DK141475B - Process for the preparation of an animal feed additive containing an antibiotic AV290 synthane complex. - Google Patents

Description

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The present invention relates to a process for the preparation of a dry animal feed additive containing solids from culture media and an antibiotic AV290 synthane complex. "Syntane" is a synthetic tanning agent, cf. below.

5 Antibiotic AV290 is produced by fermentative biosynthesis when grown under controlled conditions of Streptomyces canidus NRRL 3218 and mutants thereof. The preparation and properties of antibiotic AV290 are described in U.S. Patent No. 3,338,786. There have been major difficulties in extracting the antibiotic economically. According to said patent, adsorption is used on charcoal followed by elution and column chromatography. Such a procedure is not terribly expensive when pure antibiotic is required for medical use.

15 However, when the antibiotic is to be used in animal feed additives, the cost factor becomes very important and there is therefore a need for an inexpensive method of extracting the antibiotic for this purpose.

The process of the invention is characterized in that a) a total culture medium containing antibiotic AV290 is acidified to a pH of 1.4-5.0, b) a synthane is added to the acidified culture medium in an amount sufficient c) removing the solids from the culture medium together with the precipitated antibiotic AV290 synthane complex, and d) drying the mixture of solids from the culture medium and antibiotic AV290 synthane complex.

The process of the present invention practically causes complete removal of the antibiotic activity from the culture medium or liquid. Furthermore, the antibiotic-synthane complexes thus prepared can be used without isolating the constituents of animal feed additives, which is an important economic advantage.

The synthetic tanning agents used in the present process are condensation products of naphthalene sulfonic acids or various sulfonated phenols with formaldehyde. Almost all of these materials are available in the form of polymers, e.g. polymers having 2,3,4,5 and 6 monomer units. Most of these synthetic tanning agents are not purely isolated chemical compounds, but often mixtures. They are usually amorphous water-soluble materials available either in concentrated aqueous solution or in powder form, and the color varies from colorless to dark brown. For convenience, these 10 sulfonated synthetic tanning agents are collectively referred to as "synthans" and this term is used hereinafter.

In the present process, naphthalene synthanes produced by condensing naphthalene-1 or -2-sulfonic acid with formaldehyde are preferred. The actual structure has not been determined, most likely the product is a mixture, but the product (from naphthalene-2-sulfonic acid) may be represented by the formula s ° 3h

20 wn <! / —1, SO, H

kJJ- ch2 \ xch2 —uu -I n n is 0-3 or 4 25

In the process of the present invention, aromatic hydroxy synthanes may also be used.

There are two general methods for preparing these synthans: (1) Sulfonation of an aromatic hydroxy compound followed by condensation with formaldehyde, and (2) condensation of an aromatic hydroxy compound with formaldehyde followed by sulfonation to form a water-soluble product. The general type of compound prepared from phenol can be represented by the formula 35 3 UU76

OH OH OH

5 '' T

R R R R = H or SO, H

L J 3 n is 0-3 or 4

In addition to phenol, cresols, naphthols, bis-phenols, and the like, as well as mixtures of formed products, may be used as starting materials, depending on the condensation and sulfonation conditions. Another type of aromatic hydroxy synthanes which can be used in the process of this invention has the sulfonic acid group attached to the methylene group. These synthans are prepared by reacting a phenol (phenol, cresols, naphthols, bis-phenols, etc.) with formaldehyde and sodium bisulfite or sulfur dioxide.

With regard to the above-mentioned synthans, reference may be made to the groundbreaking article by Stiasny entitled "Syntans, New Artificial Tanning Materials", in J. Soc.

Chem. Ind., 1913, page 775, and to the article by Turley in J. Am. Leather Chem. Assoc. 40, 58 (1945). Furthermore, processes for producing synthanes are described in e.g. German Patent Nos. 262,558, 266,139, 280,233, 25,291,457, 409,984, 433,292, 493,795, 537,451 and 539,474.

Other suitable synthans which may be used in the present invention are listed below:

Trademark Manufacturer% Bioactivity "Suprak 57" American Cyanamid Co. 41 30 "Tru-tan R T N" A.J. & J.O. Pilar Co. 48 "Tru-tan RT" A.J. & J.O. Pilar Co. 28 "Tru-tan PA 65" A.J. & J.O. Pilar Co. 50 "Orotan TV" Rohm & Haas Co. 21 "Orotan WM" Rohm & Haas Co. 33 35 "Leukanol" Rohm & Haas Co. 68 "Sulframine 1298" WITCO Chemical Co. <46% 4 141475

The products of the antibiotic and synthans have been referred to as reversible antibiotic-synthane complexes.

Their exact chemical nature has not been determined, but covalent bonding is not included and the product is not a physical mixture. The chemical bonds are reversible, as antibiotic AV290 can be recovered from the complexes by adsorption on a column of cross-linked carboxymethyldextrangel followed by elution with aqueous acid.

In the process of the invention, the pH of the total culture medium is adjusted to a value in the range 1.4-5.0 with dilute aqueous acid. Suitable acids for this purpose are dilute hydrochloric acid, dilute sulfuric acid, dilute trifluoroacetic acid and the like, although even glacial acetic acid can be used. Then an aqueous solution of a synthane or mixtures of synthanes is slowly added with stirring at ambient temperature. The antibiotic and synthans form a complex that is water-insoluble and therefore precipitates. The precipitated synthane complex together with the solids from the culture medium is then removed by filtration or centrifugation and dried. The products thus obtained can be dried by (1) slurrying the moist solids into polar water-miscible solvents such as acetone, followed by filtration, rinsing and air drying, or by (2) resuspending the moist solids in water and freeze drying or spray drying.

Thus, when the products of the present invention are carefully dried under temperature conditions that do not degrade antibiotic AV290, they are usually obtained in the form of gray to brown powders or solids. In dry form, these products are extremely stable and can be stored without appreciable loss of antibiotic activity for extended periods of time. This long shelf life is of course an important practical advantage.

It is an advantage of the present invention that the amount of synthane added to precipitate the complexes with the antibiotic is not critical and that no exact stoichiometric conditions should be followed. In general, the amount of synthane used will be somewhat more than the minimum amount required to form the complex with the anti-biotic. Excess of synthane will simply remain in solution by filtration. However, the amount of synthane required is directly proportional to the concentration of the antibody in the culture medium or liquid and varies with the nature of the synthane used. The specific bioactivity of the precipitated complex also varies with the nature of the synthane used. Thus, it is indeed evident that the complexes contain varying relative amounts of antibiotic, and of course, it is quite likely that these are mixtures of complexes, since the syntheses used are not pure single chemical compounds.

The smallest amount of synthane required to form the complex with the antibiotic in any single culture medium can be readily determined as follows. A sample (conveniently 50-100 ml) of the entire culture medium is taken, which is cleared by removing the mycelium and other insoluble matter by filtration, preferably using a filter aid. The filtrate is then acidified to a pH of 1.8-2.0 with dilute aqueous mineral acids such as dilute hydrochloric acid, dilute sulfuric acid, dilute phosphoric acid or the like. This solution is then titrated with the special aqueous synthane solution to be used until no precipitation or turbidity is formed. The amount of synthane solution for the culture medium is then calculated from the titre of the sample taken, providing a small excess.

30 In recent years, the use of antibiotics in animal feed to increase growth characteristics and feed utilization has become of great economic importance. The dried solids from the culture medium containing the synthane complexes, either alone or in combination with suitable carriers, increase the rate of growth upon addition to animal feed. Furthermore, feed utilization is increased. They have the advantage that the growth rate of non-ruminants such as poultry and pigs, especially piglets taken early from the sow, is greatly increased and the feed conversion rate is greatly increased.

The present feed additive is administered in an amount sufficient to provide approximately the following dose levels in mg per milligram. individual per day:

Large ruminants 350

Small ruminants 200 10 Non-ruminants 100

Poultry 2

The number of mg of antibiotic AV290 per The weight unit of any feed additive of the invention can be readily determined by the biological specimen disclosed in United States Patent No. 3,338,786. From the data thus obtained, one can easily calculate the amount of feed supplement to be used per day. tons of feed.

Many different carriers can be used in the preparation of the feed additive containing the dried solids from the culture medium containing the synthane complexes. Suitable carriers for use in the feed additives are e.g. soybean meal, alfalfa flour, cotton seed oil, linseed oil, corn flour, cane sugar molasses, urea, bone meal and corn cob flour. The carrier promotes a uniform distribution of the complexes of the finished feed to which the additive is mixed. It thus has an important function in ensuring proper distribution of the complexes in the feed.

The process of the invention is explained in more detail below, with Examples 1-2 illustrating the preparation of synthans.

Example 1

Preparation of a Sulfonated Phenol-Formaldehyde Condensate-Synthane 35 A mixture of 2 moles of phenolic sulfonic acid and 1 mole of aldehyde, in 37.5% aqueous solution, is heated in a sealed bottle on a steam bath for 1 hour. A dark colored solution is obtained in which, after cooling overnight, a small amount of an amorphous white solid is deposited. This is easily dissolved by adding a small amount of cold water. After partial neutralization, the solution can be used for tanning.

Syntans of this general type are marketed by A.J. and J.O. Pilar Co. under the trademark "Trutan S-55" and "Trutan R108", cf. Encyclopedia of Chemical Technology, Krik-Othmer, Interscience Publishers (195.4), Volume 10 13, page 594.

Example 2

Preparation of a sulfonated naphthalene-formaldehyde condensate synthane 20 g of naphthalene is sulfonated with 10 ml of 93% hydrochloric acid for 2 hours keeping the temperature at ca. 105 ° C.

The mixture is then cooled to ca. At 35 ° C and stirring 5 ml of 37.5% aqueous forraldehyde solution. The product is then dissolved in water to a concentration of approx. 35% and the acidity is reduced to approx. 1/10 N by the addition of alkali.

20 Syntans of this general type are sold by A.J. and P.O. Pilar Co. under the trademark "Trutan OA" and by Rohm & Haas Co. under the trademark "Tamol N" ®, cf. Encyclopedia of Chemical Technology, Kirk-Othmer, Inter-science Publishers (1954), Volume 13, page 594.

Example 3

Preparation of antibiotic AV290- "Trutan RT Regular" -_ complex_ 14 liters of AV290 culture liquid containing 722 mcg AV290 antibiotic per ml is adjusted to pH 30 with sulfuric acid and filtered using diatomaceous earth. A 100 ml portion of equal parts "Trutan RT Regular" (a phenol-formaldehyde bisulfite condensate from A.J. and J.O. Pilar Co.) and water are added to 10 liters of the filtrate with stirring. The precipitate is allowed to settle, 35 and the supernatant is decanted. The precipitate is isolated by centrifugation, washed twice with 250 ml of water, suspended for approx. 1 liter of water and freeze-dried. 30.5 g of dried product are obtained which, upon testing, show an antibiotic activity of 28.3%.

Example 4

Preparation of Antibiotic AV290- "Trutan RT Regular" Complex Based on Total Culture Medium 3. Liter of AV290 Culture Medium Containing 2000 mcg of Antibiotic 290 Per ml is adjusted to pH 1.9 with sulfuric acid. To the stirred acidified culture medium is added 15 ml of "Trutane RT Regular". The mixture is centrifuged and the 10 solids are washed twice with 350 ml of water by slurry, centrifugation and decanting. The antibiotic activity remaining in the above culture broth is precipitated with an additional 20.8 ml of "Trutane RT Regular". The washed solids from the last step are added to the washed solids from the initial step, suspended in 1000 ml of water and lyophilized. 77 g of dried product is obtained, which exhibits an antibiotic activity of 6.3% in biological testing.

Toxicity study: 20 The product is tested for toxicity as follows: The product is administered to female mice (Carworth Farms CF-1) weighing 18-20 g. The product is orally administered in 0.2% agar at a dose of 1024 mg per day. . kg body weight. All mice survived a 14-day trial.

Example 5

Preparation of Antibiotic AV290- "Trutan RT Regular" _ Complex from Total Culture Medium_ 37.5 liters of culture fluid containing 3660 mcg of antibiotic AV290 per ml is adjusted to pH 2 with 30 sulfuric acid. To the acidified culture medium is added, with stirring, 1312.5 ml of "Trutane RT Regular" which has been diluted with 1312.5 ml of water. The mixture is stored at 4 ° C overnight, then filtered using 800 g of diatomaceous earth. After washing the filter cake with 35 liters of water, the moist filter mass is dried in vacuo at approx. 40 ° C. 1439 g of dried product is obtained which exhibits an antibiotic activity of 9%.

UU75 9

Example 6

Preparation of Antibiotic AV290- "Trutan RT Regular" Complex from Total Culture Medium 1500ml Culture Medium Containing 1940 mcg of Antibiotic AV290 Per ml is adjusted to pH 2 with sulfuric acid until the acidified culture medium is added with stirring 52.5 liters of "Trutane RT Regular" diluted with 52.5 liters of water. The mixture is filtered using 45 kg of diatomaceous earth as filter aid, and the filter cake is washed on the filter press with water. The moist filter cake is dried in vacuo at ca. 55 ° C. 93 kg of dried product is obtained, showing an antibiotic activity of 3.15%.

Example 7 Growth-promoting effect of antibiotic AV290- "Trutane RT Regular" complex in admixture with solids from total culture medium_

The cultivation of the feed additive prepared according to Example 4 is determined as follows: 20 day-old chicks are purchased in the specified number. The chickens are placed in a heated chicken mat and kept in a chill room at a temperature of approx. 24 ° C. All groups of chickens are weighed at the start of the trial and at the end of the trial after 28 days. The chickens have free access to feed 25 and water. In all the experiments, the following basic feed is used.

Basal feed

Ingredient g / kg

Ground Yellow Corn 514 30 Soybean Oil (44 #) 500

Corn gluten flour 50

Herring flour (60 #) 50

Fat 40 Dried alfalfa flour (17 #) 20 35 Ground lime 5

Dicalcium Phosphate 12

Sodium chloride 5 ir

Trace minerals 1

Vitamin premix ** 5 UU75 ίο χ Trace minerals - many 6%, iodine 0.12%, iron 2.0%, copper 0.2%, Cobalt 0.02%, zinc 2.0% and calcium 25.5% xx Vitamin premix - pr. kg of feed contains 5 125 mg of butylated hydroxytoluene, 500 mg of DL methionine, 3300 I.E. vitamin A, 1100 I.E. vitamin D 2, 2.2 I.E. vitamin E, 11 mcg vitamin B 2 / 4.4 mg riboflavin, 27.5 mg niacin, 8.8 mg pantothenic acid, 500 mg choline chloride, 1.43 mg folic acid and 1.1 mg menadione sodium bisulfite for 5 g ground yellow corn.

The test results are given in the following Table I.

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Example 8

Preparation of antibiotic AV290 complex from total culture medium 5 ml portions of the total culture medium from 5 a culture of AV290 containing 1000 micrograms of antibiotic AV290 is adjusted to pH 2.0 with sulfuric acid. The amounts of each precipitant listed below are then added and the complexes are centrifuged. The above liquids are tested by the paper plate diffusion plate method. The agar is seeded with Corynebacterium zerosis NRRL-B-1397.

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