JPS6287522A - Anticoccidial agent and feed efficiency improver - Google Patents

Abstract

PURPOSE:The titled agent, containing an antibiotic substance 6270 obtained from a microorganism of the genus Nocardiopsis or a salt thereof as an active constituent, effective for coccidiosis of domestic fowls and pigs and capable of promoting growth of domestic fowls and improving the utilization efficiency of feed. CONSTITUTION:An anticoccidial agent of feed efficiency improver containing an antibiotic substance 6270 [having the following physico-chemical properties; Property; Colorless needlelike crystal. Acidic substance. Melting point; 115-118 deg.C. Elementary analysis (%); C, 62.72; H, 9.49; O, 27.78. Specific rotatory power [alpha]<20>D=-11.5 (C 1.0, methanol). Solubility; Soluble in methanol, acetone, etc., and insoluble in water, etc.] or a salt thereof as an active constituent. The antibiotic substance 6270 is capable of being an excellent agent having anticoccidial activity and feed efficiency improving action in a very low concentration.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an anti-coccidiosis agent and a feed efficiency improving agent.

The present inventors tested the anti-coccidiosis and feed efficiency-improving activities of a number of antibiotics with the aim of developing new anti-coccidiosis agents and feed efficiency-improving activities, and found that they showed activity at very low concentrations. The present invention has been achieved by discovering a novel anti-coccidiosis agent and feed efficiency improving agent.

Coccidiosis is a disease caused by infection with a protozoan of the Eimeria genus, and is an infectious disease that causes diarrhea and malnutrition in poultry, eventually leading to death.

Oocysts, one generation of this protozoan, are excreted with poultry feces, form sporulations, and are transmitted one after another. The economic value of these infected items is greatly reduced.

Preventing coccidiosis is therefore of great importance for industry.

For this reason, many preventive treatments have been proposed, but none of them are sufficiently effective, and protozoa that are resistant to these have emerged, so the emergence of new effective drugs is awaited. ing.

Therefore, the present inventors conducted intensive research and development on a drug that is effective against coccidiosis in poultry and pigs, and as a result, they obtained a drug by culturing microorganisms belonging to the genus Nocardiopsis. The inventors have discovered that the antibiotic 6270 substance is effective against coccidiosis in poultry and pigs, and have completed the anti-coccidiosis agent of the present invention.

The active ingredient of the present invention is antibiotic 6270 substance.

This is an antibiotic developed by the present inventors for the first time, and its manufacturing method and physicochemical properties will be explained below.

Antibiotic 6270 substance is produced by culturing microorganisms belonging to the genus Nocardiopsis isolated from soil collected in Abashiri, Hokkaido in an appropriate medium, and the antibiotic, which exhibits high antibacterial activity against Gram-positive bacteria, accumulates in the medium. After examining its physicochemical and biological properties, it was found to be a new antibiotic.

This was named antibiotic 6270 substance (patent application 1983-
72912).

As the microorganisms used in the production of the antibiotic 6270 substance, any microorganism can be used as long as it has the ability to produce the antibiotic 6270 substance, but for example, Nocardiopsyst sp. 62 newly isolated by the present inventors.
Strain No. 70 (Accession Number: Fiber Science and Technology Research Institute No. 7569) is preferred 6 In carrying out the present invention, antibiotics! ff6
270 substance-producing bacteria can be cultured by a known release mi'i method, but from an industrial perspective, continuous λ stirring culture is preferred. As production medium, the usual raw materials used for culturing actinomycetes are used, namely various carbon sources,
A nitrogen source, organic or inorganic salts, and optionally an antifoaming agent can be used in appropriate combinations. Generally, carbon sources include glucose, MJ powder, glycerin, dextrin, sucrose, animal and vegetable oils, and nitrogen sources include soybean flour, corn steep liquor, wheat germ, ammonia, and the like. Calcium carbonate, sodium chloride, potassium chloride as needed.

Inorganic salts such as phosphates may be added, and organic or inorganic salts having the effect of aiding the growth of bacteria and promoting the production of antibiotic 6270 substances may also be added as appropriate.

The culture temperature is generally in the range of 25 to 35°C, preferably around 30°C. The culture time varies depending on various conditions, but the amount of antibiotic FF6270 substance produced and accumulated usually reaches its maximum in 72 to 148 hours.

In order to isolate and produce the antibiotic 6270 substance from the culture, the physical and chemical properties of this substance can be utilized, and known means 1, such as means that utilize the difference in solubility with impurities, ion exchange resins, or various adsorption methods. It can be carried out by appropriately selecting and combining means such as means utilizing the difference in adsorption power to the agent, extraction means using an organic solvent immiscible with water, precipitation, impurity removal 9 dialysis, drying, recrystallization, etc.

The antibiotic 6270 substance produced in the culture is preferably collected, for example, as follows.

Add filter aid 1 to the culture solution, such as diatomaceous earth or Radiolite 700.
The culture solution is filtered, and the filtrate and bacterial cells are extracted with appropriate solvents such as ethyl acetate, acetone, etc., respectively. Next, the bacterial cell extract and the filtrate extract are combined, the solvent is distilled off from this extract, and the mixture is subjected to, for example, chromatography in order to isolate the antibiotic 6270 substance from the crude crystals of the antibiotic 6270 substance. After concentrating the eluate under reduced pressure, the residue is dissolved in a suitable solvent such as ethyl acetate, treated with dilute hydrochloric acid solution, and the solvent layer is concentrated to crystallize the antibiotic 6270 substance as a free acid.

To obtain the sodium salt, the solvent layer treated with the dilute hydrochloric acid solution is then treated with the dilute sodium carbonate solution and the solvent layer is concentrated to obtain the antibiotic 627° substance as the lithium salt. The free acid type and sodium salt type antibiotic 6270 substance obtained here can be obtained as pure crystals by recrystallization with a suitable solvent such as n-hexane-ethyl acetate. The properties of the free acid type antibiotic substance 6270 thus obtained are as follows.

(1) Colorless needle-like crystals, acidic substances (2) Melting point: 115-118℃ (3) Elemental analysis value (actual value %) C62, 72%, 89.49%.

027.78ㅅ.

(4) Specific optical rotation [α] = -11,5° (C1, O,
Methanol) (5) Ultraviolet absorption spectrum: When measured in a methanol solution, no absorption maximum is shown above 210 nm.

(6) Infrared absorption spectrum (measured with potassium bromide tablets)
Characteristic absorption (am--1): 3470.2980
, 2945, 2890.1735, 1460. 13
80. 1315. 1165°1102, 1075
．． 987. 980(7) Solubility: methanol, ethanol, ethyl acetate, chloroform,
Soluble in ether, acetone, benzene, etc., insoluble in water.

(8) Color reaction: Vanillin-sulfuric acid reaction, Dragendorff reaction positive.

Negative for ninhydrin reaction. ■Colored with 2 gases.

(9) Thin layer chromatography (Merck Key (10)
)' H-NMR spectrum: Measured at 100 M H7 in deuterated chloroform using TMS as an internal standard, δppm 3.39 (3H), 3.49 (6H)
The presence of three methoxyl groups is observed in .

(11) The antibacterial spectrum is shown in Table 1.

kanamycin, gloramphenicol,
: Tetracycline natural/soil) 1 Klebsiella Eumonie PCI602
>100 a In the table, the symbol a indicates a nutrient agar medium, b indicates a glycerin nutrient agar medium, and C indicates a potato-sucrose agar medium.

Based on the above physicochemical and biological properties, this substance belongs to the polyether group of antibiotics.

From the 1111-1-N spectrum 1-, this substance has 3 in the molecule.
However, among the borie-thyl group antibiotics, known antibiotics that have three meth-xyl groups in their molecules ('2O -12
(Japanese Unexamined Patent Publication No. 53919), A-20413 (Hani 8 Nodob Tsuku of Microbiology 41)
0. Vat, 3. CRC Press), T-42
082 (JP 51-79730), T-40517
(JP-A-50-10581), 38295 (JP-A-51-12579.3), No. 6016 (JP-A-51-12579.3)
4-8457 Rocie).

X-14868C (Japanese Unexamined Patent Publication No. 56-120696), C
P-47433 (JP-A-57-154108)-47
434, (Unexamined Japanese Patent Publication No. 154187), LL-
C23024β (JP-A-58-78598) is mentioned.

However, substance 6270 differs from the above-mentioned known substances in properties such as melting point, specific optical rotation, five-element analysis value, and infrared absorption spectrum, and is a new antibiotic.

Furthermore, the present invention relates to feed efficiency improvers.

Traditionally, antibiotics have been widely added to feed to promote growth and increase egg production in livestock and poultry. There are concerns that this may have a negative impact on human medicine, and there is also the risk of unfavorable effects from ingesting the antibiotics that remain and accumulate in the animal body when livestock products are used for human consumption. This is the actual situation.

Furthermore, as a result of intensive research into feed efficiency modifiers that do not have the drawbacks mentioned above, the present inventors found that the antibiotic 6270 substance promotes I'F production in livestock and poultry, and improves feed utilization efficiency. We have now completed the present invention by demonstrating that it can be an excellent drug that can cause cancer.

, / 7・-' , // 7・-' Reference Example 1 (Manufacturing method) A composition of 6.0% glucose, 2.0% soybean flour, 1.0% to lula yeast, and 0°5% calcium carbonate. Nocardiopsis varnish in medium (pH 6,0) LQ with
P strain No. 6270 (Accession number: Microtechnical Research Institute No. 7569)
No.) and cultured at 30°C for 48 hours. This culture solution was inoculated into medium 100Q having the same composition as above, and it was heated to 30℃ for 90 minutes.
Culture with aeration in a 200Q capacity tank for 6 hours. The ventilation song is 100 Q/min, and the bell rotation speed is 25 Orpm.

Add a filter aid (trade name Niradiolite 700) to this culture solution.
was added and filtered, and the filtrate and bacterial cells were added to 9 tubes.

The filtrate is then extracted with ethyl acetate 40Q, and the bacterial cells are extracted with acetone 30Q. The acetone extract of the bacterial cells is concentrated under reduced pressure to remove acetone, and then extracted with 20Q ethyl acetate. This extract and the extract from the filtrate were combined, concentrated under reduced pressure, and the residue was converted into silica gel (trade name: Wakogel).
750 g of Ni 200) was adsorbed on a column packed with chloroform, and 750 g of chloroform 11-methanol (100:1
). The active fraction is concentrated to dryness under pressure to obtain an oily residue. Next, this was dissolved in as much acetone as possible, absorbed into a Sephadex L [(-20) column packed with acetone, developed and eluted with acetone, and the obtained active fraction was concentrated. Crude crystals of Substance 6270 are obtained. This is dissolved in ethyl acetate and shaken with dilute hydrochloric acid, and the ethyl acetate layer is concentrated under reduced pressure. The resulting crystals are recrystallized from n-hexane-ethyl acetate. 23.4 g of the free acid of Substance 6270 is obtained as colorless needle crystals.The free acid of Antibiotic 6270 Substance thus obtained exhibits the above-mentioned physicochemical and biological properties.

The present invention will be illustrated below by examples and test examples, but the present invention is not limited thereto.

Example 1 Preparation 6270 substance 1% Corn starch 99% Both substances are ground and mixed uniformly to make a premix containing 1% 6270 substance 6 Test Example 1 Anti-coccidial effect test agent and feed: Antibiotic 6270 The substance was mixed evenly with Oriental's Complete Compound Feed for Young Chicks at the test concentration, and the administration was continued from just before the second inoculation to Osis 1 until Test R"'(' B (8 days after infection). .The animals were fed ad libitum.

The comparative drug is an anti-coccidial drug! Monensin, known as -2, was used.

Test chicks: The test chicks are male chicks from the egg-laying breed Shaverstar-cross, and are completely free from coccidia infection. -, 7-day-old chickens (9 days old at the time of infection) that had been kept in captivity were used for each group of 5 birds.

Inoculation osis 1- and inoculation amount: A susceptible strain of Eimeria tenella isolated and identified at the Livestock Hygiene Laboratory of the Ministry of Agriculture, Forestry and Fisheries was used. The amount of inoculation was 3 x 10' mature oocysts per chick, which were inoculated intravenously with a metal probe.

Determination of effectiveness: The determination of effectiveness is the relative increase rate of chicks.

The anticoccidial index (A, C, 1,) was calculated from the survival rate, oocyst value, and intestinal lesion value, and judgment was made based on the index value.

Anticoccidial index (A, C, 1,) = (relative gain rate + survival rate) - (oocyst value + lesion value) 120 or less is inappropriate as an anticoccidial agent.

Below 160, use as an anti-coccidiosis agent. Less effective.

160-180 are anti-coccidial agents and moderately effective.

180 or higher is used as an anti-coccidiosis agent Extremely effective.

a) Body weight measurement The weight is determined by the amount of salary increase for each group at the end of the experiment (body weight at the end of the experiment -
The weight at the time of infection) was determined, and the relative weight gain rate was determined, setting the uninfected, unmedicated control group as 100.

b) Oocyst value On day 8 [1] after infection, oocysts in the caecum were counted by intestinal homocyne-1, and the oocyst-1 value was determined as follows.

[Method using intestinal homogenate]

Number of oocysts in the intestinal tract Oosis 1 to value 0.0 to 0
．． lX10' 0 0.1~1.0X10' 1 2.0~s, oxto' i.

6.0~to, 0XIOG 20 >11.0XIO' 40 C) Intestinal lesion degree Chicks were killed at the end of the experiment (8 days post-infection).

The intestinal tract was examined macroscopically to determine the extent of the lesion. In addition, the lesion degree was determined as follows.

(-)0: Cecum is completely normal. If there is a bleeding spot, score it as 10.

(+) 1: The shape of the cecum is normal. The contents are slightly fluid and yellowish in color. The cecum is partially slightly swollen and whitish.

(++)2: The shape of the cecum is almost normal. Swelling of the mucosa can be seen all over the area. There is no bleeding in the contents, and the mucus is yellowish and discolored. A small number of white dots of necrosis and hemorrhagic spots are seen within the mucosa.

(+++) 3: The atrophy and deformation of the cecum are clear and it becomes slightly longer than the rectum. It has no normal contents and is often filled with blood clots or off-white cheese-like degeneration. The cecal wall becomes noticeably thickened and brittle, and petechiae may still remain.

The lesion extends to the base of the cecum but does not extend into the cecum.

(++++) 4: Atrophy and deformation of the cecum are significant. Generally sausage-shaped, its length is the same as or shorter than the rectum. The lesion extends to the 173rd to 174th positions of the rectum.

Groups 1 to 3 were fed with feed containing a predetermined concentration of antibiotic 6270 substance from 2 days before inoculation with oocysts, group 4 was fed with feed containing the comparative antibiotic monensin, and group 5 was fed with feed containing oocysts. The 6th group is an infection-free, non-medication control group reared on a basal diet that does not contain inoculation or the test drug.

This test result shows that the antibiotic 6270 substance has an excellent anti-coccidial effect.

/'/'/゛' Test Example 2 Feed efficiency test for pigs Test pigs: Landrace basic feed: a) 4 weeks from the start of the test 60% grains (corn, wheat, barley), 1 soybean oil meal
5%, animal feed (skimmed milk powder, fish meal) 15%, other (enzymes, calcium carbonate, calcium phosphate, salt, etc.) 10% b) 4-12 weeks Cereals (corn, milo, wheat) 78%, soybean oil cake 13 %, fishmeal 5%, others (calcium carbonate, calcium phosphate, salt, etc.) 4% Method: 15 piglets with an average age of 35 days were divided into 3 groups of 5 piglets each so that the average weight was approximately equal, and fed as basic feed. antibiotic 62
70 substances were added at a rate of 0.12.5.25 ppm,
The mice were fed for 12 weeks, and their body weight and feed intake were measured.

In addition, the total number of days that pigs developed diarrhea during the 4-week period from the start of the test was investigated. The results are shown in Table 3.

By feeding this antibiotic 6270 substance-added feed, the feed conversion rate will improve by 5-8%, and the amount of pay will increase by 5-11%.
Two were recognized. It was also observed that the incidence of diarrhea in pigs was significantly reduced.

Test Example 3 Feed efficiency test collection for ruminants: Holstein concentrate feed: Cereals (corn, milo, barley) 71.
5%, corn gluten feet, bran,
Rice bran oil cake) 11.5%, vegetable rice cakes (large σ oil cake, linseed oil cake) 5.5%, others (alfalfa meal, molasses, calcium phosphate, salt) 11.5% Method: 8 month old Fifteen male steers were divided into three groups of five animals each so that their average body weights were approximately equal, and the antibiotic 6270 substance was added to the basal concentrate feed at a rate of 17, 5, and 15 ppm of O, and fed for 240 days. In addition, 1 kg of rice straw was fed to each animal as roughage. The results are shown in Table 4.

By feeding this antibiotic 6270 substance-added feed, the conversion rate of concentrated feed was improved by 7 to 10%, and the amount of pay increased by 9 to 11%.
% improvement was observed.

Claims (1)

[Scope of Claims] 1) An anticoccidiosis agent containing as an active ingredient an antibiotic 6270 substance or a salt thereof having the following physicochemical properties. (1) Colorless needle-like crystals, acidic substance. (2) Melting point 115-118°C (3) Elemental analysis value (actual value %) C 62.72%, H 9.49%, O 27.78% (4) Specific optical rotation [α]＾2＾0_D= -11.5° (
c1.0, methanol) (5) Ultraviolet absorption spectrum: When measured in a methanol solution, no absorption maximum is shown above 210 nm. (6) Infrared absorption spectrum (measured with potassium bromide tablets)
Characteristic absorption (cm^-^1): 3470, 298
0, 2945, 2890, 1735, 1460, 138
0, 1315, 1165, 1102, 1075, 987
, 980 (7) Solubility: methanol, ethanol, ethyl acetate, chloroform,
Soluble in ether, acetone, benzene, etc., insoluble in water. (8) Color reaction: Vanillin-sulfuric acid reaction, Dragendorff reaction positive. Negative for ninhydrin reaction. Colored with I_2 gas. (9) Thin layer chromatography (using Kieselgel GF_2_5_4 manufactured by Merck & Co.): Solvent system Rf value Chloroform-methanol (20:1) 0.49 n-hexane-ethyl acetate (1:1) 0.08 Benzene-ethyl acetate ( 1:1) 0.04 Benzene-acetone (1:1) 0.53 Chloroform-acetone (2:1) 0.36 n-hexane-ethyl acetate (1:3) 0.13 Ethyl acetate 0.22 (10 )^1H-NMR spectrum: Measured at 100 MHz in deuterated chloroform using TMS as an internal standard.
The presence of three methoxyl groups is observed at δppm 3.39 (3H) and 3.49 (6H). 2) A feed efficiency improving agent containing antibiotic 6270 substance or its salt as an active ingredient.