide antibiotics include antibiotics having antibacterial activity against intestinal harmful bacteria of various animals including livestock such as poultry and swine, for example, strains of the genus Clostridium. It is known that these compounds include neutral or basic compounds such as enramycin and nosiheptide, and daptomycin having an acidic lipopeptide structure (JP-A-55-9).
2353) and the like. Among the antibiotics of this kind, thiopeptin (Fujisawa Pharmaceutical Co., Ltd.) is already on the market as an animal growth promoter, and glycopeptide is a substance having a growth promoting effect superior to thiopeptin. System antibiotic PA-4286
7-A is disclosed (JP-A-1-190633).
On the other hand, Avilamycin (Eli.
Antibiotics such as lily) and tylosin (Shionogi Pharmaceutical) have been found to increase VFA in the digestive tract, decrease lactic acid production, and save glucose.
In particular, it is considered to be closely related to the improvement of feed utilization rate of livestock such as pigs, sheep, horses and cattle. As described above, although various antibiotics have been developed, more antibiotics that have the above-mentioned effects and are effective and safe as the target growth promoter of livestock are needed.

[0007]

[Means for Solving the Problems] The inventors of the present invention have conducted earnest studies to develop a novel growth promoting agent, especially a growth promoting agent for animals. Has the above-mentioned actions and is useful for improving feed utilization rate, and has completed the present invention. That is, the present invention provides formula I:

[Chemical 2] Select [wherein, X is L-HyPro or L-Pro, R is -CH ₃ or -CH (CH _{3) 2,} n is an integer of 9-12] from the group consisting of compounds and salts thereof represented by The present invention provides a growth promoter containing one or more compounds as an active ingredient.

Compound I, which is an active ingredient of the growth promoting agent of the present invention, is disclosed as an acyl octapeptide having a novel structure together with its salt in JP-A-3-188098. In the present specification, a compound represented by the formula I is
According to the description of the publication, it is called Plusbacin. The following eight types are known as typical plus bacin compounds. In formula I, X is L-HyPr
a compound in which o, n is 10 and R is —CH ₃ (plus bacin A ₁ ); X is L—HyPro, n is 9 and R is —CH (CH ₃ ) ₂
(Plasmacin A ₂ ); X is L-HyPro,
n is 10, R is -CH (CH _{3) 2,} compound (Purasubashin A _3); X is L-HyPro, n is 12, R is -CH ₃
(Plasmacin A ₄ ); X is L-Pro, n
But 10, R compound is -CH ₃ (Purasubashin B _1); X is L-Pro, n is 9, R is -CH (CH _{3) 2,} compound (Purasubashin B _2); X is L-Pro , N is 10 and R is —CH (CH ₃ ) ₂ (plus-basin B ₃ ); and X is L-Pro, n is 12, and R is —CH ₃ (plus-basin B ₄ ).

In the present specification, "L-HyPro" means L-trans-3-hydroxyproline, "D-HyAsp" means D-threo-β-hydroxyaspartic acid, and "L-HyPro".
“HyAsp” means L-threo-β-hydroxyaspartic acid, and “aThr” means allo-threonine. “Feed utilization rate” refers to the degree of absorption and utilization of feed, and improvement of feed utilization means that large weight gain can be obtained with a small amount of feed, and specifically, it is an energy source of animals. It is achieved by increasing the production of VFA, especially by increasing the production ratio of propionic acid, decreasing the production of lactic acid, and saving the consumption of glucose and protein.

For the purpose of the present invention, not only the above-mentioned eight kinds of plus bacin and salts thereof, but also other compounds of the plus bacins represented by the formula I and having animal growth promoting activity and salts thereof are useful. The salts of plus bacin useful in the present invention include lithium, potassium, alkali metals such as sodium, magnesium, alkaline earth metals such as calcium, or inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and hydrobromic acid, And salts with organic acids such as acetic acid, oxalic acid, maleic acid, fumaric acid, citric acid, malic acid, adipic acid and succinic acid. In the present specification, simply referring to plus bacin, each of the compounds of the formula I including plus bacin A _{1 to} A ₄ and B _{1 to} B ₄ and salts thereof, or a mixture consisting of any combination thereof, or It shall refer to all compounds of formula I.

As is clear from the results of in vitro and in vivo test examples described below, the plus bacin of the present invention is
It is a safe and effective antibiotic with a growth promoting effect on animals. That is, it shows high antibacterial activity in vitro against several Gram-positive bacteria such as C. perfringens, and
It was active against a total of 16 strains of C. perfringens derived from cattle, pigs, sheep and chickens. Further, in an in vitro test using intestinal contents collected from the large intestine (proximal colon) of pigs, the intestinal bacteria save glucose and protein consumption, suppress lactic acid production, and reduce volatile fatty acid (VF).
It has been shown to increase the production of A), in particular the proportion of propionic acid. On the other hand, when administered to broiler chicks, a higher weight gain effect was observed than in the known antibiotic PA-42867-A administration group, and it was found that the intestinal flora was not significantly affected. Furthermore, it was confirmed from the results of the acute toxicity test that it is a highly safe antibiotic. These test results show that plus bacins are safe antibiotics that have an effect of improving the feed utilization rate of animals such as livestock and poultry, and can be routinely administered to animals for the purpose of promoting animal growth. It is shown.

The plasbacins of the present invention can be obtained by culturing a bacterium having a plasbacin-producing ability in a medium, separating and purifying the culture from the culture in a conventional manner. As such a bacterium, a bacterium of the genus Pseudomonas described in JP-A-3-188098 (for example, Pseudomonas sp. PB-6250 (FE
RM BP-2938)). Purification of plus bacin from the culture depends on whether it is accumulated in the cells or secreted into the medium,
Any of these can be performed by methods known to those skilled in the art such as solvent extraction, crystallization and column chromatography. In the following production examples, a method of culturing a Pseudomonas genus in a medium to purify the produced plus bacin from inside or outside the cells is shown. However, the object of the present invention can be achieved by using plus bacin obtained from any source by any production method as long as it has an animal growth promoting action. Therefore, a growth promoting agent using a plus bacin obtained by a method other than the method described in the present specification is also included in the scope of the present invention.

When the growth-promoting agent of the present invention is administered to an animal, it may be administered orally as it is or as a formulation in an appropriate dosage form, but it is generally a commonly used carrier ( (For example, defatted rice bran, defatted soybean powder, bran, kaolin, talc, calcium carbonate, lactose, water, etc.)
It is preferable to administer a mixture of the above, or such a mixture, or plus bacin alone in admixture with animal feed or water. The plus bacin used here does not necessarily have to be a purified product,
It may be a culture of a bacterium, a crude product (for example, a partially purified product of a culture obtained by culturing a plasmocin-producing bacterium in a medium), or a bacterium itself containing plusbacin. Plusbacin can also be used in the form of the above pharmaceutically acceptable salt. When plasbacin is administered for the purpose of promoting growth, the daily dose is usually 0.1 to 5 m / kg of body weight, although it varies depending on the animal.
g, preferably 0.2-4 mg, more preferably 0.5-2.
It is 5 mg.

When the plus bacin of the present invention is mixed with feed, the feed may be any feed commonly used as animal feed. Such feeds are usually
Corn, bran, rice, wheat, cottonseed meal, milo, soybean meal, fish meal, defatted rice bran, oils and fats, alfalfa, calcium carbonate, calcium phosphate, sodium chloride, choline chloride, various vitamin preparations (vitamin A, vitamin D, vitamin E, Vitamin B1, vitamin B2, vitamin B6, vitamin B12, calcium pantothenate, nicotinamide, folic acid), inorganic salts (magnesium sulfate, iron sulfate, copper sulfate, zinc sulfate, potassium iodide, cobalt sulfate), etc. It is prepared by mixing all. Other additives permitted in the field of livestock, for example, other antibiotics, bactericides, anticoccidial agents, anthelmintics, etc. may be added to the feed.

The content of the plus bacin in the feed containing the plus bacin according to the present invention is such that when they are used alone, when any mixture thereof is used, bacterial cells containing them and an extraction crude product containing them are used. In either case, the concentration of plus bacin used is 0.5 to 1
A range of 00 ppm, preferably 1.0 to 50 ppm, and more preferably 5 to 20 ppm is suitable, but it should be calculated so that the required amount of plus bacin is contained in the feed, and be added to the feed of individual animals. Is preferred. Alternatively,
The growth promoting agent of the present invention is a dosage form suitable for ordinary oral administration used in the field of veterinary medicine, for example, solid agents such as tablets, powders, granules, capsules and pellets; water agents; oily suspension agents. Or a liquid preparation such as a syrup or an elixir. These formulations can be given as they are or as a mixture with feed. In its preparation, any of the conventional excipients, binders, lubricants, aqueous solvents, oily solvents, emulsifiers, suspending agents and the like can be used, and other additives such as preservatives and stabilizers. It may also contain agents, vitamins, other antibiotics, germicides and the like. The growth promoter of the present invention can be administered to animals in general, and examples thereof include poultry such as chicken, turkey, duck and quail, cattle, horses, pigs, sheep, goats, mink, rabbits and other ruminant or monogastric livestock. Is mentioned. As a method for raising animals, a generally used method may be used as it is.

[0016]

EXAMPLES The present invention will be described in more detail with reference to the following examples. Production Example 1 Production of Plus Bacin The method of this Production Example is substantially the same as the method described in JP-A-3-188098. (A) Fermentation production process: glucose 10 g, yeast extract 5
1L of medium consisting of 1000g of tap water (pH unadjusted)
Pseudomonas sp. PB-6250 (FERM) in a 2 L Erlenmeyer flask containing
The cells of the culture slant of BP-2938) are aseptically suspended. This is subjected to shaking culture at 250 rpm for 18 hours at 28 ° C. 7 L of this culture was added to cornstarch 10
g, potato starch 10 g, CA-1 (animal feed) 20
g, medium 20 consisting of 1000 ml of tap water (no pH adjustment)
Inoculate a 500L tank containing 0L, and aerate 200L /
Incubate at 250 rpm, 28 ° C. for 72 hours per minute.

(B) Extraction and purification steps: (1) 25 kg of salt was added to about 200 L of the above culture solution,
The pH is adjusted to 3.0 with dilute hydrochloric acid and centrifuged with a Sharpless centrifuge. The microbial cell portion is extracted twice with 54 L of 70% acetone, the extract is concentrated under reduced pressure, and most of the acetone is distilled off. 10 L of water was added to the remaining aqueous solution (15 L),
The pH is adjusted to 8.0 with dilute sodium hydroxide, and passed through a column (10 L) of Diaion HP-20 (manufactured by Mitsubishi Kasei) to adsorb plus bacin. After washing the column with water, it is eluted by a concentration gradient elution method from 30% acetone to 100% acetone. The active elution fractions were collected (10 L), and most of the acetone was distilled off under reduced pressure.
Extract with 5 L of butanol at pH 2.5 (prepared with dilute hydrochloric acid). The butanol extract was concentrated under reduced pressure, and acetone was added to add crude powder containing plasmin (hereinafter,
23.9 g was obtained.

(2) Chloroform-ethanol-10%
Silica gel column packed with acetic acid (4: 7: 2) solvent (Merck, 70-230 mesh, 1000 g).
A solution prepared by dissolving the above-mentioned crude powder (23 g) in the above-mentioned solvent is placed on, and developed and eluted in the same solvent. Collect the active elution fractions and concentrate under reduced pressure with the addition of water and butanol. From the concentrated solution, antibiotics were extracted with butanol at pH 2.0, and acetone was added to obtain 6.34 g of crude plasmin powder.

(3) The crude powder was subjected to high performance liquid chromatography under the following conditions to obtain a purified fraction. Column: YMC AP-324 S15 / 30 300
Å ODS (manufactured by Yamamura Chemical Co., Ltd.) Mobile phase: acetonitrile-50 mM phosphate buffer containing 50 mM sodium sulfate, pH 7.5 (36:64) Flow rate: 100 ml / min Detected at 220 nm by an ultraviolet absorption detector. Sample pH
It was dissolved in 8.0 water and 500 mg was injected at one time.

According to the above-mentioned chromatography, first the fractions containing plasmasin A ₁ and B ₁ and then the fractions containing plasmasin A ₂ and B ₂ and finally the plasmasine A ₃ , A ₄ , B ₃ and The fraction containing B ₄ is eluted. Each fraction is collected separately, concentrated under reduced pressure, and extracted with butanol at pH 2.5. The extract is washed with 0.1N hydrochloric acid and then with water. Further, by concentrating under reduced pressure and adding acetone, 150 mg of plasmasine A ₁ and B ₁ hydrochloride mixture, 2.14 g of A ₂ and B ₂ hydrochloride mixture and A ₃ , A ₄ , B ₃ and B ₄ hydrochloride mixture 1.0 g was obtained.

(4) Plasbacin A _{1 to} A ₄ and Plasbacin B _{1 to} B _{4 are} each prepared from the above mixture.
Separation was carried out again by high performance liquid chromatography under the following conditions. Column: Ultron 7CN 20 × 250 mm (manufactured by Shinwa Kako Co., Ltd.) Mobile phase: acetonitrile-50 mM phosphate buffer containing 50 mM sodium sulfate, pH 2.2 (26:74, A ₂ and A _{2 in} case of separation of A ₁ and B ₁ ) In the case of B ₂ , A ₃ , B ₃ , A ₄ and B ₄ , 28:72) Flow rate: 11.25 ml / min Detected at 220 nm by UV absorption detector. Sample is 50%
It was dissolved in methanol and 5 mg was injected at a time. Fractions of each component were collected, concentrated under reduced pressure, extracted with butanol at pH 2.5, and the extract was extracted with 0.1N hydrochloric acid and then with
After washing with water, the mixture was further concentrated under reduced pressure, and acetone was added to obtain a hydrochloride salt of each component. From 77 mg of the mixture of A ₁ and B ₁ , 23 mg of A ₁ hydrochloride and 6 mg of B ₁ hydrochloride were obtained. 100 mg of a mixture of A ₂ and B ₂ to A ₂ hydrochloride 6
8 mg and 4 mg of B ₂ hydrochloride were obtained. A ₃ , A ₄ , B ₃
And a mixture of B ₄ 150 mg to A ₃ hydrochloride 52 mg, A ₄
10 mg of hydrochloride, 5 mg of B ₃ hydrochloride and 2 mg of B ₄ hydrochloride were obtained. The antibacterial activity of each of the thus obtained components of plus bacin and the plus bacin group obtained in (1) was measured by the agar plate dilution method according to the minimum inhibitory concentration (MIC) measuring method of the Japanese Society of Chemotherapy Standard Method. As a result, the antibacterial activity of Plasbacin A ₃ was the highest, and 77% of the group was the Plasbacin group obtained in (1).

Production Example 2 Production of Plasbacin (a) Fermentation production step: A strain of the genus Pseudomonas that secretes the product outside the cells is selected and cultured in the same manner as in Production Example 1. (B) Extraction and purification process: 2.7 L of chloroform was added to about 265 L of the above fermentation broth (content: 347 g), and sterilization was carried out with stirring for 3 hours. Next, the pH of the fermentation broth was adjusted to 2 with 6N hydrochloric acid.
Adjusted to n-butanol / methanol (1: 1)
288 L was added and the mixture was extracted with stirring for 1 hour. A clear extract was obtained by separating the bacterial cells with a Sharpless centrifuge (Kansai Centrifuge Co., Ltd.). The extract was concentrated under reduced pressure, the acetone was distilled off, 20 kg of sodium chloride was added to the concentrate, and the mixture was salted to obtain 142 L of n-butanol extract.
Got The butanol extract was concentrated under reduced pressure while adding water,
After the distillation, 18 L of acetone was added to obtain 917 g of crude plus bacin (content of 226 g) as a yellowish brown powder. 369 g of crude plus bacin powder was suspended in 30 L of water. 2N sodium hydroxide was added with stirring to dissolve the plus bacin to a final pH of 8.2 and then Dowe
It was passed through an x 1x2 (acetic acid type) 18x40 cm column.
After washing the column with water and 0.5 M ammonium acetate solution, plus bacin was eluted from a 50% methanol / 0.5 M ammonium acetate solution. The eluate was cooled at 4 ° C. overnight, and the crystalline plus-basin that had separated was filtered off and washed with acetone. Vacuum dried and purified Plus Basin 36g
(TLC 1 spot) was obtained as a yellow powder. As plus-basin, A ₁ , B ₁ , A ₂ , B ₂ , A ₃ , A ₄ (B
₃ ), etc., containing 6 to 7 or more components.

[0023] (c) Purasubashin A in vitro antimicrobial activity from a mixture obtained in the _third separation and purification (b) above was isolated and purified high Purasubashin A _3. (1) Crude fraction of plus bacin 16 g of the above-mentioned purified plus basin (TLC 1 spot) yellow powder is dissolved in 150 ml of deionized water. 29 in advance
% Acetonitrile / 50 mM phosphate buffer (pH
7.5) Bakerbond Wp C4 (average particle size 40 micron) glass column 80 mm x 400 mm (column volume: about 2 L) equilibrated with the solution, using the same buffer, flow rate 8
Elution (12 L) was carried out with 5 ml / ml (column internal pressure: 2.5-3.6 kg / cm ² ) to fractionate the plus bacin A ₁ and B ₁ components. Next, the concentration of acetonitrile was increased to 30% and eluted (about 9 L) to fractionate the plusbasin A ₂ and B ₂ components. Finally, the acetonitrile concentration was increased to 30% and eluted (about 6 L) to fractionate the plusbasin A ₃ and A ₄ components. Acetonitrile was distilled off from each fraction, and the mixture was acidified with hydrochloric acid (pH 2) to extract with n-butanol, and acetone was added to the concentrate to give a colorless powder. Thus, 4.5 g of a mixture of plus bacin A ₃ and A ₄ was obtained.

(2) Isolation of Plasbacin A ₃ Separation of Plasbacin A ₃ component and A ₄ component by reverse phase CN HPLC 0.5 g of a mixture of Plasbacin A ₃ and A ₄ was dissolved in 6 ml of 50% methanol water, and then HPLC fraction was added. I took it. HPL
The C sorting conditions are as follows. Column: R-555-15, S-15,120A, CN 50φx500mm (YMC) Mobile phase: 28% acetonitrile / 50 mM phosphoric acid (pH
2.2) Flow rate: 100 ml / min (internal pressure 17-24 kg / cm ² G) Detection: 220 nm Plasbacin A ₃ fractions showing a single peak in analytical HPLC are collected and concentrated under reduced pressure to remove the solvent and produce. N-Butanol extraction was performed from the aqueous solution. The n-butanol extract was thoroughly washed with water, concentrated under reduced pressure, and added with acetone to give a colorless powder. Purified plus bacin A ₃ (carboxylic acid) 195m
g was obtained. (3) Purasubashin A ₃ production purification of the sodium salt Purasubashin A ₃ a (carboxylic acid) was dissolved in purified water. After adjusting the pH by the slow addition of 1N sodium hydroxide with stirring to 7.0, the solution freeze-dried As a result, 197 mg of plus bacin A ₃ sodium salt was obtained as a colorless powder.

The utility of plus bacin as an animal growth promoter was tested by the following method. Test Example 1 Acute toxicity plus bacin has antibacterial activity as described in JP-A-3-188098 and is also effective for bacterial diseases and the like. The acute toxicity of the plus bacin obtained in the production example was tested by oral or intraperitoneal administration in rats and mice according to the evaluation criteria of feed additives and the test method thereof (Japan Science Feed Association, 1992). As a result, male and female deaths of both animals did not appear at the maximum dose tested (oral administration: 300 mg / kg, intraperitoneal administration: 100 mg / kg). less than,
₃ shows the non-toxic level of orally or intraperitoneally administered plus bacin A ₃ in rats and mice.

[Table 1] The fish toxicity of the compounds according to the present invention was notified by the Ministry of Agriculture, Forestry and Agriculture Administration Bureau (40
According to the Agricultural Administration B No. 2735) acute toxicity test of Japanese medaka, "96 hours non-exchange water-stopping method", 10 ppm was administered. No abnormalities were observed in Japanese medaka during the test period. Found to have.

Test Example 2 Antibacterial activity against major bacterial disease-causing bacteria in the field of livestock The antibacterial activity of Plasbacin A ₃ against major bacterial disease-causing bacteria in the field of livestock was tested in vitro. That is,
For the antibacterial activity against 14 strains of 14 strains shown in Table 2, the minimum inhibitory concentration (MIC) was determined by the agar plate dilution method or the liquid dilution method according to the standard method of the Japanese Society of Chemotherapy. The test results are shown in Table 2.

[0027]

[Table 2] Apparently from Table 2, Plasbacin A ₃ is Staphylococcus aureus , Streptococcus agalactie ( Streptococcusagalactiae ).
iae ), C. perfringens, Streptococcus sp. and other gram-positive bacteria.

[0028] Test Example 3 C., An indicator of in vitro screening of antibiotics for the purpose of antimicrobial activity growth promotion for pel perfringens C. The activity against pel perfringens, cows, pigs, total such from sheep and chicken 16 C. perfringens spp. The test results are shown in Table 3.

[0029]

[Table 3]

Test Example 4 Plasbacin A ₃ against poultry
Growth-promoting effect The effect of plus bacin obtained in the production example on poultry was examined by the growth-promoting effect on broiler chicks and the fluctuation of intestinal flora. 1) The growth-promoting effect of plus bacin A ₃ on broiler chicks (8 days old, chunky breed) was confirmed by the known antibiotic PA.
-42867-A was investigated as a control drug. Additive concentration is feed mash without added antibacterial agent [crude protein (CP)
Concentration: 23%] was added at a concentration of 10 ppm for 10 days, and examined in a battery breeding system. The experimental method was as follows: first chicks were bred with a feed having a CP concentration of 23% until 8 days of age, and then 23 per group.
Each group was divided into 3 experimental groups so that the body weight of each group was uniform. Each experimental group contained plus bacin A ₃₁
0 ppm x 10 days, PA-42867-A 10 ppm x 10
Days, 10 days without a control group. Each of these experimental groups was bred under an environment temperature of 26 ± 2 ° C. Efficacy was evaluated from the weight gain during the test period (10 days). The test results are shown in Table 4.

[0031]

[Table 4] It is apparent from the table that the plus bacin A ₃ addition group has a significant (P <0.05) greater weight gain than the non-addition control group, and is larger than that of the control drug PA-42867-A administration group. .

2) Next, the change in the intestinal flora caused by the feeding of the feed supplemented with plus bacin A _{3 was} carried out.
Between the contents, Clostridium ( Clostridi
a ) and enterococci ( Enterococci ) were investigated. 9 times the amount of BHI (Brain
Heart infusion: Difco) suspended in medium and serially diluted at appropriate times, then CW (Cl
ostridia Welchili) agar medium was inoculated with 0.1 ml of each diluted EF (Enterococci faecalis: Nissui Pharmaceutical) agar medium for the isolation of Enterococcus.
The CW agar medium was anaerobically cultivated, and the EF agar medium was cultivated under aerobic conditions at 37 ° C. for 48 hours. The test results are shown in Table 5.

[0033]

[Table 5] The number of Clostridials in the small intestine is plus bacin A ₃ ,
Significant (P <0.0) due to feeding PA-42867-A
1) to have been reduced, reduction in the number of Enterococcus acids by Purasubashin A ₃ is not observed, Purasubashin A ₃ was found to be a compound which is less affected on the intestinal flora.

Test Example 5 Plus pocin for poultry of the group
Growth-promoting effect Antibacterial activity is 77% that of Plusbacin A ₃ Production Example 1
Using the plus bacin group obtained in (1), the growth promoting effect on broiler chicks and the effect on intestinal flora were examined in the same manner as in Test Example 4. The results are shown in Tables 6 and 7, respectively.

[Table 6] 2.2% in the plus bacin group addition group compared to the non-addition control group
You can see that the weight gain is heavy.

[Table 7] Although the number of Clostridium in the small intestine was significantly (P <0.01) decreased by the feeding of the plusbacin group, the decrease of the enterococcus number by the plusbasin group was not observed, and the effect on the intestinal bacterial flora of the plusbasin group was small. it is obvious.

Test Example 6 Effect of plus bacin on various indicators relating to feed utilization rate Various indicators relating to feed utilization rate (production of VFA and lactic acid in the intestine, and consumption of protein and glucose) of plus bacin and existing antibiotics were observed. The effects were tested in vitro. That is, by anaerobically culturing the digestive tract contents containing enterobacteria derived from the proximal colon of pigs in a liquid medium, the effect of each specimen on the action of saving nutrients or the production of energy sources, which are indicators in the medium, 1) Sample: Plus Bacin A ₃ sodium salt (1000 μg titer
/ mg), Avilamycin (Eli Lilly: 1206 unit)
s / mg), colistin [family drug: 16900 units / mg] and erythromycin (Shionogi: 940 μg titer / mg). These drugs should be concentrated at the drug substance concentration (5 mg /
ml) was prepared and diluted according to the purpose of the experiment. 2) Collection of intestinal contents: A conventional pig (1.5 months old, weight: 15 kg) purchased from Hiraoka Pig was euthanized with an anesthetic and the large intestine (proximal colon) was removed. The intestine is
It was ligated to about 10 to 15 cm using a ligature. afterwards,
Separated with scissors and stored at -80 ° C.

3) Medium and Culture Method PYG liquid medium was used for culturing the intestinal contents. In order to maintain strict anaerobic culture conditions, commercially available CO ₂ gas was passed through a reducing device (Hirasawa Seisakusho) to remove impure gas (oxygen) and then used. 0.6 g of intestinal contents (large intestine) was added to PYG liquid medium (5.4 ml) in which CO ₂ gas was sufficiently blown, and 60 μl of a drug solution containing a specimen (final concentration 10 ppm)
Was added. Then, CO ₂ gas was blown in for several minutes, and the cells were cultured under anaerobic conditions at 40 ° C. for 8 hours. Before and after the start of culture, the amounts of glucose, protein, lactic acid, and VFA as indicators in the medium were measured by the following methods to reduce the consumption of glucose and protein, suppress the production of lactic acid, and produce VFA. Each specimen was evaluated for enhancing effect.

4) Evaluation of each index (1) Conservation effect on glucose and protein consumption The culture solution obtained in 3) above after the completion of culture was centrifuged (10.0).
(00 rpm, 10 minutes), the glucose content in the supernatant was measured by a glucose measurement kit (o-toluidine boric acid method) of Wako Pure Chemical Industries, and the protein content was measured by a protein assay manufactured by Bio Rad. It measured using the kit (protein assay kit) according to the instruction manual. Glucose (unit:
The consumption-saving effect of mg / dl) and protein (unit: μg / ml) was measured using the calculation formula described below. [(AB) / A] × 100 = saving rate (%) A: control (0 hour) glucose amount ¹⁾ -control (8 hour) glucose amount ²⁾ B: control (0 hour) glucose amount ³⁾ - Addition of drug (8 hours) Glucose amount ^{4) 1)} , ²⁾ , ³⁾ , ⁴⁾ : Or protein amount results are shown in Table 8 below.

(2) Lactic acid production inhibitory action and volatile fatty acid (VFA) production increasing action 1 ml of the culture solution (0 hours and 8 hours) obtained in 3) above was used for deproteinization.
0.1 ml of 5% metaphosphoric acid and 25 μl of 50% sulfuric acid were added, and the mixture was allowed to stand at room temperature overnight. Then, centrifugation was performed at 10,000 rpm for 10 minutes, and the supernatant was used for measurement of lactic acid and VFA. Lactic acid was measured by the boron trifluoride methanol method. That is, 25 μl of an internal standard substance (1% malonic acid) was added to 1 ml of the centrifugation supernatant, and further methylation was performed.
The same amount (1 ml) of boron trifluoride methanol as the supernatant was added, and the mixture was allowed to stand at room temperature overnight. Lactic acid was extracted by adding 1 ml of chloroform, and lactic acid was measured using gas chromatography under the following conditions. The rate of inhibition of lactic acid production was measured according to the following calculation formula. [(AB) / A] × 100 = inhibition rate (%) A: control (8 hours) lactic acid amount-control (0 hour) lactic acid amount B: drug addition (8 hours) lactic acid amount-control (0 hour) Lactic acid amount

The VFA was measured by an ether extraction method using a part of the centrifugal supernatant after the deproteinization treatment. In particular,
Internal standard substance (1% crotonic acid) per 1 ml of centrifugation supernatant
Was added to 25 μl and mixed, then 1 ml of ethyl ether was added.
In addition, VFA was extracted. After centrifugation at 1.000 rpm for 3 minutes, the amount of VFA (μM / ml) contained in the ether layer was measured using gas chromatography under the following conditions. The results are shown in Table 8.

[0040]

[Table 8] Gas chromatography conditions: Shimadzu GC-14B and widebore capillary column (BP20-W12-100) were used for the gas chromatography and the column, respectively. The sample was injected using an auto-injector, and the injection amount was 1 μl. The gas chromatographic analysis conditions are as follows. Gas flow rate temperature setting Carrier (P) 600 kpa, Carrier (M) 45 kpa Column VFA 115 ° C, NVFA 90 ° C Hydrogen 60 kpa Injection 200 ° C Air 60 kpa Detector 200 ° C From Table 8, the plus bacin of the present invention is intestinal VFA. It has excellent values for all four indicators of lactic acid, lactic acid, protein and glucose, and has an action of saving glucose and protein consumption by intestinal bacteria in the large intestine, an action of increasing VFA production amount, and an action of inhibiting lactic acid production. It is clear.

Test Example 7 Effect of plus bacin on VFA production amount The effect of plus bacin and existing antibiotics on VFA production in the intestine was examined by digesting the digestive tract contents containing enterobacteria derived from the proximal colon of swine. VFA produced by anaerobic culture in a medium
The sample, intestinal contents collected, the culture conditions and the VFA measurement method examined in vitro by analyzing the above are the same as in Test Example 6. The results are shown in Table 9.

[Table 9] Table 9 shows that the production of propionic acid in the presence of Plasbacin is significantly higher than that of the other tested antibiotics, and also the production of acetic acid. From this result, it is clear that plus bacin acts on intestinal bacteria to produce a higher proportion of propionic acid from carbohydrates and improves feed utilization. The following is an example of adding the plus bacin of the present invention to feed.
In addition, the compounding ratio of the component is represented by weight%.

Example 1 Corn 46.45% Mylo 15.00% Soybean meal 5.00% Fish powder 3.00% Defatted rice bran 25.00% Alfalfa 3.00% Calcium carbonate 1.00% Calcium phosphate 0.70% Sodium chloride 0.40% Vitamin A, D, E mixture 0.05% Inorganic salt mixture * 0.1% Vitamin B group mixture ** 0.1% Plus Bacin A ₃ 10 ppm * Inorganic salt mixture: Manganese sulfate, zinc sulfate, copper sulfate,
A mixture of cobalt sulfate and potassium iodide. ** Vitamin B group mixture: Vitamin B ₁ , vitamin B ₂ ,
A mixture of vitamin B ₆ , vitamin B ₁₂ , biotin, folic acid and calcium pantothenate. Mix the components in the above proportions and mix well.

Example 2 Corn 41.00% Mylo 25.00% Soybean meal 19.10% Fish powder 8.00% Oil and fat 4.00% Calcium carbonate 1.40% Calcium phosphate 0.85% Vitamin inorganic salt mixture * 0.26% Methionine 0.10% Sodium chloride 0.29% Plus Bacin A ₃ 10 ppm * Vitamin inorganic salt mixture: vitamin A, vitamin D ₃ ,
A mixture of vitamin E, vitamin B ₁ , vitamin B ₆ , vitamin B ₁₂ , calcium pantothenate, nicotinamide, vitamin K, choline chloride, magnesium sulfate, copper sulfate, iron sulfate, zinc sulfate, cobalt sulfate and potassium iodide. . Mix the components in the above proportions and mix well.

Example 3 Corn 78.00% Soybean meal 9.00% Fish powder 10.00% Fat 3.90% Crude fiber 2.40% Crude ash 5.10% Calcium 1.07% Phosphoric acid 0.73% Mixture of alfalfa meal and calcium carbonate 3.00% Plus Bacin A ₃ 10 ppm Above Mix the components in proportions and mix well.

[0045]

The active ingredient of the growth promoting agent of the present invention, plus bacin, has an antibacterial action against harmful bacteria in the intestines of animals, and saves glucose and protein consumption in the intestines.
While suppressing lactic acid production and increasing the production ratio of VFA, which is an effective energy source, especially the production ratio of propionic acid, it is a highly safe compound that hardly affects the intestinal flora of animals. Therefore, the growth promoter of the present invention can be fed to these animals on a daily basis for the purpose of improving feed utilization rate of animals such as poultry and livestock, and promoting growth, and can contribute to efficient breeding. .

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location (C12P 21/02 C12R 1:38)

Claims (7)

[Claims] 1. Formula I: [Wherein, X is L-HyPro or L-Pro, R is -CH ₃ or -CH (CH _{3) 2,} n represents an integer of 9-12] containing a compound or a salt thereof represented by as an active ingredient Growth promoter. 2. The growth promoting agent according to claim 1, which is used for promoting the growth of animals. 3. The growth promoting agent according to claim 2, wherein the animal is poultry or livestock. 4. The growth promoting agent according to claim 3, wherein the animal is pig. 5. The growth promoting agent according to claim 1 or 2, which contains one or more compounds selected from the group consisting of the compound of formula I and salts thereof in a concentration of 0.5 to 100 ppm. 6. The growth promoting agent according to claim 1, wherein n is 9, 10 or 12. 7. X is L-HyPro, n is 10, and R
Growth promoter according to claim 1 or 2 but is -CH (CH _{3) 2.}