Antibacterial microelement chelates and the use thereof in animal feeds

Abstract

The present invention relates to a microelement organic O-chelate or N-chelate complex compound, for the inhibition of facultative pathogenic bacteria. The present invention further relates to a composition, feed additive or feed comprising the compounds, as well as methods for the preparation thereof, and for the use thereof in animal stock farming.

Claims

1. A method for the inhibition of pathogenic bacteria proliferating in the small intestine part of the digestive tract and causing diseases therein or on the surface of the skin, and/or for the treatment of a disease caused by said bacteria, said method comprising administering to an animal selected from the group consisting of poultry, swine and bovine, an effective amount of the compound or a composition comprising the compound of general formula
(M).sub.n(X).sub.m(Y).sub.o wherein M is selected from Zn, Cu, Fe, Mn or Ag; X is NH.sub.3 or H.sub.2O; Y is an amino acid, a fatty acid, a hydroxy acid and/or a polyamino-carboxylic acid; n is 1-6; m is 1-6; o is 1-8 wherein the compound is selected form the group consisting of: copper (H.sub.2O) bis-glycinate, copper (H.sub.2O).sub.2 bis-glycinate, Cu (H.sub.2O) bis-malate, copper (H.sub.2O).sub.2 ethylenediamine tetraacetic acid, zinc (H.sub.2O) bis-glycinate, zinc (H.sub.2O).sub.2 ethylenediamine tetraacetic acid, zinc (H.sub.2O).sub.2 malate, Zn(H.sub.2O) bis-alaninate, Zn(H.sub.2O) bis-aspartate, Zn(H.sub.2O) bis-butyrate, Zn(H.sub.2O) bis-glutamate, Zn(H.sub.2O) bis-propionate, Zn(H.sub.2O) bis-valerianate, Zn(H.sub.2O) bis-malate, copper (NH.sub.3).sub.2 bis-glycinate, copper (NH.sub.3).sub.2 ethylenediamine tetraacetic acid, copper (NH.sub.3).sub.2 bis-malate, Cu (NH.sub.3).sub.2 asparaginate, Cu (NH.sub.3).sub.2 bis-asparaginate, Cu (NH.sub.3).sub.2 bis-glutamate, Cu (NH.sub.3).sub.2 glutamate, zinc (NH.sub.3).sub.2 bis-malate, zinc (NH.sub.3).sub.2 ethylenediamine tetraacetic acid, zinc (NH.sub.3).sub.2 malate, zinc (NH.sub.3).sub.2 methionate, zinc (NH.sub.3).sub.4 bis-glycinate, zinc (NH.sub.3).sub.4 malate, Zn (NH.sub.3).sub.2 asparaginate, Zn (NH.sub.3).sub.2 aspartate, Zn (NH.sub.3).sub.2 bis-asparaginate, Zn (NH.sub.3).sub.2 bis-aspartate, Zn (NH.sub.3).sub.2 bis-glutamate, Zn (NH.sub.3).sub.2 bis-glycinate, Zn (NH.sub.3).sub.2 bis-histidinate, Zn (NH.sub.3).sub.2 citrate, and Zn (NH.sub.3).sub.2 glutamate, wherein the disease is selected from the group consisting of: poultry enteric diseases, swine enteric diseases, bovine enteric diseases, as well as superficial treatments, mastitis of dairy cattle, metritis of dairy cattle, hoof lesions of ungulates, enteric and superficial diseases of other animals.

2. The method according to claim 1, wherein the pathogenic bacterium is selected from the group consisting of: Salmonella enterica, Salmonella enterica subp. Enterica serovar enteritidis, Salmonella typhimurium, Salmonella infantis, Salmonella gallinarium, S. paratyphi, S. abortus-equi, S. java, S. cholerae, S. typhi-suis, S. sendai, Escherichia coli, Clostridium perfringens, Cl. barati, Cl. sordellii, Cl. botulinum A-F, Cl. novyy A, B, C, D, Cl. septicum, Cl. chuvoei, Cl. hystoliticum, Cl. sporogenes, Cl. tetani, Brachyspira hyodysenteriaea, Brachyspira pilosicoli Arcanobacterium piogenes, Staphylococcus aureus, Streptococcus agalactiae, Lawsonia intracellularis.

3. The method of claim 1 wherein the disease is selected from the group consisting of: poultry enteric diseases, swine enteric diseases, bovine enteric diseases, as well as superficial treatments, mastitis of dairy cattle, metritis of dairy cattle, hoof lesions of ungulates, enteric and superficial diseases of other animals.

4. The method of claim 1 wherein the compound is selected from the group consisting of: copper (NH.sub.3).sub.2 bis-malate, zinc (NH.sub.3).sub.2 ethylenediamine tetraacetic acid, zinc (H.sub.2O).sub.2 ethylenediamine tetraacetic acid, zinc (H.sub.2O).sub.2 malate, copper (NH.sub.3).sub.2 ethylenediamine tetraacetic acid, copper (H.sub.2O).sub.2 ethylenediamine tetraacetic acid, copper (H.sub.2O).sub.2 bis-glycinate and copper (NH.sub.3).sub.2 bis-glycinate.

5. The method of claim 1 comprising administering to the animal a composition comprising the compound together with standards additives.

6. The method of claim 1 comprising administering to the animal a composition comprising at least two, synergistically acting compounds of the general formula, optionally together with standards additives.

7. The method of claim 1, said method comprising mixing the compound or composition or a feed additive comprising the compound into standard feed.

8. The method of claim 1, wherein the compound is selected from the group consisting of: zinc tetraammine bis-glycinate chelate, zinc malate chelate, zinc diammine malate chelate, zinc tetraammine malate chelate, zinc diammine methionate chelate, copper diammine lysinate chelate, and zinc diammine aminate chelate.

9. The method according to claim 1, wherein the facultative pathogenic bacterium is selected from the group consisting of: Salmonella, E. coli, Clostridium sp., Brachyspyra sp., Arcanobacterium sp., Staphylococcus sp., Streptococcus sp., Lawsonia sp., Eimerella sp.

10. The method of claim 1, wherein X is complexed to M as an O-chelate or N-chelate ligand.

11. The method of claim 1, wherein X is connected by coordination bond to M.

12. The method of claim 1, wherein the compound is obtained from compound MCO.sub.3 or M(NH.sub.4).sub.2CO.sub.3, respectively.

13. A method for the treatment of a disease selected from the group consisting of poultry enteric diseases, swine enteric diseases, bovine enteric diseases, as well as superficial treatments, mastitis of dairy cattle, metritis of dairy cattle, hoof lesions of ungulates, enteric and superficial diseases of other animals, for the inhibition of bacteria proliferating in the small intestine part of the digestive tract or on the surface of the skin and causing diseases therein, for the treatment of a disease caused by said bacteria, or for increasing body-weight gain and/or increasing feed utilization and/or increasing egg yield and/or decreasing mortality in the population of poultry and/or swine and/or dairy cow, said method comprising administering to an animal selected from the group consisting of poultry, swine and bovine an effective amount of the compound of general formula
(M).sub.n(X).sub.m(Y).sub.o wherein M is Zn, Cu, Fe, Mn or Ag; X is NH.sub.3 or H.sub.2O; Y is an amino acid, a fatty acid, hydroxy acid and/or a polyamino-carboxylic acid; n is 1-6; m is 1-6; o is 1-8 wherein the compound is selected form the group consisting of: copper (H.sub.2O) bis-glycinate, copper (H.sub.2O).sub.2 bis-glycinate, Cu (H.sub.2O) bis-malate, copper (H.sub.2O).sub.2 ethylenediamine tetraacetic acid, zinc (H.sub.2O) bis-glycinate, zinc (H.sub.2O).sub.2 ethylenediamine tetraacetic acid, zinc (H.sub.2O).sub.2 malate, Zn(H.sub.2O) bis-alaninate, Zn(H.sub.2O) bis-aspartate, Zn(H.sub.2O) bis-butyrate, Zn(H.sub.2O) bis-glutamate, Zn(H.sub.2O) bis-propionate, Zn(H.sub.2O) bis-valerianate, Zn(H.sub.2O) bis-malate, copper (NH.sub.3).sub.2 bis-glycinate, copper (NH.sub.3).sub.2 ethylenediamine tetraacetic acid, copper (NH.sub.3).sub.2 bis-malate, Cu (NH.sub.3).sub.2 asparaginate, Cu (NH.sub.3).sub.2 bis-asparaginate, Cu (NH.sub.3).sub.2 bis-glutamate, Cu (NH.sub.3).sub.2 glutamate, zinc (NH.sub.3).sub.2 bis-malate, zinc (NH.sub.3).sub.2 ethylenediamine tetraacetic acid, zinc (NH.sub.3).sub.2 malate, zinc (NH.sub.3).sub.2 methionate, zinc (NH.sub.3).sub.4 bis-glycinate, zinc (NH.sub.3).sub.4 malate, Zn (NH.sub.3).sub.2 asparaginate, Zn (NH.sub.3).sub.2 aspartate, Zn (NH.sub.3).sub.2 bis-asparaginate, Zn (NH.sub.3).sub.2 bis-aspartate, Zn (NH.sub.3).sub.2 bis-glutamate, Zn (NH.sub.3).sub.2 bis-glycinate, Zn (NH.sub.3).sub.2 bis-histidinate, Zn (NH.sub.3).sub.2 citrate, and Zn (NH.sub.3).sub.2 glutamate.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1. IR spectrum of zinc mono-glycinate chelate.

(2) FIG. 2. IR spectrum of copper mono-glycinate chelate.

(3) FIG. 3. Clostridium perfringens forms an inhibition zone on TSA agar medium.

EXAMPLE 1PREPARATION OF ZINC BIS-GLYCINATE CHELATE

(4) To 1 mol ZnO (79.5 g), 200 ml distilled water, 2.1 mol NH.sub.4OH, and 48.5 g CO.sub.2 is added. The reaction product is Zn(NH.sub.4).sub.2CO.sub.3 at 120 C and 10-12 bar pressure, after 4 hours of reaction time. The pH of the obtained chelate compound is set with CO.sub.2 to a value of 8.0. At this time, ZnCO.sub.3 precipitate is formed. The precipitate obtained is filtered, then reacted with 2 mol glycine. This way, the following compound with structure (I) is formed:

(5) ##STR00002##
(M)(Glycine).sub.2, wherein M is Zn or Cu. A preferred example of the compound of formula is Zn(Glycine).sub.2.

(6) The drying step for the preparation of the compound is preferably carried out so as to keep the water content of the microelement chelate compound by retaining the appropriate water content. In the case when the product is dried to a water content of 12-14%, a powder formulation of the compound of formula (I) is obtained. If the product dried to near zero, to about 3% water content, then it loses its water content connected to the central metal atom by dative bond. The biological activity of this latter product is different, it will be significantly lower than that of compound of formula (I). The product is a thick, hygroscopic, viscous compound that has high solubility in water.

EXAMPLE 2PREPARATION OF ZINC DIAMMONIUM BIS-GLYCINATE CHELATE

(7) To 1 mol ZnO (79.5 g), 150 ml distilled water, 2.2 mol NH.sub.4OH, and 48.5 g CO.sub.2 is added. The reaction product is Zn(NH.sub.4).sub.4CO.sub.3 at 120 C and 10-12 bar pressure, after 4 hours of reaction time. The obtained chelate compound is directly reacted with 2 mol glycine. The following compound with structure (II) is obtained:

(8) ##STR00003##
(M)(NH.sub.4).sub.2(glycine).sub.2, wherein M is Zn or Cu. The glycine may be replaced by any amino acid, for example methionine, lysine, aspartic acid, etc. A preferred compound according to formula (II) is Zn(NH.sub.4).sub.2(glycine).sub.2.

EXAMPLE 3PREPARATION OF ZINC TETRAAMMONIUM BIS-GLYCINATE CHELATE

(9) To 1 mol ZnO (79.5 g), 100 ml distilled water, 4.2 mol NH.sub.4OH, and 48.5 g CO.sub.2 is added. The reaction product is Zn(NH.sub.4).sub.4CO.sub.3 at 120 C and 10-12 bar pressure, after 4 hours of reaction time. The obtained chelate compound is directly reacted with 2 mol glycine. The following compound with structure (III) is obtained:

(10) ##STR00004##
(M)(NH.sub.4).sub.4(glycine).sub.2, wherein M is Zn, Cu or Fe. The glycine may be replaced by any amino acid, for example methionine, lysine, aspartic acid, etc. A preferred compound according to formula (III) Zn(NH.sub.4).sub.4(glycine).sub.2.

EXAMPLE 4PREPARATION OF ZINC MALATE CHELATE

(11) To the ZnCO.sub.3 compound prepared as described in Example 1, 2.0 mol malic acid is added in aqueous solution. The following compound with structure (IV) is obtained:

(12) ##STR00005##
Zn malate. The malic acid may be replaced by any hydroxy acid, for example glycolic acid, lactic acid, hydroxy-butyric acid, citric acid, etc. Zinc may be replaced with another trace element, for example copper, etc.
The drying step for the preparation of the compound is preferably carried out so as to keep the water content of the microelement chelate compound by retaining the appropriate water content. In the case when the product is dried to a water content of 10-12%, a powder formulation of the compound of formula (IV) is obtained. If the product dried to near zero, to about 3% water content, then it loses its water content connected to the central metal atom by dative bond. The biological activity of this latter product is different, it will be significantly lower than that of compound of formula (IV).

EXAMPLE 5PREPARATION OF ZINC DIAMMONIUM MALATE CHELATE

(13) To the Zn(NH.sub.4).sub.2CO.sub.3 compound prepared as described in Example 2, 2.0 mol malic acid is added in aqueous solution. The following compound is obtained:

(14) ##STR00006##
Zn diammonium malate. The malic acid may be replaced by any hydroxy acid, for example glycolic acid, lactic acid, hydroxy-butyric acid, citric acid, etc. Zinc may be replaced with another trace element, for example copper, etc.

EXAMPLE 6PREPARATION OF ZINC TETRAAMMONIUM MALATE CHELATE

(15) To the Zn(NH.sub.4).sub.4CO.sub.3 compound prepared as described in example 3, 2.0 mol malic acid added in aqueous solution. The following compound with structure (VI) is obtained:

(16) ##STR00007##
Zn tetraammonium malate. The malic acid may be replaced by any hydroxy acid, for example glycolic acid, lactic acid, hydroxy-butyric acid, citric acid, etc. Zinc may be replaced with another trace element, for example copper, etc.

EXAMPLE 7PREPARATION OF ZINC DIAMMONIUM METHIONATE CHELATE

(17) To the Zn(NH.sub.4).sub.2CO.sub.3 compound prepared as described in Example 2, 2 mol methionine is added in aqueous solution.

(18) ##STR00008##
Zn diammonium methionate.

EXAMPLE 8PREPARATION OF ZINC DIAMMONIUM LYSINATE CHELATE

(19) To 1 mol of the Zn(NH.sub.4).sub.2CO.sub.3 compound prepared as described in Example 2, 2 mol lysine is added.

(20) ##STR00009##
Zn diammonium lysinate.

EXAMPLE 9PREPARATION OF TRACE ELEMENT AMINATE CHELATE

(21) To 1 mol of the ZnCO.sub.3 or CuCO.sub.3 compound prepared as described in Example 1, 2 mol amino acid of choice is added. The compound obtained is Zn(aminate).sub.2 chelate.

(22) In the case when the product is dried to a water content of 12-14%, then Zn(H.sub.2O)(aminate).sub.2 is formed. If the product dried to near zero, about 3% water content, then it loses its water content connected to the central metal atom by dative bond. The biological activity of this latter product is different, it will be significantly lower than that of the compound Zn(H.sub.2O)(aminate).sub.2 compound.

EXAMPLE 10PREPARATION OF TRACE ELEMENT DIAMMONIUM CHELATE

(23) To 1 mol of the Zn(NH.sub.4).sub.2CO.sub.3 compound prepared as described in Example 2, 2 mol amino acid of choice is added. The compound obtained is Zn diammonium aminate chelate.

EXAMPLE 11PREPARATION OF TRACE ELEMENT EDTA CHELATE

(24) To 1 mol of the Zn(CO3) compound prepared as described in Example 1, 1 mol EDTA is added in aqueous solution. After the completion of the reaction, the final product, preferably applied onto a carrier, is dried to 10% water content, to obtain the following O-chelate compound.

(25) ##STR00010##

EXAMPLE 12PREPARATION OF TRACE ELEMENT DIAMMONIUM EDTA CHELATE

(26) One mole of the Zn(NH4)2CO3 compound prepared as described in Example 2 is reacted with 1 mol EDTA, to obtain the following compound. Zinc may be replaced with copper, iron or manganese as chelate forming trace element.

(27) ##STR00011##

EXAMPLE 13PREPARATION OF ZINC MONO-GLYCINATE CHELATE ON A FACTORY SCALE

(28) We performed the preparation of a chelate in 8000 liter reactors and thus produced trace element chelate. To 4000 liter water, 2875 kg zinc sulfate heptahydrate and 750 kg glycine was added. After reacting for 4 hours at 90 C, a zinc mono-glycinate product is formed, that was converted to a micro-granulate in a fluid bed dryer, thus obtained the product.

(29) The formation of chelate bonds in the product obtained were verified by structure determining techniques (complexometric metal ion analysis, calefaction experiments, acid-base titration, recording of central IR spectra) and by recording the far IR spectra of the obtained product. FIG. 1 shows the IR spectrum of the zinc mono-glycinate chelate. Evaluation of the IR spectrum:

(30) 2000-4000 cm.sup.1:

(31) Two distinct bands appear with band maximums of 3160-3211 cm.sup.1 and 3456-3390 cm.sup.1, respectively. In the case of the latter bands, there is a shoulder in the higher wave number range. The previous band can be assigned to the NH valence vibrations of the NH.sub.3.sup.+ group (based on the spectrum of glycine, glycine HCl). The latter band(s) can be assigned to the coordinated water molecules. The presence of the shoulder indicates that there are water molecules bound with varying strengths (e.g. coordinated and non-coordinated, bound by only hydrogen bridges).

(32) Bands at 1600, 1400 cm.sup.1 and the proximity thereof:

(33) In both cases, one sharp and intensive band maximum can be observed (1643, 1643, 1649, 1652, 1651 cm.sup.1) and (1412, 1411, 1410, 1409, 1410 cm.sup.1), respectively. This indicates that the carboxylate group of the glycine is coordinated in each case. The differences of the respective band maximums (231, 222, 239, 243, 241 cm.sup.1) indicates bidentate coordination, i.e. both oxygens are coordinated. A bridge type bond may be presumed, as it was suggested in the case of Cu(Glycine)SO.sub.4x2H.sub.2O, Zn(Glycine)SO.sub.4x2H.sub.2O complexes, based on X-ray diffraction data [3].

(34) 1500 cm.sup.1 and the proximity thereof:

(35) In each samples, a medium intensity band may be observed with a maximum around 1500 cm.sup.1 (1492, 1480, 1478, 1478, 1476 cm.sup.1), which clearly indicates the presence of protonated glycine(NH.sub.3.sup.+), in accordance with what is observed in the 2000-4000 cm.sup.1 range.

(36) 1100, and 600 cm.sup.1, and the proximity thereof:

(37) IN each samples, intensive bands may be observed near wave numbers 1100 and 600 cm.sup.1 (1100, 1081, 1108, 1113, 1113, 1114 and 631, 617, 618, 618, 617 cm.sup.1, respectively). At the same time, a shoulder may be observed at the higher wave number range, which indicates that the sulfate ion is coordinated to the metal ion as an ion or/and in monodentate manner (with a single oxygen).

(38) In the place of glycine, any other amino acid may be present, such as methionine, lysine, aspartic acid, etc. Zinc may be replaced with another trace element, for example copper, manganese, etc.

EXAMPLE 14PREPARATION OF COPPER MONO-GLYCINATE CHELATE ON A FACTORY SCALE

(39) To 1.8 cubic meter water, 375 kg glycine was added at 60-70 C. To this solution, 1250 kg crystalline CuSO.sub.4 is added while heating it to 80 C., then cooled for 30 minutes. The reaction product is dried into a micro-granulate in a fluid bed dryer.

(40) The Cuchelateglycine coordination of the factory product is clearly visible on the IR spectrum. The glycine molecule is coordinated through the carboxylic group, and the amino group remains protonated. The carboxylate group is coordinated as a bidentate bridge. Two types of glycine molecules are visible as coordinated in different environments. The glycine is coordinated through the carboxylic group, as a bidentate bridge ligand. The amino group of the glycine remains protonated. The sulfate ion is coordinated in monodentate and/or bidentate manner. FIG. 2 shows the IR spectrum of the copper mono-glycinate chelate.

(41) 2000-4000 cm.sup.1; band maximum is visible at wave numbers 3138 and 3192 cm.sup.1, respectively, with several smaller maximums in the lower wave number range.

(42) 1600, 1400 cm.sup.1 and the proximity thereof: 2 of each band maximums are visible, 1647, 1580 (in both cases) and 1458, 1410 and 1463, 1409 cm.sup.1 wave number values, respectively.

(43) 1500 cm.sup.1 and the proximity thereof: there is a medium intensity band is visible at a value of 1512 and 1502 cm.sup.1, respectively.

(44) 1100 and 600 cm.sup.1 and the proximity thereof: there are 3 and 2 band maximums are visible, respectively, with shoulders.

(45) Conclusion: the carboxylic group of glycine is coordinated, the amino group is protonated. The sulfate ion binds to the copper ion in bidentate manner.

EXAMPLE 15PREPARATION OF A MIXTURE OF ZINC AND COPPER MONO-GLYCINATE CHELATE

(46) A 1000 liters of the Zn mono-glycine chelate prepared in Example 11 is mixed together with 250 liters of the Cu mono-glycine chelate prepared in Example 12 at 60 C in a blade mixer reactor, then the thus obtained mixture is dried into a micro-granulate in fluid bed dryer.

EXAMPLE 16DETERMINATION OF MIC AND CID CONCENTRATIONS OF MICROELEMENT CHELATE COMPOUNDS ON FACULTATIVE PATHOGEN MICROORGANISMS AND ON NORMAL COMPONENTS OF THE ENTERAL FLORA

(47) The Conventional methods for studying the antimicrobial activityfrom a practical, curative medical viewpoint, the resistance spectrum of the antimicrobial agentare the disk diffusion, or agar well diffusion assays, and dilution experiments done in liquid cultures. In a standardized (NCCLS, DIN, etc.) disk or hole diffusion assay, the effects of an antimicrobial agent is studied on the growth of a test microorganism spread onto the surface of an agar culture dish. In an agar diffusion assay e.g. a bacterial lawn is prepared on the agar dish, then according to the procedure developed in the laboratory, the inoculated plates are punched out in the middle with an agar disk cutting device. Then 10-100 pa assay sample is placed into the hole. The test compound diffuses from the test disk placed onto the bacterial lawn or from a hole made in the agar dish into the culture medium, and thus forms a growth-propagation inhibition zone around the disk or hole. Based on the agar diffusion test, the sensitivity of the strain studied can be unambiguously determined against the test compounds. According to the clinical practice, similarly to the standardized assays of antibiotic agents (where the diameter of the zone is proportional to the concentration of the agent), we classify according to the sensitivity zones formed around the disk holes. In the case of each agents, the microorganisms are qualified as resistant or sensitive to the given agent based on the zone diameter. The use of new antimicrobial agents requires high level of caution. Namely, it is not known what influences the diffusion of the antimicrobial agent (in the agar diffusion assay), such as the pH of the medium, the concentration of dissolved O.sub.2 or CO.sub.2, interactions with the components of the medium), therefore the appropriate techniques and mediums suitable for both the given agent and microorganisms must be selected based on multiple factors. Suitable concentrations of the microorganisms were prepared in liquid cultures in culture dilution tests, and the progress of growth and propagation was verified by microscope and/or optical techniques. This test allows the determination of the minimal inhibitory concentration (MIC) of the agent of interest on a given microorganism. Further, in the case when the death of the microorganisms within the tubes not showing growth is also checked, to determined whether the agent has only a static (growth inhibitor) or a cid (i.e. having microbe killing activity) effect, then the minimal cid concentration (MCC) may also be determined. The ratio of MCC/MIC gives an essential information on the in vivo efficacy of the agent: if this value is high, the in vivo usefulness of the agent is probably low.

(48) TABLE-US-00002 TABLE 2 The strains examined in the study: Pathogen strains: Salmonella enterica subp. Enterica serovar enteritidis (SALMO) Facultative Escherichia coli ATCC 35218 (COLI) pathogen Micrococcus luteus NCAIM B 01072 (MCC) strains: Brachyspira hyodysenteriaea (own isolate, B/06) Staphylococcus aureus NCAIM B.01065.sup.T Streptococcus agalactiae NCAIM B.01882.sup.T Clostridium perfringens NCAIM B.01417.sup.T Components of Lactococcus lactis subsp. Lactis the normal NCAIM B 02070.sup.T enteral flora, Lactobacillus casei v. rhamnosus Dderlein LCR 35 lactic acid Leuconostoc mesenteroides producers: (LACTIC ACID PRODUCERS) Fungi: Saccharomyces cerevisiae (LSE)

(49) Until using in the assays, the strains were stored in suspended form in TSB broth (Scharlau Microbiology) with 25% sterile glycerol frozen at 80 C. or lyophilized.

(50) Isolation of B. hyodysenteriaea Strain (B/06)

(51) B. hyodysenteriae strains were isolated from growing and store-pigs showing the clinical symptoms of swine dysentery, raised in different regions of Hungary. After slaughtering at the slaughterhouse, the colon snares of the pigs showing clinical signs were tied down and were transported to the laboratory within six hours. After opening the colon sections, the sample was provided as scraping from the colon mucosa. In every case, the mucosal scrapings on a streaker were spread onto the surface of freshly prepared TSA (tryptone-soy) agar (Scharlau Microbiology) supplemented with 10% defibrinated bovine blood and 400 g/ml spectinomycin (Sigma-Aldrich Kft.). After inoculation, the cultures were incubated for 96 hours at 42 C. under strictly anaerobic conditions. The anaerobic conditions were achieved by using anaerobic gas generating pouches (Oxoid, Gas Generating Kit, Anaerobic system BR0038B) and anaerobic culturing jars (Oxoid, Anaerobic jar). The determination of the primary and secondary biochemical properties and the identification of the isolated strains was carried out by standard techniques (Quinn et al., 1994).

(52) In Vitro Inhibition Experiments

(53) Addition of a given amount (ppm, mg/kg, g/ml) of trace element to the microbial culture medium from stock solution. The sample solutions with varying compositions were 2 serially diluted in liquid culture medium on a 24-well Greiner tissue culture plate, then 5 l suspension of the sample bacterium in 0.5 MacFarland density prepared with physiological saline was measured into the different concentration liquid culture mediums. In the case of assaying combinations of active ingredients, a cross dilution technique was used, when the two different solutions were diluted across the surface of the plate from two directions. The plates were incubated for 24 hours at the atmospheric conditions required by the bacterium assayed. The results were then read, and the Minimal Inhibitory Concentration (MIC) of the test compounds was thus determined. In the cases where the Minimal Lethal concentration (CID) was assayed, then from the wells not showing opacity after the incubation period, 10 l solution was inoculated onto the surface of blood agar and checked whether any bacterial growth occurred on the surface of the culture medium after the incubation period.

(54) Agar Gel Diffusion

(55) Clostridium perfringens: The strain grown in aerobic or anaerobic vessel was propagated on sterile agar, from which a suspension with 0.5 MacFarland density was prepared with sterile physiological saline, then inoculated onto the surface of culture media with cotton swabs. A hole was welled into inoculated media with specialized puncturing tool, then 50 l of the test solution of the microelement chelate complex in varying concentrations were measured into the holes. Evaluation of the cultures was carried out by visual inspection. The presence of an inhibition zone formed around the holes is indicative of the efficacy of the tested solution.

(56) B. hyodysenteriae (B/06): In these assays, precultures were prepared from the thawed bacterial mass on blood agar (modified tryptone soy agar, supplemented with 5-10% defibrinated bovine blood). From the visibly well-hemolyzed preculture agar plates, 8-10, nearly same sized (5% difference) agar block inoculates were excised, then were spread onto 90 mm diameter freshly prepared agar plates with a sterile glass rod with rounded end about 5 mm in diameter. The plates were dried in covered form for 5 minutes.

(57) According to the procedure developed in the laboratory, the inoculated plates were punched out in the middle with an agar disk cutting device. 100 l of the given dilution of the test compound was placed into the hole with 5 mm diameter. After dripping the solution with known concentration, the plates were placed into an anaerostat (Oxoid anaerobic pouch and jar) within 15 minutes, and incubated at 37 C. for 4-5 days in oxygen free atmosphere.

(58) The bacterial culture of Clostridium perfringens is inhibited by the microelement organic chelate placed into the hole at the middle of the agar dish. The compound diffused into the agar forms a well-defined, concentrically shaped inhibition zone depending on the concentration, and due to the effect of dilution, the inhibitory effect ceases after a certain distance and the culture shows growth beyond the inhibition zone (see FIG. 3).

(59) Determination of the CID Value

(60) It is the concentration of the active ingredient that causes the complete death of the microbes. Under sterile conditions within a laminar box, a dilution series was prepared from the liquid culture mediums that were incubated for 24 hours, then from the dilution series 100 l each was placed onto agar dishes and spread with a sterile glass rod. The incubation period is 24 hours under conditions favorable for the growth of the microbe, then the number of colonies on the agar dishes are counted.

(61) Two options were taken into consideration for the evaluation

(62) 1. No colonies are visible on the agar dishes.

(63) 2. There are visible colonies on the agar dishes making the counting possible.

(64) In the case where no colonies are visible on the culture medium after the incubation, it indicates the microbe killing effect (CID) of the trace element chelate. The number of countable colonies grown on the agar dishes gives the actual MIC value of the given trace element chelate.

EXAMPLE 17MICROBIOLOGICAL MIC AND CID RESULTS OF MICROELEMENT (ZINC, COPPER, IRON, MANGANESE) CHELATE (MONO-, DI-, AND BIS-AMINATE, FATTY ACID, AND HYDROXY FATTY ACID, POLYAMINO CARBOXYLIC ACID) COMPOUNDS

(65) The microbiological assay system showed biological activity at different concentrations with the microelement chelate complex samples studied.

(66) It can be seen from the results of the experiments performed that the assay samples have different MIC values. The pathogens and facultative pathogens examined have significantly higher sensitivity than that of the components of the normal intestinal flora, fungi and lactic acid producers. It can be concluded from the in vitro results that mixing these test compounds into the feed would be preventive, it prevents the growth of pathogens and facultative pathogens, in addition to keeping the equilibrium. It was determined that the chelate compounds studied have different MIC values.

(67) TABLE-US-00003 TABLE 3 Minimal inhibitory concentration (MIC) of the microelement chelate compounds in ppm (mg/kg). Micrococcus Escherichia Salmonella LACTIC ACID Saccharomyces Assayed chelates luteus coli enterica PRODUCERS cerevisiae Zinc mono- 60 100 100 800 800 glycinate Zinc di-glycinate 60 100 120 800 800 Zinc bis-glycinate 440 360 320 800 800 Zinc (H2O) bis- 60 120 220 800 500 glycinate Copper mono- 200 400 400 800 800 glycinate Copper di- 200 340 450 800 800 glycinate Copper bis- 280 500 600 800 800 glycinate Copper (H2O) bis- 60 300 400 800 800 glycinate Iron mono- 100 350 450 1000 1000 glycinate Manganese mono- 100 300 300 1000 1000 glycinate Zn diammine NA 80 120 500 500 bis-malate Zn bis-malate NA 280 360 600 600 Zn(H2O)-bis- NA 90 120 500 600 malate Cu diammine NA 262 262 NA NA bis-malate Zn diammine NA 115 115 NA NA bis-glycinate Zn tetraammine NA 140 140 NA NA bis-glycinate Zn(H.sub.2O) bis- NA 100 110 NA NA alaninate Zn(H.sub.2O) bis- NA 160 160 NA NA propionate Zn(H.sub.2O) bis- NA 120 120 NA NA valerianate Zn(H.sub.2O) bis- NA 110 110 NA NA butyrate Zn bis-salycilate n.a. >4000 >4000 n.v n.a. Zn bis-benzoate n.a. >4000 >4000 n.v n.a. Zn nitrolotriacetate n.a. 40 40 n.v n.a. Cu(H.sub.2O) bis- n.a. 120 210 n.a. n.a. malate Zn diammine n.a. 92 184 n.a. n.a. aspartate Zn diammine n.a. 123 247 n.a. n.a. bis-aspartate Zn diammine n.a. 114 228 n.a. n.a. tri-aspartate Zn mono-aspartate n.a. 74 74 n.a. n.a. Zn(H.sub.2O) bis- n.a. 103 103 n.a. n.a. aspartate Zn(H.sub.2O) tri- n.a. 75 70 n.a. n.a. aspartate Zn diammine n.a. 167 83 n.a. n.a. glutamate Zn diammine n.a. 123 123 n.a. n.a. bis-glutamate Zn diamine n.a. 117 117 n.a. n.a. tri-glutamate Zn mono- n.a. 70 115 n.a. n.a. glutamate Zn(H.sub.2O) bis- n.a. 82 82 n.a. n.a. glutamate Zn(H.sub.2O) tri- n.a. 70 280 n.a. n.a. glutamate Zn diammine n.a. >4000 >4000 n.a. n.a. bis-histidinate Zn bis-histidinate n.a. >4000 >4000 n.a. n.a. Cu diammine n.a. 620 620 n.a. n.a. asparaginate Cu diammine n.a. 1025 2050 n.a. n.a. bis-asparaginate Cu diammine n.a. 1400 >4000 n.a. n.a. tri-asparaginate Cu diammine n.a. 688 343 n.a. n.a. glutamate Cu diammine n.a. 775 775 n.a. n.a. bis-glutamate Cu diammine n.a. 1950 1950 n.a. n.a. tri-glutamate Zn diammine n.a. 48 98 n.a. n.a. citrate Staphylococcus Streptococcus Clostridium Brachyspira Arcanobacterium aureus agalactiae perfringens hyodysenteriae piogenes Zn diammine 80 20 340 2315 21 bis-malate Zn(H.sub.2O) bis- 160 180 360 4400 n.a. malate Cu(H.sub.2O) bis- NA NA 50 88 n.a. malate Cu diammine 262 131 65 143 n.a. (malate).sub.2 Cu mono-glycinate n.a. NA 50 90 92 Cu di-glycinate 2 145 50 n.a. n.a. Cu bis-glycinate 110 420 360 320 260 Cu(H2O) bis- 5.3 171 100 96 32 glycinate Zinc mono- 40 80 210 >4000 n.a. glycinate Zn n.a. n.a. 1000 450 n.a. ethylenediamine- tetraacetic acid Zn (H.sub.2O).sub.2- n.a. n.a. 400 320 n.a. ethylenediamine tetraacetic acid Cu(H.sub.2O).sub.2- n.a. n.a. 60 100 n.a. ethylenediamine tetraacetic acid Cu nitrolo triacetic n.a. n.a. >4000 450 n.a. acid Cu n.a. n.a. 160 320 n.a. ethylenediamine tetraacetic acid Zn diaminonitrolo n.a. n.a. 400 600 n.a. triacetic acid Zn diamino n.a. n.a. 100 >4000 n.a. ethylenediamine tetraacetic acid Cu diaminonitrolo n.a. n.a. 120 200 n.a. triacetic acid Cu diamino n.a. n.a. 20 80 n.a. ethylenediamine tetraacetic acid Zn(H.sub.2O) bis- n.v n.v >4000 >4000 n.a. alaninate Zn bis-salycilate n.v n.v >4000 n.v n.a. Zn bis-benzoate n.v n.v >4000 n.v n.a. Zn mono-glycinate 20 40 210 >4000 n.a. Zn di-glycinate 20 20 165 >4000 n.a. Zn(H.sub.2O) bis- 4 29 180 >4000 n.a. glycine Cu(H.sub.2O) bis- n.a. n.a. 50 88 n.a. malate Zn diammine 46 23 369 n.a. n.a. asparaginate Zn diammine 62 15 494 n.a. n.a. bis-asparaginate Zn diammine 114 14 456 n.a. n.a. tri-asparaginate Zn asparaginate 18 37 297 n.a. n.a. Zn bis-asparaginate 26 51 412 n.a. n.a. Zn tri-asparaginate 18 19 450 n.a. n.a. Zn diammine 61 15 245 n.a. n.a. glutamate Zn diammine 42 21 334 n.a. n.a. bis-glutamate Zn triammine 58 15 469 n.a. n.v tri-glutamate Zn glutamate 36 18 475 n.a. n.a. Zn(H.sub.2O) bis- 21 10 331 n.a. n.a. glutamate Zn(H.sub.2O) tri- 35 17 281 n.a. n.a. glutamate Zn diammine >4000 12 1950 n.a. n.a. bis-histidinate Zn bis-histidinate >4000 4 650 n.a. n.a. Cu diammine 620 20 38 n.a. n.a. asparaginate Cu diammine 1025 16 32 n.a. n.a. bis-asparaginate Cu diammine 1400 43 44 n.a. n.a. tri-asparaginate Cu diammine 343 11 43 n.a. n.a. glutamate Cu diammine 387 24 97 n.a. n.a. bis-glutamate Cu diammine 487 8 30 n.a. n.a. tri-glutamate Zn diammine 49 49 n.a. n.a. n.a. citrate Ag glycinate n.v n.v n.v n.v 184

(68) Several conclusions can be drawn from the above results. Then minimal inhibitory concentration (MIC) of the microelement chelate compounds studied was determined for one or more facultative pathogenic microorganisms. It was determined that without exception, all was capable of inhibiting the pathogen microorganism. In concentrations of 60-200 ppm Micrococcus luteus, and/or in concentrations of 70-400 ppm Escherichia coli, and/or in concentrations of 80-488 ppm Salmonella enterica, and/or in concentrations of 40-498 ppm Staphylococcus aureus, and/or in concentrations of 10-427 ppm Streptococcus agalactiae, and/or in concentrations of 65-167 ppm Clostridium perfringens, and/or in concentrations of 90-494 ppm Brachyspira hyodysenteriae, and/or in concentrations of 21-184 ppm Arcanobacterium piogenes was inhibited.

(69) The components of the normal intestinal flora, lactic acid producers Lactococcus lactis subsp. Lactis, Lactobacillus casei v. rhamnosus and Leuconostoc mesenteroide and/or Saccharomyces cerevisiae (LSE) were only inhibited at or above a concentration of 500 ppm, and preferably at concentrations of 800-1000 ppm.

EXAMPLE 18DOUBLE SYNERGISTIC EXAMINATION OF MICROELEMENT (ZINC, COPPER, IRON, MANGANESE) MONO-GLYCINATE CHELATES. SYNERGIC STUDIES OF THE MICROELEMENT COMPONENT

(70) There is a synergy between two or more test compounds when the biological effects thereof is enhanced by their interactions. Knowing the MIC and CID values, it was deemed important to ensure the selectivity of the trace element chelates. To achieve this goal, the experiments were continued with studying the microelement chelates together, looking for synergies.

(71) TABLE-US-00004 TABLE 4 Compositions without showing synergy and antagonistic Double synergistic MIC values (ppm) of microelement chelates E. coli Salmonella Chelates assayed LSE and inhibitory effect inhibitory effect Brachyspira in double synergy Clostridium Lactococcus on each other on each other hyodysenteriae Zn:Cu mono- Zn 360:Cu 50 Zn 800:Cu 800 Zn 43.7:Cu22 glycinate Zn:Fe mono- Zn 350:Fe 1000 Zn 800:Fe 1000 Zn 180:Fe 110 glycinate Fe:Mn mono- Fe 1000:Mn 500 Fe 1000:Mn 1000 glycinate Zn:Mn mono- Zn 350:Mn 500 Zn 800:Mn 1000 Zn 10:Mn 1940 Zn 10:Mn 1940 glycinate Cu:Mn mono- Cu 100:Mn 500 Cu 800:Mn 1000 glycinate Cu:Fe mono- Cu 100:Fe 1000 Cu 800:Mn 1000 glycinate Zn maleinate - Cu Zn 90:Cu 23 Zn 90:Cu 23 mono-glycinate

(72) TABLE-US-00005 TABLE 5 Compositions showing synergy Double synergistic MIC values (ppm) of microelement chelates Chelates assayed in double synergy E. coli Salmonella Zn:Cu mono-glycinate Zn 90:Cu 50 Zn 90:Cu 50 Zn:Fe mono-glycinate Zn 30:Fe 260 Fe:Mn mono-glycinate Fe 250:Mn 240 Fe 230:Mn 240 Zn:Mn mono-glycinate Cu:Mn mono-glycinate Cu 210:Mn 240 Cu 210:Mn 240 Cu:Fe mono-glycinate Cu 50:Fe 230 Cu 50:Fe 230

(73) Based on these results, the minimal inhibitory concentration (MIC) of 6 trace element chelate was determined for the pathogenic Salmonella enteritidis. These trace element chelate concentrations have practically no effect on the normal components of the intestinal flora (LSE and Lactococcus) (800 ppm). In the case of the facultative pathogenic E. coli, however, five different compositions of these six trace element chelate have effective synergistic MIC value.

EXAMPLE 19MULTIPLE SYNERGISTIC EXAMINATION OF MICROELEMENT (ZINC, COPPER) AND ANION (AMINO ACIDS, ORGANIC AND HYDROXY ACIDS, ETC.) CHELATES

(74) In addition to what was shown in Example 18, synergy was not only found when the microelements were combined, but synergies were recognizable when the anion part of the compounds, i.e. the amino acids, acids were combined (Table 5).

(75) It was concluded based on the data of the table that a synergy exists beyond the base compounds if the chelate compounds consisting different metals are optimally mixed, as well as the anion parts are optimally mixed, and an aggregate effect can be obtained if chelates comprising NH.sub.4.sup.+, H.sub.2O ligands are mixed.

(76) TABLE-US-00006 TABLE 6 Compositions showing sextuple synergy Escherichia Salmonella Staphylococcus Streptococcus Clostridium Synergy of aminates coli enteritidis aureus agalactiae perfringens Zn animate.sub.6 195 ppm 97 49 97 390 (gly.sub.2, asp.sub.2, glu.sub.2, his.sub.2, lys.sub.2, ala.sub.2) Zn(NH4).sub.2 animate.sub.6 75 150 37 78 298 (gly.sub.2, asp.sub.2, glu.sub.2, his.sub.2, lys.sub.2, ala.sub.2) Cu (NH4).sub.2 animate.sub.6 29 117 15 30 117 (gly.sub.2, asp.sub.2, glu.sub.2, his.sub.2, lys.sub.2, ala.sub.2)
Explanation:

(77) The trace element anionic chelates with the synergy of zinc ammonium carbonate aminates gly (glycine), asp (aspartic acid), glu (glutamic acid), his (histidine), lys (lysine) and ala (alanine) sextuple combination in concentrations of 49-390 ppm, the trace element anionic chelate zinc aminates also in sextuple synergy at concentrations 37-238 ppm and the copper ammonium aminates in sextuple synergy at concentrations of 15-117 ppm are useful in vitro to prevent the possible diseases caused by E. coli and Salmonella enteritidis, Staphylococcus aureus, Streptococcus agalactiae and Clostridium perfringens by interfering with the proliferation of the facultative pathogenic microbes.

EXAMPLE 20MULTIPLE SYNERGISTIC EXAMINATION OF MICROELEMENT (ZINC, COPPER, IRON) MONO-GLYCINATE CHELATES SYNERGIC STUDIES OF THE MICROELEMENT COMPONENT

(78) Knowing that the double synergies of the trace element chelates gave 50-80% results on the proliferation of microbes, further triple synergy studies were carried out. Next to the trace element chelates in the double synergies, a third trace element chelate was added. For the determination of triple synergy, all assay methods were utilized, and the final results obtained is shown when the two liquid as well as the microplate assay gave the same result.

(79) TABLE-US-00007 TABLE 7 Compositions without showing triple synergy Triple synergistic MIC values (ppm) of microelement chelates Triple synergy E. coli Salmonella Clostridium variations Zn mono-glycinate:Cu mono-glycinate:Fe mono-glycinate 1 Zn 20:Cu 100:Fe 20 Zn 90:Cu 100:Fe 60 2 Zn 90:Cu 10:Fe 40 Zn 20:Cu 110:Fe 140 3 Zn 10:Cu 60:Fe 260

(80) TABLE-US-00008 TABLE 8 Compositions showing triple synergy Triple synergistic MIC values (ppm) of microelement chelates Triple synergy Coli Salmonella Clostridium variations Zn mono-glycinate:Cu mono-glycinate:Fe-glycinate 1 Zn 40:Cu 30:Fe 70 2 Zn 40:Cu 30:Fe 20

(81) The trace element chelates are useful in vitro for the prevention of enteral diseases caused by the examined E. coli and Salmonella in double synergistic combination, and for the examined Clostridium microbes in triple synergistic combination, by selectively prohibiting the proliferation of the facultative pathogenic microbes, while not inhibiting the lactic acid bacteria and yeast forming the normal intestinal flora.

EXAMPLE 21DETERMINATION OF THE EFFECTIVE CID OR STATIC CONCENTRATIONS OF MICROELEMENT (ZINC, COPPER, IRON, MANGANESE) MONO-GLYCINATE CHELATES

(82) The minimal lethal concentration (CID) is determined as described in Example 14.

(83) TABLE-US-00009 TABLE 9 LACTIC ACID COLI SALMO PRODUCERS LSE Fe 318 ppm Zn 70 Fe 318 ppm Zn 70 Fe 318 ppm Zn 70 Fe 318 ppm Zn 70 Cu 206 Mn 242 Cu 206 Mn 242 Cu 206 Mn 242 Cu 206 Mn 242 Cu 52 Zn 88 Cu 52 Zn 88 Cu 52 Zn 88 Cu 52 Zn 88 CID concentrations of different microelement chelates (ppm) Microelement chelates Salmonella S. tiphy Clostridium Lactococcus LSE Zinc mono- 3000 3000 800 800 glycinate Copper mono- 200 800 800 glycinate

(84) In the case of Salmonella strains, the CID value of the zinc chelate is 30-fold higher than the MIC value, whereas in the case of Clostridium strains, the CID value of the copper chelate is only twice as much as the MIC value. This difference may also be explained with the different sensitivity of the microbe species. However, it should be considered in the case of the components of the normal intestinal flora that both chelate have the same concentration for the MIC and CID values, the results are being indicative of that in the case of the components of the normal intestinal flora, the proliferation of the microbe is not inhibited at these concentrations, but the microbes are killed.

EXAMPLE 22PILOT FEEDING EXPERIMENTS WITH A COMPOSITION CONTAINING ZINC (H.SUB.2.O).SUB.2 .ETHYLENEDIAMINE TETRAACETIC ACID AND COPPER (H.SUB.2.O).SUB.2 .ETHYLENEDIAMINE TETRAACETIC ACID HELATES IN COMBINATION

(85) Experimental feeding was performed with growing pigs on two occasion, under pilot environmental conditions on a stock carrying Brachyspira hyodysenteriae.

(86) The control and experimental groups both were comprised of 100 store-pigs. The latter group received the experimental composition, in the 1 kg/ton feed dose as determined in laboratory tests. In the first study, the control group received a preventive dose of an antibiotic in its feed (Table 10). In the second study, only the pigs suffering from dysentery were individually treated (Table 11). The parameters studied: number of treatments, development of losses, average slaughter weight, and feed utilization.

(87) TABLE-US-00010 TABLE 10 Effect of the composition on the yield and health of store-pigs (The control group received preventive antibiotic treatment) Control group Experimental Designation (antibiotic) Group Number of animals 100 100 Initial average weight (kg) 31.5 31.7 Die off 0 0 Average slaughter weight (kg) 109.5 112.3 Feed uptake (kg) 25 877 25 071 Feed utilization (kg/kg) 3.31 3.11

(88) TABLE-US-00011 TABLE 11 Effect of the composition on the yield of store-pigs (The control group received individual antibiotic treatment by injection) Control Experimental group group Number of animals 100 100 Initial average weight kg 28.4 28.1 Individuals treated by intramuscularly 21 2 Die off during the experiment 0 0 Slaughter weight kg 105.8 109.8 Feed utilization kg/kg 3.23 2.96

(89) In the first experiment, (Table 10), after the same length of fattening time, the slaughter weight in the experimental group was 2.8 kg higher, i.e. 2.6% on average, compared to the control group, and the feed amount necessary to produce 1 kg live weight was 0.2 kg less, which means 6% increase in feed utilization.

(90) In the second experiment (Table 11) the slaughter weight was 3.9% higher in the experimental group, while the feed utilization was better by a value of 9.1% in the experimental group that that of the control group.

EXAMPLE 23PILOT FEEDING EXPERIMENT WITH A COMPOSITION CONTAINING COPPER (H.SUB.2.O).SUB.2 .BIS-GLYCINATE

(91) Experimental feeding was performed with growing pigs, under pilot environmental conditions with the animal paired methods on a stock infected with the pathogen Lawsonia intracellularis. The control group received the usual antibiotic supplement mixed into the feed. The feed of the experimental group was supplemented with a composition of copper diammonium bis-glycinate on a carrier, in a rationing of 1 kg/t.

(92) The parameters studied: number of treatments, development of losses, average slaughter weight, and feed utilization.

(93) TABLE-US-00012 TABLE 12 Effect of the treatment on the yield in paired-animal experiment. Experimental group Control Number of animals 326 331 Days of fattening 101 101 Initial average weight kg 30.86 33.74 Added weight kg 76.15 71.73 Average slaughter weight kg 107.01 105.47 Feed utilization kg/kg 3.10 3.31

(94) The slaughter weight in the experimental group was higher by 1.6%. The added weight in the experimental group was higher by 6.2%. The feed utilization in the experimental group was better by a value of 6.8% in the experimental group that that of the control group.

EXAMPLE 24PILOT EXPERIMENTS WITH COPPER AMMONIA BIS-GLYCINATE CHELATE ON A FARM SHOWING THE SYMPTOMS OF NECROTIC ENTERITIDIS CAUSED BY CLOSTRIDIUM PERFRINGENS INFECTION

(95) The experiments were carried out in two barns, holding 17 100 ROSS-308 baby chicks each. The farm is plagued continually with Clostridium perfringens infection. It requires antibiotic treatments in batches.

(96) The first barn held the individuals of the control group, whereas the second barn held the individuals of the experimental group. The control group did not receive supplement in its feed. The flock held in the experimental barn had a feed supplemented with copper diammonium bis-glycinate applied onto a carrier in a dose of 1.0 kg/t.

(97) The results are summarized in the Table 13.

(98) TABLE-US-00013 TABLE 13 Effect of feeding the experimental composition on raising broilers Control Experimental Parameter studied group group Difference Days of raring 42 42 Chicks at start 17 100 17 100 Chicken shipped 16 242 16 238 4 Total living weight, kg 31 005 32 395 1390 (4.5%) Total weight gain, kg 30 298 31 681 1383 (4.6%) Total feed use, kg 59 260 59 880 620 Mean slaughter weight, g 1909 1995*** 86 (4.5%) Relative feed utilization kg/kg 1.96 1.89 3.6% ***= the difference is significant at P 0.001

(99) The weight gain of the experimental group exceeded that of the control group by 4.6% while the experimental meat hybrid chicken required 3.6% less feed for the unit live weight gain.

(100) The faces of the animals in the experimental group was well formed, no signs of a change indicating diarrhea was visible. The litter was somewhat drier in this group, the ammonia level of the air was perceptionally lower than that of the control.

(101) No adverse side effects were observed when feeding the experimental composition.

EXAMPLE 25EFFECT OF FEEDING COPPER AMMONIUM ETHYLENEDIAMINE TETRAACETIC ACID IN A FLOCK OF EGG LAYING HENS AFTER CLOSTRIDIUM PERFRINGENS CHALLENGE

(102) Under laboratory conditions 108, 32 week old egg laying hens were placed into cages, with an animal density of 3 hens par cage. Before entering the birds into the experiment, cloacal swabs were used to test for the pathogen Clostridium perfringens. It was established that the flock is moderately infected by Clostridium perfringens.

(103) Three equal groups were formed from the 108 birds with 36 egg laying hen in each.

(104) 1. Negative control group I C. perfringens negative 2. Positive control group II C. perfringens positive, inoculated by a gastric probe with 2 ml 10.sup.6 CFU/ml C. perfringens bacterial culture. 3. Supplemented, treatment group III C. perfringens positive, inoculated by a gastric probe with 2 ml 10.sup.6 CFU/ml C. perfringens bacterial culture.
The results are shown in Table 14.

(105) TABLE-US-00014 TABLE 14 Yield parameters of egg laying hens challenged by C. perfringens. Negative control Positive control Treatment Data group I group II group III Total eggs (pc.) 1750 1526 1868 No. of eggs/hen 48.60 42.38 51.80 Total egg weight (kg) 114.07 103.99 125.70 Weight of egg g/pc. 65.1 g 68.1 g 67.2 g

(106) The egg yield decreased by more than 13% in the positive control group, compared to the negative control group, due to the C. perfringens infection. In response to the treatment, the egg yield increased by 6.7% compared to the negative control group and by 22.4% compared to the positive control group. The total egg weight showed similar tendency during the experiment. There was a different trend in the measurement of individual eggs, because the individual egg weight was the largest in the positive control group.

EXAMPLE 26FEEDING EXPERIMENT TO PREVENT DIARRHEAL DISEASES OCCURRING AT WEANING BY ADMINISTERING ZINC (H.SUB.2.O).SUB.2 .MALEINATE

(107) 32 days old weaned piglets were fed by a feed supplemented with zinc ammonium maleinate. The experimental animals had E. coli infection. The control group received the standard antibiotic supplement in the feed to control the E. coli infection.

(108) The production yields are shown in Table 15.

(109) TABLE-US-00015 TABLE 15 Production yield parameters during the experiment Control Experimental group group No. of animals 28 31 Initial weight (kg) 9.11 9.19 Duration of the experiment (days) 45 38 Die off during the experiment 1 0 Body weight at the end of the experiment (kg) 21.26 24.05 Daily weight gain (g) 270 391 Feed utilization (kg/kg) 2.31 2.04

(110) The average daily weight gain was 45% higher in the experimental group than in the control group. The feed utilization was 13% better in the experimental group than in the control group. There was no die off in the experimental group, however, in the control group 1 animal (3.6%) died during the experimental period.

REFERENCES

(111) Chu, B. C, Garcia-Herrero, A., Johanson, T. H., Krewulak, K. D., Lau. C. K., Peacock, R. S., Slavinskaya, Z, Vogel, H. J.: Siderophore uptake in bacteria and the battlefor iron with the host; a bird'eye view. Biometals(2010)23(4), 601-611. Du, Z. (1994): Bioavailabilities of copper in copper proteinate, copper lysine and cupric sulfate, and copper tolerances of Holstein and Jersey cattle. Ph. D. Thesis, University of Kentucky, Lexington, Ky. Kakukk Tibor, dr. Schmidt Janos, 1988. Takarmnyozastan 3.4.:122-145 Leong, J. (1986) Siderophores: their biochemistry and possible role in the biocontrol of plant pathogens. Annu. Rev. Phytopathol., 24: 187-209. Mahan et al. (1994): Biotechnology in the feed industry: Proc. of Alltech's Tenth Annual Symposium. p. 323-333 Miethke and Marahiel, (2007) SIDEROPHORE-BASED IRON ACQUISITION AND PATHOGEN CONTROL, Microbiol. Mol. Biol Rev. 71(3), 413-451 Neilands, J. B. (1981) Microbial iron compounds. Annu. Rev. Biochem., 50: 715-731. Neilands, J. B., Konopka, K., Schwyn, B., Coy, M., Francis, R. T., Paw, H., Bagg, A. (1987) Comparative biochemistry of microbial iron assimilation. In: Iron Transport in Microbes, Plants and Animals. (Winkelmann, G., van der elm, D., Neilands, J. B. Eds.), (VCH Verlagsgesellschaft, Weinheim, Germany) pp. 3-33. Quinn P. J., Carter M. E., Markey B. K., Carter G R., 1994. Clinical Veterinary Microbiology. Wolfe Publishing, London Shi et al. (1995): Influence of iron oxide, iron sulfate and iron proteinate on Cu bioavailabilities from Cu sulfate and Cu proteinate. J. Dairy Sci. 78: 187 (Suppl. 1). Weger, L. A. de, Boxtel, R. van, Burg, B. van der, Gruters, R., Geels, F. P., Schippers, B., Lugtenberg, B. (1986) Siderophores and outer membrane proteins of antagonistic, plant-growth-stimulating, root-colonizing Pseudomonas spp. J. Bacteriol., 165: 585-594. Whittaker et al. (1993) Regul Toxicol Pharmacol., 18(3), 419-27