Composition for controlled release of physiologically active substances and process for its preparation

Abstract

The present invention relates to a rumen-resistant composition in the form of microgranules, a process for its production and a feedstuff containing such composition.

Claims

1. A rumen-resistant composition in the form of microgranules, each microgranule containing: i) a core comprising: a) one or more physiologically active substances selected from the group consisting of amino acids, vitamins, enzymes, proteins, carbohydrates, probiotic microorganisms, prebiotic foods, mineral salts, choline derivatives of choline and organic acids; b) a matrix comprising substances selected from the group consisting of binding substances, inert substances and extrusion adjuvants; and c) a disintegrant agent selected from the group consisting of amides in dry form, vegetal lecithins, ethoxylated oils, monoglycerides, diglycerides of fatty acids, agar agar in dry form, effervescent mixtures of bicarbonate of an alkali metal and a polycarboxylic acid, effervescent mixtures of bicarbonate of ammonium and a polycarboxylic acid, cellulose in dry form, and methacrylate copolymers; and ii) at least two coating layers of the core, each having a different composition; wherein a first coating layer proximal to the core comprises (i) a hydrophobic substance selected from the group consisting of: fats, hydrogenated oils, monoglycerides of fatty acids, diglycerides of fatty acids, fatty acid esters, and fatty acid alcohols, and (ii) at least one pollutant substance selected from the group of emulsifying substances, fatty acids, and methacrylate copolymers; wherein a second coating layer, arranged on the outside of the first layer, comprises (i) at least one pollutant substance selected from the group of emulsifying substances, fatty acids, and methacrylate copolymers, and (ii) a hydrophobic substance selected from the group consisting of microcrystalline waxes, paraffin waxes, vegetal waxes and edible synthetic waxes.

2. The composition according to claim 1, wherein said emulsifying substance is selected from the group consisting of soy lecithin, sunflower lecithin, ethoxylated castor oil, alkaline metal alginates, magnesium alginates, and a combination thereof.

3. The composition according to claim 1, wherein said polluting fatty acid is selected from the group consisting of palmitic acid, oleic acid, linoleic acid, linolenic acid, stearic acid, and a combination thereof.

4. The composition according to claim 1, wherein said methacrylate copolymer is polymethyl methacrylate (PMMA).

5. The composition according to claim 1, wherein said microgranule comprises at least two coating layers; and each coating layer comprises an amount of polluting substance between 0.01% and 40% by weight with respect to the weight of the coating layer.

6. The composition according to claim 5, wherein each coating layer comprises an amount of emulsifying substance between 0.1% and 6% by weight with respect to the weight of the coating layer.

7. The composition according to claim 5, wherein each coating layer comprises an amount of polluting fatty acid between 10% and 35%.

8. The composition according to claim 1, wherein one or each coating layer comprises palmitic acid, oleic acid, linoleic acid, linolenic acid, stearic acid or a combination thereof.

9. The composition according to claim 1, wherein one or each coating layer comprises soy or sunflower lecithin.

10. The composition according to claim 1, wherein one or each coating layer comprises ethoxylated castor oil.

11. The composition according to claim 1, wherein one or each coating layer comprises sodium or magnesium alginate.

12. The composition according to claim 1, wherein one or each coating layer comprises polymethyl methacrylate (PMMA).

13. The composition according to claim 1, wherein the two coating layers comprise different amounts of pollutants.

14. The composition according to claim 1, wherein said two coating layers comprise different amounts of emulsifying substances, fatty acids, or methacrylate copolymers.

15. The composition according to claim 1, wherein the first coating layer comprises (i) one or more hydrogenated vegetable oil in an amount that is about 50% to about 90% by weight with respect to the weight of the first coating layer, and (ii) a fatty acid and/or an emulsifying agent in an amount that is about 5% to about 50% by weight with respect to the weight of the second coating layer.

16. The composition according to claim 1, wherein in each microgranule the total weight of the coating layers is between 10% and 50% of the microgranule weight.

17. The composition according to claim 1, wherein said core comprises a disintegrant agent in an amount by weight between 1.5% and 6.5%.

18. The composition according to claim 1, wherein said core has a cylindrical shape, the height of which is comprised between 0.5 mm and 2 mm or a spheroidal shape, the diameter of which is comprised between 0.5 mm and 2 mm.

19. A premixture for animal feedstuff comprising the composition according claim 1.

20. A feedstuff comprising the premixture according to claim 19.

21. A process for the preparation of a composition according to claim 1, comprising the steps of: obtaining microgranules by extruding a mixture comprising: a) one or more physiologically active substances selected from the group consisting of amino acids, vitamins, enzymes, proteins, carbohydrates, probiotic microorganisms, prebiotic foods, mineral salts, choline derivatives of choline and organic acids; b) one or more matrix substances selected from the group consisting of binding substances, inert substances, and extrusion adjuvants; and c) a disintegrant agent selected from the group consisting of amides in dry form, vegetal lecithins, ethoxylated oils, monoglycerides, diglycerides of fatty acids, agar agar in dry form, effervescent mixtures of bicarbonate of an alkali metal and a polycarboxylic acid, effervescent mixtures of bicarbonate of ammonium and a polycarboxylic acid, cellulose in dry form, and methacrylate copolymers; optionally subjecting the microgranule to spheronization; forming at least two coating layers on the microgranules, each having a different composition, wherein a first coating layer proximal to the microgranule comprises (i) a hydrophobic substance selected from the group consisting of: fats, hydrogenated oils, monoglycerides of fatty acids, diglycerides of fatty acids, fatty acid esters, and fatty acid alcohols, and (ii) at least one pollutant substance selected from the group of emulsifying substances, fatty acids, and methacrylate copolymers; wherein a second coating layer, arranged on the outside of the first layer, comprises (i) at least one pollutant substance selected from the group of emulsifying substances, fatty acids, and methacrylate copolymers, and (ii) a hydrophobic substance selected from the group consisting of microcrystalline waxes, paraffin waxes, vegetal waxes and edible synthetic waxes.

Description

EXAMPLES

(1) For the following examples, the following analysis and inspection tools were used: Sieves, arranged at the end of each single production step, for the microgranule dimension line check; Microscope for visual checks; Melting point gauge for thermal resistance check; Penetrometer for hardness and mechanical resistance check; Automatic titrator or HPLC for the quantitative determination of the active ingredients (concentration); Pharmaceutical dissolver for determining the degree of ruminal bypass. The experimental conditions of use were 39° C., 15 rpm, 8 hours presence in the solution with “ruminal” pH of about 6.8. Daisy.sup.II ANKOM: commercially available laboratory artificial rumen, for determining the degree of ruminal bypass. The experimental conditions of use were 39° C., 8 hours presence in ruminal conditions, buffer solution at “ruminal” pH with insertion of “ruminal inoculum” and re-creation of ruminal anaerobiosis according to a university procedure known to those skilled in the art. The pH normally tested in the quality control phase is 6.8. Pharmaceutical dissolver for determining the degree of post-ruminal digestibility. The experimental conditions of use followed the procedure known to experts in the field as “Boisen Test” and, in particular, they were: 39° C., 30 rpm, 2 h, buffer solution with “gastric” pH (2.0)+pepsin inoculum. 39° C., 30 rpm, 4 h, buffer solution with “intestinal” pH (6.8)+pancreatine inoculum. 39° C., 30 rpm, 18 h, buffer solution with “intestinal” pH (6.8)+lipase and bile extract inoculum.

Example 1—Preparation of Microgranules Containing Choline Chloride

Example 1.1

(2) 425 kg of choline chlorine with a purity of 99% were mixed with 10 kg of spray rice wax, 15 kg of zinc stearate, 10 kg of soy lecithin and 40 kg of silica. The mixture was extruded using an extruder with sectors at different temperature gradients according to the following program:

(3) TABLE-US-00001 Sector 1 Sector 2 Sector 3 Sector 4 Sector 5 Sector 6 85° C. 85° C. 50° C. 50° C. 45° C. 65° C.

(4) The cores thus obtained had a concentration of 85% in choline chloride. The cores were subsequently subjected to coating in a pan.

(5) A first coating layer was formed, coating 400 kg of microgranules with 120 kg of a coating mixture comprising: 65% by weight of hydrogenated palm oil; 32% by weight of palmitic acid; 3% by weight of soy lecithin.

(6) A second coating layer was then formed, coating the microgranules coated by the first layer with 40 kg of a coating mixture comprising: 50% by weight of hydrogenated palm oil; 50% by weight of rice wax.

(7) All the above indicated percentages are percentages by weight based on the total weight of the covering layer.

(8) The microgranules thus obtained had a concentration of 60% by weight of choline chloride with respect to the total weight of the microgranules.

(9) Then the microgranules were subjected to an evaluation of the degree of ruminal by-pass, post-ruminal digestibility and total bioavailability using the Boisen method. For a complete description of the Boisen method, reference is made to the publication S. Boisen, J. A. Fernàndez “Prediction of total tract digestibility of energy feedstuffs and FIG. diets by in vitro analyses” Animal Feed Science Technology 68, 277-286, 1997.

(10) The in vitro results are highlighted in Table 1.

(11) TABLE-US-00002 TABLE 1 Sam- Content Degree of ruminal bypass Digestibility Bioavailability ple (%) (*) (**) (**) 1 59.4 80.3 89.3 71.7

Example 1.2

(12) 425 kg of choline chlorine with a purity of 99% were mixed with 10 kg of spray rice wax, 15 kg of zinc stearate, 10 kg of soy lecithin, 5 kg of citric acid, 5 kg of sodium bicarbonate and 30 kg of silica. The mixture was extruded using an extruder with sectors at different temperature gradients according to the following program:

(13) TABLE-US-00003 Sector 1 Sector 2 Sector 3 Sector 4 Sector 5 Sector 6 70° C. 75° C. 80° C. 80° C. 80° C. 85° C.

(14) The cores thus obtained had a concentration of 85% in choline chloride. The cores were subsequently subjected to coating in a pan.

(15) A first coating layer was formed, coating 400 kg of microgranules with 100 kg of a coating mixture comprising: 75% by weight of hydrogenated palm oil; 23% by weight of palmitic acid; 2% by weight of ethoxylated castor oil.

(16) A second coating layer was then formed, coating the microgranules coated by the first layer with 60 kg of a coating mixture comprising: 50% by weight of hydrogenated rapeseed oil; 45% by weight of carnauba wax; 5% by weight of polymethyl methacrylate.

(17) All the above indicated percentages are percentages by weight based on the total weight of the covering layer.

(18) The microgranules thus obtained had a concentration of 60% by weight of choline chloride with respect to the total weight of the microgranules.

(19) Then the microgranules were subjected to an evaluation of the degree of ruminal by-pass, post-ruminal digestibility and total bioavailability using the Boisen method. For a complete description of the Boisen method, reference is made to the publication S. Boisen, J. A. Fernàndez “Prediction of total tract digestibility of energy feedstuffs and FIG. diets by in vitro analyses” Animal Feed Science Technology 68, 277-286, 1997.

(20) The in vitro results are highlighted in Table 2.

(21) TABLE-US-00004 TABLE 2 Sam- Content Degree of ruminal bypass Digestibility Bioavailability ple (%) (*) (**) (**) 2 59.7 78.2 92 71.9

Example 1.3

(22) The extruded cores obtained with the procedure of Example 1.2 were spheronized with the use of an aqueous 75% choline chloride solution as an adjuvant for spheronization and subsequently they were coated in a pan.

(23) A first coating layer was formed, coating 400 kg of spheroidal microgranules with 125 kg of a coating mixture comprising: 72% by weight of hydrogenated palm oil; 25% by weight of palmitic acid; 3% by weight of ethoxylated castor oil.

(24) A second coating layer was then formed, coating the microgranules coated by the first layer with 20 kg of a coating mixture comprising: 50% by weight of hydrogenated palm oil; 50% by weight of rice wax.

(25) Finally, a third coating layer of 20 kg containing 100% of polymethyl methacrylate was formed.

(26) All the above indicated percentages are percentages by weight based on the total weight of the covering layer.

(27) The microgranules thus obtained had a concentration of 60% by weight of choline chloride with respect to the total weight of the microgranules.

(28) The in vitro results are highlighted in Table 3.

(29) TABLE-US-00005 TABLE 3 Sam- Content Degree of ruminal bypass Digestibility Bioavailability ple (%) (*) (**) (**) 3 61.4 86.4 85.0 73.4

Example 2—Preparation of Microgranules Containing Lysine HCl

Example 2.1

(30) 400 kg of micronized lysine hydrochloride were mixed with 60 kg of granulated rice wax, 48 kg of powdered powder, and 12 kg of soy lecithin. The mixture was extruded using an extruder with sectors at different temperature gradients according to the following program:

(31) TABLE-US-00006 Sector 1 Sector 2 Sector 3 Sector 4 Sector 5 Sector 6 85° C. 65° C. 50° C. 50° C. 50° C. 50° C.

(32) The cores thus obtained had a concentration of 75% in lysine hydrochloride. The cores were subsequently subjected to coating in a pan.

(33) A first coating layer was formed, coating 350 kg of microgranules with 100 kg of a coating mixture comprising: 95% by weight of hydrogenated palm oil; 5% by weight of sunflower lecithin.

(34) A second coating layer was then formed, coating the microgranules coated by the first layer with 75 kg of a coating mixture comprising: 50% by weight of hydrogenated rapeseed oil; 48% by weight of carnauba wax; 2% by weight of sunflower lecithin.

(35) All the above indicated percentages are percentages by weight based on the total weight of the covering layer.

(36) The microgranules thus obtained had a concentration of 50% by weight of lysine hydrochloride with respect to the total weight of the microgranules.

(37) Then the microgranules were subjected to an evaluation of the degree of ruminal by-pass, post-ruminal digestibility and total bioavailability using the Boisen method. For a complete description of the Boisen method, reference is made to the publication S. Boisen, J. A. Fernàndez “Prediction of total tract digestibility of energy feedstuffs and FIG. diets by in vitro analyses” Animal Feed Science Technology 68, 277-286, 1997. The in vitro results are highlighted in Table 4.

(38) TABLE-US-00007 TABLE 4 Sam- Content Degree of ruminal bypass Digestibility Bioavailability ple (%) (*) (**) (**) 4 50.2 73.4 86 63.1

Example 2.2

(39) 324 kg of micronized lysine hydrochloride were mixed with 64 kg of rice wax spray and 12 kg of soy lecithin. The mixture was extruded using an extruder with sectors at different temperature gradients according to the following program:

(40) TABLE-US-00008 Sector 1 Sector 2 Sector 3 Sector 4 Sector 5 Sector 6 85° C. 85° C. 80° C. 80° C. 80° C. 80° C.

(41) The cores thus obtained had a concentration of 80% in lysine hydrochloride. The cores were spheronized, using a 50% lysine hydrochloride solution in water as a spheronization adjuvant, and subsequently subjected to coating in a pan.

(42) A first coating layer was formed, coating 350 kg of microgranules with 90 kg of a coating mixture comprising: 75% by weight of hydrogenated palm oil; 23% by weight of palmitic acid; 2% by weight of ethoxylated castor oil.

(43) A second coating layer was then formed, coating the microgranules coated by the first layer with 50 kg of a coating mixture comprising: 48% by weight of hydrogenated rapeseed oil; 52% by weight of rice wax; 2% by weight of sunflower lecithin.

(44) A third coating layer was then formed, coating the microgranules coated by the second layer with 40 kg of a coating mixture identical to that of the first layer.

(45) A fourth coating layer was then formed, coating the microgranules coated by the third layer with 30 kg of a coating mixture identical to that of the second layer.

(46) All the above indicated percentages are percentages by weight based on the total weight of the covering layer.

(47) The microgranules thus obtained had a concentration of 50% by weight of lysine hydrochloride with respect to the total weight of the microgranules.

(48) Then the microgranules were subjected to an evaluation of the degree of ruminal by-pass, post-ruminal digestibility and total bioavailability using the Boisen method. For a complete description of the Boisen method, reference is made to the publication S. Boisen, J. A. Fernàndez “Prediction of total tract digestibility of energy feedstuffs and FIG. diets by in vitro analyses” Animal Feed Science Technology 68, 277-286, 1997.

(49) The in vitro results are highlighted in Table 5.

(50) TABLE-US-00009 TABLE 5 Sam- Content Degree of ruminal bypass Digestibility Bioavailability ple (%) (*) (**) (**) 5 49.5 78 91 71

Example 3—Preparation of Microgranules Containing DL-Methionine

Example 3.1

(51) 430 kg of DL-methionine were mixed with 30 kg of rice wax spray, 25 kg of hydrogenated palm oil, 5 kg of silica, 5 kg of citric acid and 5 kg of sodium bicarbonate. The mixture was extruded using an extruder with sectors at different temperature gradients according to the following program:

(52) TABLE-US-00010 Sector 1 Sector 2 Sector 3 Sector 4 Sector 5 Sector 6 85° C. 45° C. 40° C. 40° C. 35° C. 60° C.

(53) The cores thus obtained had a concentration of 85% DL-methionine. The cores were subsequently subjected to coating in a pan.

(54) A first coating layer was formed, coating 400 kg of microgranules with 40 kg of a coating mixture comprising: 70% by weight of hydrogenated rapeseed oil; 30% by weight of linolenic acid.

(55) A second coating layer was then formed, coating the microgranules coated by the first layer with 35 kg of a coating mixture comprising: 35% by weight of hydrogenated palm oil; 65% by weight of carnauba wax.

(56) A third coating layer was then formed, coating the microgranules coated by the second layer with 10 kg of a coating mixture identical to that of the first layer.

(57) All the above indicated percentages are percentages by weight based on the total weight of the covering layer.

(58) The microgranules thus obtained had a concentration of 70% by weight of DL-Methionine with respect to the total weight of the microgranules.

(59) Then the microgranules were subjected to an evaluation of the degree of ruminal by-pass, post-ruminal digestibility and total bioavailability using the Boisen method. The in vitro results are highlighted in Table 6.

(60) TABLE-US-00011 TABLE 6 Sam- Content Degree of ruminal bypass Digestibility Bioavailability ple (%) (*) (**) (**) 6 69.3 82.3 75.0 61.7

Example 3.2

(61) Further 400 kg of extruded granules obtained following the same procedure described in the first part of the previous example were coated in a pan using 3 coating layers.

(62) A first coating layer was formed, coating 400 kg of microgranules with 35 kg of a coating mixture comprising: 82% by weight of hydrogenated palm oil; 18% by weight of linolenic acid.

(63) A second coating layer was then formed, coating the microgranules coated by the first layer with 35 kg of a coating mixture comprising: 52% by weight of hydrogenated rapeseed oil; 45% by weight of carnauba wax; 3% by weight of sunflower lecithin.

(64) A third coating layer was then formed, coating the microgranules coated by the second layer with 15 kg of a coating mixture comprising: 50% by weight of hydrogenated palm oil; 49% by weight of rice wax; 1% of ethoxylated castor oil.

(65) All the above indicated percentages are percentages by weight based on the total weight of the covering layer.

(66) The microgranules thus obtained had a concentration of 70% by weight of DL-Methionine with respect to the total weight of the microgranules.

(67) Then the microgranules were subjected to an evaluation of the degree of ruminal by-pass, post-ruminal digestibility and total bioavailability using the Boisen method. The in vitro results are highlighted in Table 7.

(68) TABLE-US-00012 TABLE 7 Sam- Content Degree of ruminal bypass Digestibility Bioavailability ple (%) (*) (**) (**) 7 69.2 78.8 90 70.9

Example 4—Preparation of Microgranules Containing L-Lysine, Nicotinic Acid and DL-Methionine

Example 4.1

(69) 180 kg of 99% choline chloride were mixed with 240 kg of 99% L-lysine hydrochloride, 65 kg of nicotinic acid (vitamin PP) and 145 kg of D, L-methionine. 20 kg of citric acid, 20 kg of sodium bicarbonate, 5 kg of sunflower lecithin and 225 kg of rice wax spray were added. The mixture was extruded.

(70) The cores thus obtained had a concentration of 20% of choline chloride, 26.7% of L-lysine hydrochloride, 7.2% of nicotinic acid, 16.1% of D, L-methionine.

(71) The nuclei were subjected to coating in a pan.

(72) A first coating layer was formed, coating 400 kg of microgranules with 200 kg of a coating mixture comprising: 66% by weight of hydrogenated rapeseed oil; 34% by weight of palmitic acid.

(73) A second coating layer was then formed, coating the microgranules coated by the first layer with 100 kg of a coating mixture comprising: 48% by weight of hydrogenated palm oil; 50% by weight of rice wax; 2% by weight of soy lecithin.

Example 5

(74) Using the above mentioned analytical methods (pharmaceutical dissolver), in vitro tests were carried out to determine the degree of by-pass and the bioavailability of choline chloride: a product A consisting of choline chloride, 99% pure; a product B consisting of microencapsulated choline chloride granules obtained by means of spray-cooling technology, containing 25% by weight of choline chloride with respect to the total weight of the granule a product C consisting of microgranules obtained according to Example 4 of the patent EP1791532, containing 50% by weight of choline chloride with respect to the total weight of the granule; a product D consisting of microgranules obtained according to Example 1.3 described above.

(75) Test results are shown in the following Tables 8 and 9.

(76) Table 8 shows the results obtained considering a theoretical administration of 200 g of product.

(77) TABLE-US-00013 TABLE 8 Choline Choline Chloride Bypassed Bioavailable Choline Chloride lost in the Choline Choline Chloride Prod- administered rumen in 8 h Chloride Chloride in faeces uct (g) (g) (g) (g) (g) A 198 198 0 0 0 B 50 37.5 12.5 12.5 0 C 100 20 80 40 40 D 120 17 103 88 15
Table 8 shows the results obtained a theoretical administration of 100 g of choline chloride.

(78) TABLE-US-00014 TABLE 9 Choline Chloride Bypassed Bioavailable Choline Product to be lost in the Choline Choline Chloride Prod- administered rumen in 8 h Chloride Chloride in faeces uct (g) (g) (g) (g) (g) A 101 101 0 0 0 B 400 75 25 25 0 C 200 20 80 40 40 D 167 13.5 86.5 73 13.5

Example 6

(79) Using the above mentioned analytical methods (pharmaceutical dissolver), in vitro tests were carried out to determine the degree of by-pass and the bioavailability of choline chloride: a product A consisting of choline chloride, 99% pure; a product B consisting of microencapsulated chlorine choline granules obtained by means of spray-cooling technology, containing 25% by weight of choline chloride with respect to the total weight of the granule a product C consisting of microgranules obtained according to Example 4 of the patent EP1791532, containing 50% by weight of choline chloride with respect to the total weight of the granule; a product D consisting of microgranules obtained according to Example 1.3 described above; a product E consisting of microgranules obtained according to the extruded form of Example 1.3, but coated with coating layers devoid of pollutants; a product F consisting of microgranules obtained according to the extruded form of Example 1.3, in which the portions of disintegrants were replaced by rice wax spray (binder), but the coating layers were “polluted” with twice the amounts of pollutant compared to Example 1.3.

(80) The results of the tests are shown in the following Tables 10 and 11. The tables show that the mere presence of the disintegrant is effective in making the choline bioavailable in the post-ruminal phase.

(81) Table 10 shows the results obtained considering a theoretical administration of 200 g of product.

(82) TABLE-US-00015 TABLE 10 Choline Choline Chloride Bypassed Bioavailable Choline Chloride lost in the Choline Choline Chloride Prod- administered rumen in 8 h Chloride Chloride in faeces uct (g) (g) (g) (g) (g) A 198 198 0 0 0 B 50 37.5 12.5 12.5 0 C 100 20 80 40 40 D 120 17 103 88 15 E 120 12 108 54 54 F 120 45 75 55 20

(83) Table 11 shows the results obtained considering a theoretical administration of 100 g of choline chloride.

(84) TABLE-US-00016 TABLE 11 Choline Chloride Bypassed Bioavailable Choline Product to be lost in the Choline Choline Chloride Prod- administered rumen in 8 h Chloride Chloride in faeces uct (g) (g) (g) (g) (g) A 101 101 0 0 0 B 400 75 25 25 0 C 200 20 80 40 40 D 167 13.5 86.5 73 13.5 E 167 9.6 90.4 47 43.4 F 167 36.9 63.1 45.3 17.8