Method of protecting active ingredients from degradation during pelleting

Abstract

A system for the protection against degradation during pelleting of one or more physiologically or pharmacologically active substances, comprising compositions in the form of micro particles or granules, particularly for use in the zootechnical and/or veterinary field. The micro particles include a core which contains one or more substances having a pharmacological action, food supplements or diagnostic media, said one or more substances being characterized by the presence, within their chemical structure, of a basic functional group, specifically including an amine functional group. The core comprises also one or more carboxylic acids and/or their salts and eventually one or more excipients. Said core is coated by an outer layer of fats or waxes, and preferably by a mixture of glyceride of fatty acids.

Claims

1. A method of feeding a pelleted feed product to ruminants, the method comprising: providing the ruminants a pelleted feed product, the pelleted feed product comprising a micro particle and at least one additional feed component, the micro particle comprising a mixture of at least one active substance and at least one carboxylic acid and/or salt thereof, wherein the mixture is embedded within a fatty substance, further wherein: the at least one active substance is selected from a group consisting of isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, arginine, histidine, betaine, carnitine, thiamine, pyridoxine, streptomycin, colistin, tiamulin, neomycin, arginine, glucosamine, niacinamide and their salts, betaine hydrochloride, lysine hydrochloride, thiamine hydrochloride, thiamine mononitrate, pyridoxine hydrochloride, colistin sulfate, and tiamulin fumarate, the at least one carboxylic acid and/or salt thereof is characterized by the presence of an acidic functional group and at least one lipophilic functional group, the fatty substance comprises one or more of a composition selected from a group consisting of waxes, fats, vegetable oil, and glycerides of fatty acids, and the at least one additional feed component is selected from a group consisting of dehulled soybean meal, corn gluten meal, corn distillers, dried grains, and extruded soybeans, wherein the at least one active substance includes a basic functional group that interacts with the acidic functional group of the carboxylic acid and/or salt thereof, and the lipophilic functional group adheres to the fatty substance such that the fatty substance defines an outer fat layer that protects the at least one active substance from the at least one additional feed component in the pelleted feed product, wherein, in response to ingesting the pelleted feed product, the protected at least one active substance remains protected from ruminal degradation and available for absorption in an intestine, and wherein the micro particle has a size between 0.1 and 5000 microns.

2. The method as defined in claim 1, wherein the at least one active substance is present in an amount between 50 and 100% by weight of the mixture.

3. The method as defined in claim 1, wherein the at least one active substance is present in an amount between 60 and 100% by weight of the mixture.

4. The method as defined in claim 1, wherein the at least one carboxylic acid and/or salt thereof is present in an amount between 0.1 and 50% by weight of the mixture.

5. The method as defined in claim 1, wherein the at least one carboxylic acid and/or salt thereof is present in an amount between 0.1 and 40% by weight of the mixture.

6. The method as defined in claim 1, wherein the fatty substance has a melting point of between 55 C. and 62 C.

7. The method as defined in claim 1, wherein the amount of fatty substance is between 15 and 50% of the final weight of the active substance, the at least one carboxylic acid and/or salt thereof, and the fatty substance.

8. The method as defined in claim 1, wherein the at least one active substance content is between about 0.35% and about 1% by final weight of the pelleted feed product on an as-fed basis.

9. The method as defined in claim 1, wherein the at least one carboxylic acid and/or salt thereof is selected from the group consisting of short, medium and long chain saturated and unsaturated fatty acids and their salts consisting of butyric, myristic, lauric, palmitic, stearic, oleic and arachidic acids and their salts, benzoic acid and its salts, and dicarboxylic acids consisting of adipic and sebacic acids and their salts.

10. The method as defined in claim 1, wherein the mixture further comprises excipients.

11. The method as defined in claim 10, wherein the excipients comprise a basic functional group.

12. The method as defined in claim 10, wherein the excipients are selected from the group consisting of clay, cob meal, silica, silicates, microcrystalline cellulose, polyvinylpyrrolidone, calcium phosphate, calcium carbonate, magnesium oxide, starch, alginate, amino modified starches, amino modified silica, amino modified clays, amino modified cellulose, acrylic polymers with amine groups, chitosan, and gelatin.

13. The method as defined in claim 10, wherein said excipients are present in an amount between 0.1 and 40% by weight of the mixture.

14. The method as defined in claim 1, wherein the at least one active substance is present in an amount between 30 and 100% by weight of the mixture.

15. The method as defined in claim 1, wherein the at least one carboxylic acid and/or salt thereof is present in an amount between 0.1 and 70% by weight of the mixture.

16. The method as defined in claim 1, wherein the fatty substance has a melting point of between 50 C. and 80 C.

17. The method as defined in claim 1, wherein the glycerides are mixtures of hydrogenated fatty acid glycerides.

18. The method as defined in claim 17, wherein said hydrogenated fatty acid glycerides comprise a C-16 fatty acid triglyceride content of between 40 and 70 weight % and a C-18 fatty acid triglyceride content of between 30 and 50 weight % of a total fatty acids content.

19. The method as defined in claim 1, wherein the amount of the fatty substance is between 10 and 60% of the final weight of the active substance, the at least one carboxylic acid and/or salt thereof, and the fatty substance.

Description

EXAMPLE 1. Controlled-Release Formulation Based on a Core that Contains Active Ingredients and Carboxylic Acid Intimately Mixed in it

(1) TABLE-US-00001 TABLE 1 Composition: Core Active ingredient L-Lysine monohydrochloride 12.6 kg (ADM, Decatur Illinois, USA) L-Lysine in aqueous solution 2 kg at 50% (ADM, Decatur Illinois, USA) Carboxylic acid Stearic acid 2 kg (BBC srl, Torre Boldone BG, IT) Coating Fat Vegetoil S, hydrogenated 4.4 kg layer vegetable oil (BBC srl, Torre Boldone BG, IT)

(2) The dry L-lysine monohydrochloride was mixed with liquid basic L-Lysine and stearic acid at 70 C. in a ploughshare mixer for 30 minutes. The core was then cooled to 40 C. and the coating layer was applied at 65 C. by spraying it in a pan coater. The microparticles were then cooled to under 45 C.

(3) A release dissolution test was performed with a USP paddle apparatus (Apparatus 2) at 38 C. and 100 r.p.m, in 700 ml of distilled water.

(4) After 24 hours the released L-lysine was 18.7% with a standard deviation of 0.8.

EXAMPLE 2. Controlled-Release Formulation Based on a Core that Contains Active Ingredients and Carboxylic Acid Added on their Surface

(5) TABLE-US-00002 TABLE 2 Composition: Core Active ingredients L-Lysine monohydrochloride 240.38 kg (ADM, Decatur Illinois, USA) L-Lysine in aqueous solution 9.62 kg at 50% (ADM, Decatur Illinois, USA) Carboxylic acid Stearic acid 44.6 kg (BBC srl, Torre Boldone BG, IT) Coating layer Vegetoil S, hydrogenated 62.4 kg vegetable oil (BBC srl, Torre Boldone BG, IT)

(6) The dry L-lysine monohydrochloride was mixed with liquid basic L-Lysine at 45 C. in a ribbon blender. The liquid was sprayed on the dry L-lysine monohydrochloride using a spraying nozzle at 2 barr pressure over a time of 5 minutes. The granules were then dried in a fluid bed. Stearic acid at 70 C. was added to the surface of the preformed microgranules by spraying it in a pan coater. The core was then cooled to 40 C. and the coating layer was applied at 65 C. by spraying it in a pan coater. The microparticles were then cooled to under 45 C.

(7) A release dissolution test was performed with a USP paddle apparatus (Apparatus 2) at 38 C. and 100 r.p.m, in 700 ml of distilled water.

(8) After 24 hours the released L-lysine was 6.0% with a standard deviation of 0.6.

EXAMPLE 3. Use of an Excipient in the Coating Layer

(9) TABLE-US-00003 TABLE 3 Composition: Core Active L-Lysine monohydrochloride 6523.8 g ingredients (ADM, Decatur IL, USA) L-Lysine in aqueous solution 476.2 g at 50% (ADM, Decatur IL, USA) Carboxylic acid Stearic acid 1000 g (BBC srl, Torre Boldone BG, IT) coating layer fat Vegetoil S, hydrogenated 1980 g vegetable oil (BBC srl, Torre Boldone BG, IT) excipient Soy lecithin 20 g

(10) The dry L-lysine monohydrochloride was mixed with liquid L-lysine at 45 C. in a rotary granulating machine. The granules were then dried in a fluid bed. Stearic acid at 70 C. was added to the preformed microgranules by spraying it in a pan coater. The core was then cooled at 40 C. and coated at 65 C. with the liquid fat containing the lecithin. The microparticles were then cooled to under 45 C.

(11) A release dissolution test was performed with a USP paddle apparatus (Apparatus 2) at 38 C. and 100 r.p.m, in 700 ml of distilled water.

(12) After 24 hours the released L-lysine was 13.8% with a standard deviation of 0.4.

COMPARATIVE EXAMPLE 4. Formulation without the Carboxylic Acid in the Core

(13) In order to demonstrate the importance of the interaction between the basic function of the active with the carboxylic acid a formulation without the carboxylic acid and with the coating fat layer only was also produced and tested for release of the active ingredient.

(14) TABLE-US-00004 TABLE 4 Composition: Core Active ingredients L-Lysine monohydrochloride 7810 g (ADM, Decatur Illinois, USA) Coating fat Vegetoil PH, hydrogenated 3190 g layer vegetable oil (BBC srl, Torre Boldone BG, IT)

(15) The dry L-lysine monohydrochloride was coated with liquid hydrogenated vegetable oil 70 C. This was accomplished in a pan coater. The micro particles were than cooled to under 45 C.

(16) A release dissolution test was performed with an USP paddle apparatus at 38 C. and 100 r.p.m, in 700 ml of distilled water.

(17) After 24 hours the released L-lysine was 80%.

EXAMPLE 5. Controlled-Release Formulation Based on a Core that Contains Active Ingredient and Carboxylic Acid Salt Intimately Mixed in it

(18) TABLE-US-00005 TABLE 5 Composition: Core Active ingredient Taminizer C 210 kg and carboxylic (Taminco N.V., Gent, acids salt Belgium) Coating layer fat Vegetoil S, hydrogenated 199 kg vegetable oil (BBC srl, Torre Boldone BG, IT) excipient Soy lecithin 1 kg

(19) Taminizer C is a commercial brand product made according to patent application WO2010072842 A1 and containing choline chloride and at least one salt of medium to long chain fatty acid. Taminizer C was used as a core and it was coated at 65 C. with the liquid fat, containing the lecithin, by spraying it in a pan coater. The microparticles were then cooled to under 4 5 C.

(20) A release dissolution test was performed with a USP paddle apparatus (Apparatus 2) at 38 C. and 100 r.p.m, in 700 ml of distilled water.

(21) After 24 hours the released choline chloride was 12.9% with a standard deviation of 0.6.

EXAMPLE 6. Controlled-Release Formulation Based on a Core that Contains an Excipient at Very Low Concentration

(22) TABLE-US-00006 TABLE 6 Composition: Core Active DL-methionine 9730 g ingredient (Sumitomo Chemical, Tokyo, Japan) excipient Starch licatab M 270 g (Roquette, Freres 62136 Lestrem France) water FU distilled water 675 ml ACEF spa (Fiorenzuola, PC, IT) Carboxylic Stearic acid 1785 g acid (BBC srl, Torre Boldone BG, IT) coating fat Vegetoil S, hydrogenated vegetable oil 2500 g layer (BBC srl, Torre Boldone BG, IT)

(23) The dry DL-methionine was mixed with starch and water in the chamber of a rotary granulating machine. The granules were then dried in a fluid bed. Afterwards the stearic acid at 70 C. was added to the preformed microgranules by spraying it in a pan coater. The core was then cooled to 40 C. and the coating layer was applied at 65 C. The microparticles were then cooled to under 45 C.

(24) A release dissolution test was performed with an USP paddle apparatus (Apparatus 2) at 38 C. and 100 r.p.m, in 700 ml of distilled water.

(25) After 24 hours the released DL-methionine was 11.4% with a standard deviation of 1.2.

EXAMPLE 7. Controlled-Release Formulation Based on a Core that Contains an Excipient at High Concentration

(26) TABLE-US-00007 TABLE 7 Composition: Core Active Choline Chloride 70% on cereal 4000 g ingredient carrier (Balchem Corporation, and NY, USA) excipient L-lysine in aqueous solution at 50% 143 (ADM, Decatur Illinois, USA) carboxylic Stearic acid 857 g acid (BBC srl, Torre Boldone BG, IT) coating fat Vegetoil S, hydrogenated vegetable 3489.5 g layer oil (BBC srl, Torre Boldone BG, IT) excipient Soy lechitin 10.5 g

(27) Choline Chloride 70% is a dry commercial product containing choline chloride on a cereal carrier. It was mixed with liquid basic L-Lysine and stearic acid at 70 C. in a ploughshare mixer for 30 minutes. The core was then cooled to 40 C. and the coating layer was applied at 65 C. by spraying it in a pan coater. The microparticles were then cooled to under 45 C.

(28) A release dissolution test was performed with an USP paddle apparatus (Apparatus 2) at 38 C. and 100 r.p.m, in 700 ml of distilled water.

(29) After 24 hours the released choline chloride was 4.0% with a standard deviation of 2.1.

EXAMPLE 8. Protection Against Degradation During Pelleting

(30) The micro particles were prepared as in Example 3 and are referred to in this example as USA Lysine. The study is a replicated 44 Latin Square design using eight steers (27624 kg) that were fed a TMR twice daily, (14.9% CP, 2.75 Mcal/kg ME) ad libitum with 60% of the TMR offered at 8:00 a.m. and the remainder 12 h later. On the 7.sup.th day, plasma was collected through jugular veinipuncture at 0, 2, 4, 6 and 8 h post-feeding. Each plasma sample was analyzed for L-lysine content, [p-Lys]. Dietary Lys treatments were offered as USA Lysine and supplied as 3 types of incorporations into the grain mix: hand mixed (50 and 100 g; HSO and H100), mechanical mixed (100 g; M100) and as pellets (100 g; P100). The results of HSO did not increase [p-Lys] over the basal diet. The [p-Lys] concentrations of H100, M100 and P100 were different from the basal diet (P<0.0001). Mechanical mixing and pelleting of USA Lysine in the top-dress did not affect [p-Lys] compared with hand mixed. Regardless of dietary incorporation method, the regression equation applied to the [p-Lys] of steers fed 100 g of USA Lysine, predicts at least 50% bioavailability of Lys from USA Lysine. Specifically, 53.88% was bioavailable from the H100 incorporation, 54.76% from the M100 incorporation and 51.31% from the P100 incorporation. That the bioavailability of the active ingredient did not significantly diminish even though the micro particles had been processed into a pelleted feed was surprising.

EXAMPLE 9. Protection Against Degradation During Pelleting

(31) Materials and Methods

(32) A commercially available protected form of lysine, LysiPEARL (Kemin Industries, Inc., Des Moines, Iowa) was added to a concentrate feed and pelleted according to industry recommendations. Two physical forms of feed were used: (1) concentrate feed, meal form, as a control; and (2) concentrate feed, pellet form. The product was added to the meal form of the concentrate feed comprised of dehulled soybean meal, corn gluten meal, corn distillers dried grains and extruded soybeans, having 85.5% dry matter, 38.0% crude protein, 5.0% crude fat and 6.0% crude fiber. The mixture was pelleted using a standard pellet mill for ruminant feed. Based on the typical diet inclusion percentage for LysiPEARL and the feeding percentage for a complete grain mix, the concentration of LysiPEARL in the feed was approximately 0.35% on an as-fed basis (AF). A slightly greater concentration could be justified by arguing that LysiPEARL could be included in a lower inclusion product base such as a protein mix rather than a complete grain mix. This product base would then increase the practical concentration to a value close to 1.00% AF.

(33) The background concentration of lysine was expected to be 2.61% in the basis feed (not supplemented) and 2.78% in those treatments supplemented with 0.35% (AF) LysiPEARL. It is estimated that the amount of lysine that would be present after ruminal incubation would be approximately 50% of the values stated above. Thus, if 16 g of the treatment were incubated (8 replications multiplied by 2 g for each replication), then there would be 220 mg of lysine after 16 hours of ruminal incubation.

(34) The experimental design produced 5 treatments (Table 8).

(35) TABLE-US-00008 TABLE 8 Treatments Treatment ID Description 1 Concentrate, meal form 2 Concentrate, pellet form 3 Concentrate, meal form with LysiPEARL 4 Concentrate, pellet form with LysiPEARL 5 LysiPEARL product form

(36) Approximately 2 g of each treatment was placed into each IV bag at 8 replications. The extent of in-vitro (IV) ruminal digestion was measured after 16 hours. The constituents monitored were dry matter and lysine. Approximately 3 L of rumen fluid and 200 g of fiber mat were collected from each of 4 donor Jersey steers at 1 hour post-prandial. The 12 L of ruminal contents were composited and aliquots were mixed with Van Soest buffer so that 600 ml of the buffered ruminal contents were distributed among the IV vessels and volume raised to 1800 ml using distilled water. All procedures were under a stream of carbon dioxide in each IV vessel. After the ruminal contents and buffer were mixed, the IV bags were added to the IV vessels which remained under carbon dioxide. The IV vessels were then placed into an air-jacketed, anaerobic incubator at a pH of 6.5, 38 C. and an environment of 3% carbon dioxide and artificially ruminated each 3 hours.

(37) Post-ruminal or gastric/intestinal IV incubations were conducted in three phases: Phase 1, gastric; Phase 2, illeal; and Phase 3, wash out. The Phase 1 solution consisted of 50% hydrochloric acid in distilled water plus 0.1 g pepsin in 500 ml then raised to 1800 ml. Phase 2 solution consisted of pH 8 phosphate buffer plus a semi-purified enzymatic cocktail consisting of pancreatic amylase (0.75 g), lipase (0.75 g) and pancreatin (0.75 g) then raised to 1800 ml. Phase 3 was soaking in a water solution of sodium azide and sodium lauryl sulfate at 11 C. to remove adhering micro-organisms.

(38) After each IV incubation, the IV bags were dried in a forced-air oven at 20 C. until a constant weight. The dry bags were sent for analysis of lysine. Lysine concentration in the dry contents of the appropriate IV bags (digestive residue) was measured by AOAC 994.12.

(39) Statistics were performed using the appropriate models in XLSTAT (release 2007.5, updated in 2012, Addinsoft USA, NY).

(40) Discussion

(41) The main objective of this study was to quantify the effect of physical form (meal versus pellet) of a concentrate or complete feed on the digestive properties of a commercial protected lysine supplement. Rumen escape of the active molecule is the measurement of the amount of the molecule that is not degraded (rumen undegraded) during ruminal incubation (in this study, 16 hours was the length of ruminal incubation).

(42) Rumen undegraded values for LysiPEARL containing rumen-protected lysine are shown in Tables 9 (meal form) and 10 (pellet form) along with intestinal digestibility (amount of digestion (gastric and illeal) of the material that is not ruminally degraded (the digestibility of rumen undegraded lysine). Rumen undegraded material can also be called: rumen bypass material, or rumen escape material.

(43) TABLE-US-00009 TABLE 9 Digestive properties of lysine in meal Treatment Initial Ruminal IV Rumen Intestinal IV Total tract IV Total tract ID % lysine % lysine Undegraded % lysine % lysine Undegraded 1 1.71 24.62 75.38 73.05 73.11 26.89 5* 37.19 71.42 26.58 92.51 92.56 7.44 3 2.08 35.84 64.16 72.87 72.96 27.04 *LysiPEARL

(44) TABLE-US-00010 TABLE 10 Digestive properties of lysine in pellets Treatment Initial Ruminal IV Rumen Intestinal IV Total tract IV Total tract ID % lysine % lysine Undegraded % lysine % lysine Undegraded 2 1.76 18.95 81.05 67.68 66.85 33.15 5* 37.19 71.42 28.58 93.38 93.43 6.57 4 2.07 33.03 66.97 72.54 72.63 27.37 *LysiPEARL

(45) A comparison of the data in Tables 9 and 10 reveals that the amount of lysine available at the intestinal level of the animal, thereby by-passing the rumen (Total tract IV % lysine) is almost the same in the pellet form (72.63) as it is in the meal form (72.96). Accordingly, the lysine in LysiPEARL was sufficiently protected to avoid degradation by the heat and pressures of pelleting.

(46) The foregoing description and drawings comprise illustrative embodiments of the present inventions. The foregoing embodiments and the methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method. The foregoing description and drawings merely explain and illustrate the invention, and the invention is not limited thereto, except insofar as the claims are so limited. Those skilled in the art that have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention.