Water and feed antimicrobial preservative

Abstract

Antimicrobial compositions containing buffered propionic or acetic acid mixed with pelargonic acid.

Claims

1. An antimicrobial composition for extending the shelf-life of water, feed or feed ingredients, against Salmonella, the antimicrobial composition comprising an organic carboxylic acids (RCOOH) aqueous solution that comprises: a buffered short-chain acid aqueous solution having a pH in a range of 1-5, wherein the buffered short-chain acid aqueous solution comprises a buffering agent and a short-chain acid, wherein the short-chain acid is at a concentration in a range of 80 to 95 wt. % of the total organic carboxylic acid content in the organic carboxylic acids aqueous solution, and wherein the short-chain acid is propionic acid, and wherein the buffering agent is ammonium hydroxide; and pelargonic acid (CH.sub.3(CH.sub.2).sub.7COOH)at a concentration in a range of 5 to 20 wt. % of the total organic carboxylic acid content in the organic carboxylic acids aqueous solution.

2. The antimicrobial composition of claim 1, wherein the buffered short-chain acid aqueous solution has a pH in a range of 1-3.

3. The antimicrobial composition of claim 1 further comprising a surfactant at a concentration not greater than 20 wt. % of the antimicrobial composition, wherein the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, polysorbate 40, polysorbate 60, polyglyceryl ester, polyglyceryl monooleate, decaglyceryl monocaprylate, propylene glycol dicaprilate, triglycerol monostearate, sorbitane monooleate, ethoxylated castor oil surfactants, and mixtures thereof.

4. The antimicrobial composition of claim 3, wherein the surfactant is at a concentration that is in a range of 0.5 to 5 wt. % of the antimicrobial composition.

5. The antimicrobial composition of claim 1 further comprising a terpene at a concentration not greater than 20 wt. % of the antimicrobial composition, wherein the terpene is selected from the group consisting of allyl disulfide, citral, pinene, nerol, geraniol, carvacrol, eugenol, carvone, anethole, camphor, menthol, limonene, farnesol, carotene, thymol, borneol, myrcene, terpenene, linalool, and mixtures thereof.

6. The antimicrobial composition of claim 5, wherein the terpene is selected from the group consisting of allyl disulfide, thymol, citral, eugenol, carvacrol, carvone, and mixtures thereof.

7. The antimicrobial composition of claim 6, wherein the terpene concentration is in a range of 0.5-10 wt. % of the antimicrobial composition.

8. A method for extending the shelf-life of water, feed or feed ingredients, against Salmonella, the method comprising spray-treating or admixing to the water, feed or feed ingredients an effective amount of an antimicrobial composition that comprises an organic carboxylic acids (RCOOH) aqueous solution, wherein the organic carboxylic acids aqueous solution comprises: a buffered short-chain acid aqueous solution having a pH in a range of 1-5, wherein the buffered short-chain acid aqueous solution comprises a buffering agent and a short-chain acid, wherein the short-chain acid is at a concentration in a range of 80 to 95 wt. % of the total organic carboxylic acid content in the organic carboxylic acids aqueous solution, and wherein the short-chain acid is propionic acid, and wherein the buffering agent is ammonium hydroxide; and pelargonic acid (CH.sub.3(CH.sub.2).sub.7COOH) at a concentration in a range of about 5 to about 20 wt. % of the total organic carboxylic acid content in the organic carboxylic acids aqueous solution.

9. The method of claim 8, wherein the buffered short-chain acid aqueous solution has a pH in a range of 1-3.

10. The method of claim 8, wherein the antimicrobial composition further comprises a surfactant at a concentration not greater than 20 wt. % of the antimicrobial composition, wherein the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, polysorbate 40, polysorbate 60, polyglyceryl ester, polyglyceryl monooleate, decaglyceryl monocaprylate, propylene glycol dicaprilate, triglycerol monostearate, sorbitane monooleate, ethoxylated castor oil surfactants, and mixtures thereof.

11. The method of claim 10, wherein the surfactant is at a concentration that is in a range of 0.5 to 5 wt. % of the antimicrobial composition.

12. The method of claim 8, wherein antimicrobial composition further comprises a terpene at a concentration not greater than 20 wt. % of the antimicrobial composition, wherein the terpene is selected from the group consisting of allyl disulfide, citral, pinene, nerol, geraniol, carvacrol, eugenol, carvone, anethole, camphor, menthol, limonene, farnesol, carotene, thymol, borneol, myrcene, terpenene, linalool, and mixtures thereof.

13. The method of claim 12, wherein the terpene is selected from the group consisting of allyl disulfide, thymol, citral, eugenol, carvacrol, carvone, and mixtures thereof.

14. The method of claim 13, wherein the terpene concentration is in a range of 0.5-10 wt. % of the antimicrobial composition.

15. The method of claim 8, wherein said antimicrobial composition contains 20% to 90 wt. % of said organic carboxylic acids (RCOOH) in aqueous solution.

16. The antimicrobial composition of claim 1, wherein said organic carboxylic acids (RCOOH) in aqueous solution is 20% to 90 wt. % of the antimicrobial composition.

17. An antimicrobial composition for extending the shelf-life of water, feed or feed ingredients, against Salmonella, the antimicrobial composition comprising: (a) an organic carboxylic acids (RCOOH) aqueous solution having a total organic carboxylic acid content that is in a range of 20 to 95 wt. % of the antimicrobial composition, wherein the organic carboxylic acids (RCOOH) aqueous solution comprises: (i) a buffered short-chain acid aqueous solution having a pH in a range of 1-3, wherein the buffered short-chain acid aqueous solution comprises a buffering agent and a short-chain acid, wherein the short-chain acid is at a concentration in a range of 80 to 95 wt. % of the total organic carboxylic acid content in the organic carboxylic acids aqueous solution, and wherein the short-chain acid is propionic acid, and wherein the buffering agent is ammonium hydroxide; and (ii) pelargonic acid (CH.sub.3(CH.sub.2).sub.7COOH) at a concentration in a range of 5 to 20 wt. % of the total organic carboxylic acid content in the organic carboxylic acids aqueous solution; (b) a terpene at a concentration in a range of 0.5 to 20 wt. % of the antimicrobial composition, wherein the terpene is selected from the group consisting of allyl disulfide, citral, pinene, nerol, geraniol, carvacrol, eugenol, carvone, anethole, camphor, menthol, limonene, farnesol, carotene, thymol, borneol, myrcene, terpenene, linalool, and mixtures thereof; and (c) a surfactant at a concentration that is in a range of 0.5 to 20 wt. % of the antimicrobial composition wherein the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, polysorbate 40, polysorbate 60, polyglyceryl ester, polyglyceryl monooleate, decaglyceryl monocaprylate, propylene glycol dicaprilate, triglycerol monostearate, sorbitane monooleate, ethoxylated castor oil surfactants, and mixtures thereof.

18. A method for extending the shelf-life of water, feed or feed ingredients, against Salmonella, the method comprising spray-treating or admixing to the water, feed or feed ingredients an effective amount of an antimicrobial composition that comprises: (a) an organic carboxylic acids (RCOOH) aqueous solution having a total organic carboxylic acid content that is in a range of 20 to 95 wt. % of the antimicrobial composition, wherein the organic carboxylic acids (RCOOH) aqueous solution comprises: (i) a buffered short-chain acid aqueous solution having a pH in a range of 1-3, wherein the buffered short-chain acid aqueous solution comprises a buffering agent and a short-chain acid, wherein the short-chain acid is at a concentration in a range of 80 to 95 wt. % of the total organic carboxylic acid content in the organic carboxylic acids aqueous solution, and wherein the short-chain acid is propionic acid, and wherein the buffering agent is ammonium hydroxide; and (ii) pelargonic acid (CH.sub.3(CH.sub.2).sub.7COOH) at a concentration in a range of 5 to 20 wt. % of the total organic carboxylic acid content in the organic carboxylic acids aqueous solution; (b) a terpene at a concentration in a range of 0.5 to 20 wt. % of the antimicrobial composition, wherein the terpene is selected from the group consisting of allyl disulfide, citral, pinene, nerol, geraniol, carvacrol, eugenol, carvone, anethole, camphor, menthol, limonene, farnesol, carotene, thymol, borneol, myrcene, terpenene, linalool, and mixtures thereof; and (c) a surfactant at a concentration that is in a range of 0.5 to 20 wt. % of the antimicrobial composition wherein the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, polysorbate 40, polysorbate 60, polyglyceryl ester, polyglyceryl monooleate, decaglyceryl monocaprylate, propylene glycol dicaprilate, triglycerol monostearate, sorbitane monooleate, ethoxylated castor oil surfactants, and mixtures thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Definitions:

(2) An organic acid of the invention is a carboxylic acid compound having straight or branched C.sub.1 to C.sub.18 hydrocarbon chain, e.g., formic acid, acetic acid, propionic acid, butyric acid and pelargonic acid.

(3) A buffered solution is one which resists changes in pH when small quantities of an acid or an alkali are added. Buffer capacity is a quantitative measure of the resistance of a buffer solution to pH change on addition of hydroxide ions. Buffering systems of the invention include

(4) HCl, Sodium citrate pH=1-5

(5) Citric acid, Sodium citrate pH=2.5-5.6

(6) Acetic acid, Sodium acetate pH=3.7-5.6

(7) NH.sub.4Cl, NH.sub.4OH pH 1 to 11

(8) An antimicrobial terpene of the invention may be allyl disulfide, citral, pinene, nerol, geraniol, carvacrol, eugenol, carvone, anethole, camphor, menthol, limonene, farnesol, carotene, thymol, borneol, myrcene, terpenene, linalool, or mixtures thereof. Preferred terpenes are allyl disulfide, thymol, citral, eugenol, carvacrol, and carvone, or mixtures thereof.

(9) The term effective amount of a compound means that amount which is capable of performing the function of the compound or property for which the effective amount is expressed, such as a non-toxic but sufficient amount to provide an antimicrobial effect. Thus an effective amount may be determined by one of ordinary skill in the art using routine experimentation.

(10) Formulations can vary not only in the concentration of major components i.e. organic acids, but also in the type of terpenes, type of surfactant and water concentration used. The invention can be modified by adding or deleting terpenes and surfactants from the formulations.

(11) The term synergistic effect or synergy means an improved preservative effect when the ingredients are used as a mixture compared to the expected additive effect based on each component used alone.

(12) Compositions of the present invention comprise organic acids having 1 to 18 carbons, containing an effective amount of a C.sub.2:C.sub.9 or a C.sub.3:C.sub.9 mixture, resulting in a synergistic preservative effect. In general, an aqueous solution of the short-chain acids is buffered to a pH between 1 and 5, preferably between pH 1 and 3, then the C.sub.9 (Pelargonic) acid is added in an amount from 2 to 20 wt. % along with optional terpenes and surfactants and other additives.

(13) Antimicrobial terpenes, plant extracts or essential oils containing terpenes can be used in the invention as well as the more purified terpenes. Terpenes are commercially available or can be produced by various methods known in the art, such as solvent extraction or steam extraction/distillation or chemically synthesized.

(14) The surfactant can be non-ionic, cationic, or anionic. Examples of surfactants include polysorbate 20, polysorbate 80, polysorbate 40, polysorbate 60, polyglyceryl ester, polyglyceryl monooleate, decaglyceryl monocaprylate, propylene glycol dicaprilate, triglycerol monostearate, Tween 20, Span 20, Span 40, Span 60, Span 80, ethoxylated castor oil surfactants or mixtures thereof.

(15) The total composition can comprise 1% to 100% by weight organic acids, preferably 20-95%. Of the organic acid component, 2% to 20% by weight is pelargonic acid and the remaining 98% to 80% by weight is acetic acid, propionic acid or a mixture thereof. The composition may contain 0 to 20% by weight terpenes, preferably 0.5-10%, and 0 to 20% by weight surfactant, preferably 0.5-5%. The total composition may contain 0 to 99 wt. % water.

(16) The present invention is effective against any of these classifications of infective agents present in water, feed and major feed ingredients, in particular, bacteria, mycoplasma, viruses and fungi. Examples of these infective agents are Staphylococcus aureus, Aspergillius fumigatus, Mycoplasma iowae, Sclerotima homeocarpa, Rhizocionia solani, Colletotrichum graminicola, Penicilhun sp., Mycoplasma pneumoniae, E. coli, Salmonella sp., Clostridia sp., Campylobacter sp. and others. The compositions and methods of the present invention are effective in preventing many, if not all, of these infections in a great variety of subjects, including humans, other mammals and avians.

(17) The present invention includes a method to disinfect water, feed and feed ingredients. The method comprises administering the composition by a variety of means. For example, sprayed onto feed, sprayed onto water, mixed in drinking water, applied to surfaces where water and feed are stored for future uses or consumed daily, added drop wise through a standard medicator or water disinfector, for example in starter, grower and finisher animal houses.

(18) The composition of the present invention can be used safely and effectively as a preservative for water and feed for all commercially grown animals, for human consumption and external use, for companion animals, and other animals where a low microbial concentration is desired in the feed or water supply.

(19) Throughout this application, various publications are referenced. The disclosures of these publications are hereby incorporated by reference in their entireties into this application.

Example 1

Evaluation of Buffered Organic Acids

(20) Purpose: To determine the Effect of pH on the Antimicrobial Activity of Acetic and Propionic Acid

(21) Treatments:

(22) 1) Control (negative control)

(23) 2) Formic acid:Propionic acid (90:10 ratio; positive control)

(24) 3) Acetic acid (pH 1)

(25) 4) Acetic acid (pH 2)

(26) 5) Acetic acid (pH 3)

(27) 6) Acetic acid (pH 4)

(28) 7) Acetic acid (pH 5)

(29) 8) Acetic acid (pH 6)

(30) 9) Acetic acid (pH 7)

(31) 10) Propionic acid (pH 1)

(32) 11) Propionic acid (pH 2)

(33) 12) Propionic acid (pH 3)

(34) 13)Propionic acid (pH 4)

(35) 14) Propionic acid (pH 5)

(36) 15) Propionic acid (pH 6)

(37) 16) Propionic acid (pH 7)

(38) Procedure:

(39) Propionic and acetic acids were buffered with ammonium hydroxide to pHs ranging from 1 to 7. Acid contents of the buffered solutions were determined by a weight to weight ratio calculation to obtain the same acid content in the test solutions. Solutions are added to sterile deionized water to provide a 0.025%, 0.05%, 0.075% and 0.1% acid solution. The pHs of the deionized water solutions were recorded and any problems with solubility noted.

(40) 100 ul of a nutrient broth culture of Salmonella typhimurium was added to each dilution tube. After addition, tubes were vortexed and allowed to stand. At 4 and 24 hours after addition of the inoculum, 100 ul of the solution was plated on Standard Plate agar (triplicate plates). Plates were incubated at 37 C. for 24 hours prior to enumeration. The minimum effective dose of each acid was determined by linear regression.

(41) Results:

(42) TABLE-US-00001 TABLE 1 Effect of pH Buffering on the Effectiveness of Acetic Acid against Salmonella Salmonella at time Interval Product 4 hr 24 hr Test Product Conc. cfu/g % reduction cfu/g % reduction Control N/A 1505 0 1180 0 Formic acid:Propionic acid (90:10) 0.025 203 87 0 100 Formic acid:Propionic acid (90:10) 0.05 50 97 0 100 Formic acid:Propionic acid (90:10) 0.075 20 99 0 100 Formic acid:Propionic acid (90:10) 0.1 3 100 0 100 Acetic pH 1 0.025 883 41 107 91 Acetic pH 1 0.05 750 50 7 99 Acetic pH 1 0.075 617 59 17 99 Acetic pH 1 0.1 520 65 7 99 Acetic pH 2 0.025 920 39 170 86 Acetic pH 2 0.05 817 46 50 96 Acetic pH 2 0.075 673 55 20 98 Acetic pH 2 0.1 670 55 17 99 Acetic pH 3 0.025 1100 27 300 75 Acetic pH 3 0.05 843 44 117 90 Acetic pH 3 0.075 927 38 90 92 Acetic pH 3 0.1 873 42 43 96 Acetic pH 4 0.025 1067 29 543 54 Acetic pH 4 0.05 1167 22 407 66 Acetic pH 4 0.075 1097 27 263 78 Acetic pH 4 0.1 1167 22 183 84 Acetic pH 5 0.025 1267 16 993 16 Acetic pH 5 0.05 1533 0 873 26 Acetic pH 5 0.075 1367 9 805 32 Acetic pH 5 0.1 1300 14 597 49 Acetic pH 6 0.025 1500 0 1167 1 Acetic pH 6 0.05 1767 0 1400 0 Acetic pH 6 0.075 1667 0 1400 0 Acetic pH 6 0.1 1633 0 1433 0 Acetic pH 7 0.025 1567 0 1300 0 Acetic pH 7 0.05 1600 0 1433 0 Acetic pH 7 0.075 1467 2 1433 0 Acetic pH 7 0.1 1567 0 1500 0

(43) TABLE-US-00002 TABLE 2 Effect of pH Buffering on the Effectiveness of Propionic Acid against Salmonella Salmonella at time Interval Product 4 hr 24 hr Test Product Conc. cfu/g % reduction cfu/g % reduction Control N/A 1505 0 1180 0 Formic acid:Propionic acid (90:10) 0.025 203 87 0 100 Formic acid:Propionic acid (90:10) 0.05 50 97 0 100 Formic acid:Propionic acid (90:10) 0.075 20 99 0 100 Formic acid:Propionic acid (90:10) 0.1 3 100 0 100 Propionic pH 1 0.025 1200 20 133 89 Propionic pH 1 0.05 923 39 37 97 Propionic pH 1 0.075 530 65 23 98 Propionic pH 1 0.1 450 70 10 99 Propionic pH 2 0.025 1067 29 70 94 Propionic pH 2 0.05 733 51 10 99 Propionic pH 2 0.075 477 68 13 99 Propionic pH 2 0.1 380 75 7 99 Propionic pH 3 0.025 1467 2 190 84 Propionic pH 3 0.05 847 44 83 93 Propionic pH 3 0.075 973 35 60 95 Propionic pH 3 0.1 603 60 27 98 Propionic pH 4 0.025 1367 9 615 48 Propionic pH 4 0.05 1200 20 293 75 Propionic pH 4 0.075 943 37 187 84 Propionic pH 4 0.1 1167 22 163 86 Propionic pH 5 0.025 >1505 0 793 33 Propionic pH 5 0.05 1400 7 943 20 Propionic pH 5 0.075 1167 22 630 47 Propionic pH 5 0.1 817 46 557 53 Propionic pH 6 0.025 >1505 0 1450 0 Propionic pH 6 0.05 1400 7 1067 10 Propionic pH 6 0.075 >1505 0 1233 0 Propionic pH 6 0.1 1700 0 1333 0 Propionic pH 7 0.025 >1505 0 1667 0 Propionic pH 7 0.05 1700 0 1367 0 Propionic pH 7 0.075 >1505 0 1700 0 Propionic pH 7 0.1 1600 0 1367 0

(44) TABLE-US-00003 TABLE 3 Minimum Inhibitory Concentrations Treatment MIC at 4 Hr MIC at 24 HR Formic:Propionic 0.067 <0.025 Acetic, pH 1 0.129 0.065 Acetic, pH 2 0.142 0.067 Acetic, pH 3 0.176 0.073 Acetic, pH 4 0.207 0.096 Acetic, pH 5 0.238 0.210 Acetic, pH 6 ND ND Acetic, pH 7 ND ND Propionic, pH 1 0.131 0.066 Propionic, pH 2 0.120 0.064 Propionic, pH 3 0.149 0.069 Propionic, pH 4 0.237 0.091 Propionic, pH 5 0.170 0.165 Propionic, pH 6 ND ND Propionic, pH 7 ND ND NDMIC can not be determined due to a lack of effect at the highest dose rate.
Conclusions: Buffering of acetic or propionic acid with ammonia decreased the effectiveness of the product to Salmonella. The break point appears to be between a pH of 3-4.

Example 2

Evaluation of Individual Organic Acids

(45) Purpose: To Determine the Effect of Carbon Chain Length of Organic Acids on Antimicrobial Activity

(46) Treatments:

(47) 1) Control

(48) 2) Formic acid:Propionic acid (90:10 ratio; positive control)

(49) 3) Formic acid

(50) 4) Acetic acid

(51) 5) Propionic acid

(52) 6) Butyric acid

(53) 7) Valeric acid

(54) 8) Caproic acid

(55) 9) Oenanthic acid

(56) 10) Caprylic acid

(57) 11) Pelargonic acid

(58) 12) Lauric acid

(59) 13) Potassium hydroxide

(60) Procedure:

(61) In this experiment, the effect of free fatty acids was evaluated. Several long chain fatty acids (caprylic, pelargonic and lauric) were not soluble in water and KOH was used to get these acids into solution in water (final solution contained equal amounts by weights of acid and KOH. Acid content of the solutions was determined by a weight to weight ratio calculation (weight of acid/total weight of buffered solution). Solutions are added to sterile deionized water to provide a 0.025%, 0.05%, 0.075% and 0.1% acid solutions. The pH of the deionized water solutions was recorded and any problems with solubility noted

(62) 100 ul of a nutrient broth culture of Salmonella typhimurium was added to each dilution tube. After addition, tubes were vortexed and allowed to stand. At 4 and 24 hours after addition of the inoculum, 100 ul of the solution was plated on agar (triplicate plates). Plates were incubated at 37 C. for 24 hours prior to enumeration. The minimum effective dose of each acid was be determined by linear regression:

(63) TABLE-US-00004 TABLE 4 Effect of Organic Acids against Salmonella Salmonella at time Interval Product 4 hr 24 hr Test Product Conc. cfu/g % reduction cfu/g % reduction Control 1600 0 1700 0 Formic acid:Propionic acid (90:10) 0.025 160 90 0 100 Formic acid:Propionic acid (90:10) 0.05 20 99 0 100 Formic acid:Propionic acid (90:10) 0.075 0 100 0 100 Formic acid:Propionic acid (90:10) 0.1 0 100 0 100 Formic acid 0.025 83 95 0 100 Formic acid 0.05 7 100 0 100 Formic acid 0.075 0 100 0 100 Formic acid 0.1 0 100 0 100 Acetic acid 0.025 917 43 80 95 Acetic acid 0.05 840 48 13 99 Acetic acid 0.075 677 58 10 99 Acetic acid 0.1 513 68 15 99 Propionic acid 0.025 1167 27 170 90 Propionic acid 0.05 900 44 40 98 Propionic acid 0.075 877 45 25 99 Propionic acid 0.1 773 52 30 98 Butyric acid 0.025 1060 34 170 90 Butyric acid 0.05 833 48 57 97 Butyric acid 0.075 977 39 30 98 Butyric acid 0.1 547 66 10 99 Valeric acid 0.025 1233 23 533 69 Valeric acid 0.05 1267 21 73 96 Valeric acid 0.075 990 38 37 98 Valeric acid 0.1 657 59 17 99 Caproic acid 0.025 1267 21 30 98 Caproic acid 0.05 1433 10 7 100 Caproic acid 0.075 523 67 0 100 Caproic acid 0.1 27 98 0 100 Oenanthic acid 0.025 1103 31 10 99 Oenanthic acid 0.05 0 100 0 100 Oenanthic acid 0.075 0 100 0 100 Oenanthic acid 0.1 0 100 0 100 Caprylic acid/KOH 0.025 1567 2 1400 18 Caprylic acid/KOH 0.05 1333 17 797 53 Caprylic acid/KOH 0.075 1100 31 77 95 Caprylic acid/KOH 0.1 0 100 0 100 Pelargonic acid/KOH 0.025 7 100 0 100 Pelargonic acid/KOH 0.05 0 100 0 100 Pelargonic acid/KOH 0.075 0 100 0 100 Pelargonic acid/KOH 0.1 0 100 0 100 Lauric acid/KOH 0.025 670 58 20 99 Lauric acid/KOH 0.05 0 100 0 100 Lauric acid/KOH 0.075 0 100 0 100 Lauric acid/KOH 0.1 0 100 0 100 KOH 0.025 0 100 0 100 KOH 0.05 0 100 0 100 KOH 0.075 0 100 0 100 KOH 0.1 0 100 0 100

(64) TABLE-US-00005 TABLE 5 Minimum Inhibitory Concentration of Organic Acids against Salmonella Treatment MIC at 4 HR MIC at 24 FIR Formic acid:Propionic acid 0.065 <0.025 Formic acid 0.064 <0.025 Acetic acid 0.129 0.064 Propionic acid 0166 0.066 Butyric acid 0.142 0.066 Valeric acid 0.174 0.070 Caproic acid 0.103 0.063 Oenanthic acid 0.075 0.063 Caprylic acid 0.109 0.090 Pelargonic acid 0.063 <0.025 Lauric acid 0.072 <0.025 Potassium hydroxide <0.025 <0.025
Conclusions: No direct relationship was observed to occur between the efficacy against Salmonella and organic acid chain length. This contrasts the effects reported for acid chain length and anti-fungal efficacy. The activity of caprylic, pelargonic and lauric can not be compared to the shorter chain acids due to the use of KOH.

Example 3

Buffered Organic Acids Mixtures

(65) Purpose: Of the long chain organic acids, pelargonic was observed to be the most effective based on prior studies. This experiment is to determine if there is a synergistic effect when buffered propionic or acetic acid is combined with pelargonic acid.

(66) Test Products:

(67) 1) Control

(68) 2) Formic acid : Propionic acid (90:10 ratio; positive control)

(69) 3) Acetic acid

(70) 4) Acetic acid: pelargonic acid (80:20: wt/wt)

(71) 5) Acetic acid: pelargonic acid (60:40: wt/wt)

(72) 6) Acetic acid: pelargonic acid (40:60: wt/wt)

(73) 7) Acetic acid: pelargonic acid (20:80: wt/wt)

(74) 8) Propionic acid

(75) 9) Propionic acid: pelargonic acid (80:20: wt/wt)

(76) 10)Propionic acid: pelargonic acid (60:40: wt/wt)

(77) 11) Propionic acid: pelargonic acid (40:60: wt/wt)

(78) 12)Propionic acid: pelargonic acid (20:80: wt/wt)

(79) 13) Pelargonic acid

(80) Procedure: Propionic and acetic acids were buffered with ammonium hydroxide to a pH of 3 and combined with pelargonic in the ratios above. Acid content of the buffered solutions was determined by weight to weight ratio calculation (weight of acid/total weight of buffered solution) and adjusted to provide an equal acid value for each treatment. The treatments above were added to sterile deionized water to make at a 0.025%, 0.05%, 0.075% and 0.1% total acid solutions. The pHs of the deionized water solutions were recorded and any problems with solubility noted.

(81) 100 ul of a nutrient broth culture of Salmonella typhimurium was added to each dilution tube. After addition, tubes were vortexed and allowed to stand. At 4 and 24 hours after addition of the inoculum, 100 ul of the solution was plated on Standard Plate agar (triplicate plates). Plates were incubated at 37 C. for 24 hours prior to enumeration. The minimum effective dose of each acid was determined by linear regression.

(82) TABLE-US-00006 TABLE 6 Effect of Pelargonic acid on the effectiveness of acetic or propionic against Salmonella Salmonella at time Interval Product 4 hr 24 hr Test Product Conc. cfu/g % reduction cfu/g % reduction Control N/A 1517 0 1344 0 Formic acid:Propionic acid (90:10) 0.025 200 87 0 100 0.05 67 96 0 100 0.075 20 99 2 100 0.1 10 99 0 100 100% Propionic pH 3 0.025 1133 25 70 95 0.05 880 42 17 99 0.075 1133 25 20 99 0.1 857 44 13 99 80% Propionic pH 3:20% Pelargonic 0.025 0 100 3 100 0.05 0 100 0 100 0.075 0 100 3 100 0.1 0 100 0 100 60% Propionic pH 3:40% Pelargonic 0.025 0 100 0 100 0.05 0 100 0 100 0.075 0 100 3 100 0.1 0 100 0 100 40% Propionic pH 3:60% Pelargonic 0.025 0 100 0 100 0.05 0 100 0 100 0.075 0 100 3 100 0.1 0 100 0 100 20% Propionic pH 3:80% Pelargonic 0.025 0 100 0 100 0.05 0 100 0 100 0.075 0 100 3 100 0.1 0 100 0 100 100% Acetic pH 3 0.025 943 38 123 92 0.05 1007 34 120 92 0.075 1007 34 77 95 0.1 967 36 83 95 80% Acetic pH 3:20% Pelargonic 0.025 0 100 0 100 0.05 0 100 0 100 0.075 0 100 3 100 0.1 0 100 0 100 60% Acetic pH 3:40% Pelargonic 0.025 0 100 0 100 0.05 0 100 0 100 0.075 0 100 3 100 0.1 0 100 0 100 40% Acetic pH 3:60% Pelargonic 0.025 0 100 0 100 0.05 0 100 0 100 0.075 0 100 3 100 0.1 0 100 0 100 20% Acetic pH 3:80% Pelargonic 0.025 0 100 0 100 0.05 0 100 0 100 0.075 0 100 0 100 0.1 0 100 0 100 Pelargonic 0.025 0 100 0 100 0.05 0 100 0 100 0.075 0 100 0 100 0.1 0 100 0 100

(83) TABLE-US-00007 TABLE 7 Minimum Inhibitory Concentration of Pelargonic when mixed with acetic or propionic acid Treatment MIC at 4 HR MIC at 24 HR Formic acid:Propionic acid 0.067 <0.025 Propionic acid 0.179 0.064 Propionic acid:pelargonic acid (80:20) <0.025 0.063 Propionic acid:pelargonic acid (60:40) <0.025 <0.025 Propionic acid:pelargonic acid (40:60) <0.025 <0.025 Propionic acid:pelargonic acid (20:80) <0.025 <0.025 Acetic acid 0.171 0.068 Acetic acid:pelargonic acid (80:20) <0.025 <0.025 Acetic acid:pelargonic acid (60:40) <0.025 <0.025 Acetic acid:pelargonic acid (40:60) <0.025 <0.025 Acetic acid:pelargonic acid (20:80) <0.025 <0.025 Pelargonic acid <0.025 <0.025

(84) Conclusion: The addition of pelargonic acid to propionic or acetic acid resulted in an increase in efficacy.

(85) Study 4

(86) Propionic and acetic acids were buffered with ammonium hydroxide to a pH of 3 and combined with pelargonic in the ratios listed. Acid content of the buffered solutions was determined by weight to weight ratio calculation (weight of acid/total weight of buffered solution) and adjusted to provide an equal acid value for each treatment. The treatments above were added to sterile deionized water to make at a 0.025% and 0.05% total acid solutions. The pHs of the deionized water solutions were recorded and any problems with solubility noted.

(87) 100 ul of a nutrient broth culture of Salmonella typhimurium was added to each dilution tube. After addition, tubes were vortexed and allowed to stand. At 4 and 24 hours after addition of the inoculum, 100 ul of the solution was plated on Standard Plate agar (triplicate plates). Plates were incubated at 37 C. for 24 hours prior to enumeration.

(88) TABLE-US-00008 TABLE 8 Effect of Pelargonic acid on the effectiveness of acetic or propionic against Salmonella Salmonella at time Interval Product 4 hr 24 hr Test Product Concentration (cfu/g) % reduction (cfu/g) % reduction Formic acid:Propionic acid (90:10) 0.025 600 57 <10 100 0.05 170 88 <10 100 100% Propionic pH 3 0.025 990 29 130 91 0.05 1000 29 50 96 99% Propionic pH 3:1% Pelargonic 0.025 1100 21 100 93 0.05 620 56 <10 100 98% Propionic pH 3:2% Pelargonic 0.025 1100 21 60 96 0.05 560 60 <10 100 95% Propionic pH 3:5% Pelargonic 0.025 780 44 <10 100 0.05 50 96 <10 100 90% Propionic pH 3:10% Pelargonic 0.025 220 84 <10 100 0.05 <10 100 <10 100 80% Propionic pH 3:20% Pelargonic 0.025 <10 100 <10 100 0.05 <10 100 <10 100 100% Acetic pH 3 0.025 1000 29 60 96 0.05 950 32 20 99 99% Acetic pH 3:1% Pelargonic 0.025 1200 14 90 94 0.05 820 41 <10 100 98% Acetic pH 3:2% Pelargonic 0.025 1100 21 40 97 0.05 710 49 <10 100 95% Acetic pH 3:5% Pelargonic 0.025 690 51 <10 100 0.05 40 97 <10 100 90% Acetic pH 3:10% Pelargonic 0.025 280 80 <10 100 0.05 <10 100 <10 100 80% Acetic pH 3:20% Pelargonic 0.025 <10 100 <10 100 0.05 <10 100 <10 100 Control 1400 1400

(89) Conclusion: The addition of pelargonic acid (1-20%) to propionic or acetic acid resulted in an increase in efficacy against salmonella.

(90) Study 5

(91) Propionic, acetic and pelargonic acids by themselves or in combination as listed were added to sterile deionized water to make at a 0.05%, 0.04%, 0.03%, 0.02% and 0.01% total acid solutions. The pHs of the deionized water solutions were recorded and any problems with solubility noted. 100 ul of a nutrient broth culture of Salmonella typhimurium was added to each dilution tube. After addition, tubes were vortexed and allowed to stand. At 24 hours after addition of the inoculum, 100 ul of the solution was plated on Standard Plate agar (triplicate plates). Plates were incubated at 37 C. for 24 hours prior to enumeration.

(92) TABLE-US-00009 TABLE 9 Effect of Pelargonic acid on the effectiveness of acetic or propionic against Salmonella Salmonella 24 hr afterTreatment (cfu/g) Test Product (%) 0.05 0.04 0.03 0.02 0.01 Formic acid:Propionic acid (90:10) 0 10 20 60 500 Pelargonic Acid 850 1500 UD 1300 1400 Propionic Acid 560 910 810 870 1200 Acetic Acid 1100 1100 UD 1100 UD Prop/Pelargonic (95/5) 0 30 240 360 1400 Acetic/Pelargonic (95/5) 20 130 UD 940 1100 Control 1400 UD = Unable to determine

(93) Conclusion: Test showed increased efficacy by mixing Propionic/Pelargonic (95/5) and Acetic/Pelargonic (95/5) 24 hours after treatment.

(94) Study 6

(95) Capric acid (5%, 10% or 20%) diluted in acetic acid or propionic acid was tested to determine its effectiveness against salmonella in feed.

(96) Commercial poultry feed amended with Salmonella typhimurium was treated with 1 or 3 kg/MT of the solutions listed below. Twenty four hours after treatment, 10 gr of feed was added to 90 ml Butterfield buffer, mixed and then 100 ul of the solution was plated on Standard Plate agar (triplicate plates). Plates were incubated at 37 C. for 24 hours prior to enumeration.

(97) TABLE-US-00010 TABLE 10 Effect of Capric/Acetic acids against Salmonella in feed. % Treatment cfu/g Reduction Control 5733 Formic:Acetic (1 kg/ton) 17 99.7 Acetic Acid (1 kg/ton) 3367 41.3 Acetic Acid (3 kg/ton) 2600 54.6 5% Capric in acetic acid (1 kg/ton) 3200 44.2 5% Capric in acetic acid (3 kg/ton) 3733 34.9 10% Capric in acetic acid (1 kg/ton) 3233 43.6 10% Capric in acetic acid (3 kg/ton) 2900 49.4 20% Capric in acetic acid (1 kg/ton) 3200 44.2 20% Capric in acetic acid (3 kg/ton) 4500 21.5

(98) TABLE-US-00011 TABLE 11 Effect of Capric/Propionic acids against Salmonella in feed. % Treatment cfu/g Reduction Control 4500 Formic:Prop 1 kg/ton 4100 8.9 Formic:Prop 3 kg/ton 2067 54.1 Prop Acid 1 kg/ton 4633 0 Prop Acid 3 kg/ton 5633 0 5% Capric in propionic acid 1 kg/ton 3233 28.2 5% Capric in propionic acid 3 kg/ton 3400 24.4 10% Capric in propionic acid 1 kg/ton 2367 47.4 10% Capric in propionic acid 3 kg/ton 4033 10.4 20% Capric in propionic acid 1 kg/ton 4067 9.6 20% Capric in propionic acid 3 kg/ton 3700 17.8

(99) Addition of capric acid to acetic or propionic acid at concentrations of 5-20% did not appear to significantly improve the efficacy of the organic acid against Salmonella in feed.

(100) Study 7

(101) Myristic acid (5%, 10% and 20%) diluted in propionic acid was tested to determine its effectiveness against salmonella in feed. Myristic acid was not soluble in acetic acid.

(102) Commercial poultry feed amended with Salmonella typhimurium was treated with 1 or 3 kg/MT of the solutions listed below. Twenty four hours after treatment, 10 gr of feed was added to 90 ml Butterfield buffer, mixed and then 100 ul of the solution was plated on Standard Plate agar (triplicate plates). Plates were incubated at 37 C. for 24 hours prior to enumeration.

(103) TABLE-US-00012 TABLE 12 Effect of Myristic/Propionic acids against Salmonella in feed. % Treatment cfu/g Reduction Control 40000 Formic:Prop 1 kg/MT 23533 41.17 Formic:Prop 3 kg/MT 7167 82.08 Prop acid 1 kg/MT 3967 90.08 Prop acid 3 kg/MT 233 99.42 5% Myristic in propionic acid 1 kg/MT 7767 80.58 5% Myristic in propionic acid 3 kg/MT 1500 69.25 10% Myristic in propionic acid 1 kg/MT 22567 43.58 10% Myristic in propionic acid 3 kg/MT 23333 41.67 20% Myristic in propionic acid 1 kg/MT 22667 43.33 20% Myristic in propionic acid 3 kg/MT 22967 42.58

(104) No beneficial effect in efficacy was observed when myristic acid was added at 5-20% to propionic acid compared to the standard propionic acid combination.

(105) Study 8

(106) Lauric acid (5%, 10%, 20%) diluted with propionic acid was tested to determine its effectiveness against salmonella in feed.

(107) Commercial poultry feed amended with Salmonella typhimurium was treated with 1 or 2 kg/MT of the solutions listed below. Twenty four hours after treatment, 10 gr of feed was added to 90 ml Butterfield buffer, mixed and then 100 ul of the solution was plated on Standard Plate agar (triplicate plates). Plates were incubated at 37 C. for 24 hours prior to enumeration.

(108) TABLE-US-00013 TABLE 13 Effect of Lauric/Propionic acids against Salmonella in feed. % Treatment cfu/g Reduction Control 33333 Formic:Prop 1 kg/MT 24633 26.1 Formic:Prop 3 kg/MT 10500 68.5 Prop Acid 1 kg/MT 2567 92.3 Prop Acid 3 kg/MT 0 100.0 5% Lauric in propionic acid 1 kg/MT 8767 73.7 5% Lauric in propionic acid 3 kg/MT 433 98.7 10% Lauric in propionic acid 1 kg/MT 7800 76.6 10% Lauric in propionic acid 3 kg/MT 833 97.5 20% Lauric in propionic acid 1 kg/MT 9100 72.7 20% Lauric in propionic acid 3 kg/MT 2333 93.0

(109) No beneficial effect was observed when lauric acid (5-20%) was added to propionic acid compared to the standard propionic acid combination.

(110) It will be apparent to those skilled in the art that a number of modifications and variations may be made in the present invention without departing from the scope of the invention. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.