PHYTASE FORMULATION

Abstract

A liquid enzyme formulation, an enzyme granule formulation, methods for manufacturing enzyme granules using a fluid bed dryer, wherein the enzyme granules are thermostable without the need for a thermostable coating is provided. The enzyme granules are phytase granules used in the manufacturing of an animal feed, wherein the phytase granule is thermostable without the need for a thermostable coating and the phytase retains about 63% to about 134% of its activity after pelleting at 80 C.

Claims

1. A granulated enzyme formulation comprising: (a) a carrier; (b) a primary solution comprising a phytase, a buffer, a stabilizer, a binding agent, a first plasticizer, an anti-microbial, or any combination thereof; and (c) a secondary solution comprising an enhancer, a second plasticizer, or any combination thereof.

2. The granulated enzyme formulation of claim 1, wherein the carrier is selected from the group consisting of: (a) a flour; (b) a wheat flour; (c) a bleached flour (d) a wheat flour and a maltodextrin; (e) a wheat flour and a pre-gelatinized starch; and (f) a starch.

3. The granulated enzyme formulation of claim 1, wherein the buffer is a sodium citrate.

4. The granulated enzyme formulation of claim 1, wherein the stabilizer is selected from the group consisting of: a sucrose, a sorbitol, a mannitol, a glycerol, a trehalose, a sodium chloride, a sodium sulphtate, kaolin, aluminum silicate, magnesium silicate, and any combination thereof.

5. The granulated enzyme formulation of claim 1, wherein the binding agent is selected from the group consisting of: a guar gum, a xanthan, a sodium algenate, a locust bean gum, a carrageenan gum, a pre-gelatinized modified starch, a maltodextrin, gelatin, a methyl celluloase, a hydroxypropyl cellulose, a hydroxypropyl methyl celluloase, a carboxymethyl celluloase, and any combination thereof.

6. The granulated enzyme formulation of claim 1, wherein the first plasticizer is selected from the group consisting of: a glycerol, polyethylene glycol, triethyl citrate, triacetin, and acetyl tri ethyl citrate.

7. The granulated enzyme formulation of claim 1, wherein the anti-micorbial is selected from the group consisting of: a potassium sorbate, sodium sorbate, sodium benzoate, sodium propionate, calcium propionate, ammonium propionate, methyl paraben, benzoic acid, sorbic acid, and any combination thereof.

8. The granulated enzyme formulation of claim 1, wherein the enhancer is selected from the group consisting of: pre-gelatinized modified starch, maltodextrin, sodium algenate, carregeenan with CaCl2, carregeenan without CaCl2, guar gum with sodium borate, guar gum without sodium borate, gelatin; methyl cellulase; hydroxypropyl cellulase; hydroxypropyl methyl cellulase; carboxymethyl cellulase; sodium chloride; sodium sulphate; kaolin; aluminum silicate, magnesium silicate, and any combination thereof.

9. The granulated enzyme formulation of claim 1, wherein the second plasticizer is selected from the group consisting of a glycerol, polyethylene glycol, triethyl citrate, triacetin, and acetyl tri ethyl citrate.

10. The granulated enzyme formulation of claim 1, further comprising a flowing aid, wherein the flowing aid is selected from the group consisting of: silicon dioxide, magnesium stearate, kaolin, talc, diatomaceous earth, and any combination thereof.

11. A method for manufacturing an enzyme granule in a fluidized bed, wherein the enzyme retains at least 63% activity at 75 C. comprising: (a) loading a dry carrier in a fluid bed granulator; (b) adding a primary enzyme solution to the dry carrier; and (c) drying the combination of (a) and (b) to a desired moisture content.

12. The method of claim 11, further comprising: (i) adding a secondary granulation solution to (a) and (b).

13. The method of claim 11, further comprising adding a flowing aid to the fluid bed dryer or dry blender after drying.

14. The method of claim 12, wherein the secondary granulation solution further comprises a flowing aid.

15. The method of claim 11, wherein the enzyme is selected from the group consisting of: a Phytase, a cellulase, lactase, lipase, protease, catalase, xylanase, beta-glucanase, mannanase, amylase, amidase, epoxide hydrolase, esterase, phospholipase, transaminase, amine oxidase, cellobiohydrolase, ammonia lyase, and any combination thereof.

16. The method of claim 11, wherein the desired moisture content is between about 3% to about 10%.

17. A formulation for phytase wherein the phytase retains an activity at a temperature comprising: (a) at least 63% activity at 80 C.; (b) at least 58% activity at 85 C.; (c) at least 43% activity at 88 C.; (d) at least 27% activity at 90 C.; and (e) at least 6% activity at 93 C.

Description

EXAMPLES

Example 1

Comparative Example of Non-Granulated Phytase

[0055] Formulation 1A liquid concentrate phytase formulation containing 10% sodium chloride and 10% sorbitol was dried by lyophilization. The dried enzyme powder was then diluted by mixing with calcium carbonate and rice hulls to form a rice hull enzyme product.

[0056] Formulation 2A liquid concentrate phytase formulation containing 10% sodium chloride and 10% sorbitol was soaked on the rice hulls. The resulting wet blend was dried by lyophilization. The dried material was diluted by mixing with calcium carbonate to form a rice hull enzyme product.

[0057] Pelleting TrialsThe rice hull enzyme product was blended with poultry feed mash at 25-100 ppm concentration, and pelleted in a California Pellet Mill at various controlled temperatures in the range of 75 C. to 93 C. The mash mixture was first conditioned by direct steam at said temperature for 15 seconds, and then pelleted through the pelleting press. The pellets were subsequently dried.

[0058] Enzyme Stability DeterminationThe starting mash and finished pellets (which were milled down to mash) were extracted in a Tris buffer. Phytase activities in the extracts were determined by a modified AOAC method. The modified AOAC method is the AOAC buffer comprising distilled water plus 0.01% Tween 20 (GIZZI, J. of AOAC International, Vol. 91, No. 2, 2008) and was modified to a composition comprising: 50 mM Tris pH 8.0, 0.01% Tween20, 10 mM CaCl.sub.2.

[0059] The percent recovery of the enzyme activity of pelleted product at each temperature was compared to that of the starting mash. The results are shown in Table 4.

TABLE-US-00004 TABLE 4 Residual Phytase activities (%) in poultry feed after pelleting at different conditioning temperatures Sample # 75 C. 80 C. 85 C. 88 C. 90 C. 93 C. Formulation 1 70% 63% 55% 49% 16% 6% Formulation 2 63% 64% 51% 43% 27% 13%

[0060] The results show that phytase activity extracted from the pelleted feed was lower at high conditioning temperatures for both formulation 1 and formulation 2.

Example 2

[0061] Formulation 3250 g of rice hulls was loaded in a GEA Aeromatic-Fielder Strea-1 fluid bed granulator; 50 ml of a liquid concentrate phytase formulation containing 10% sorbitol and 10% sodium chloride was sprayed onto the rice hulls through a top spray two-fluid nozzle. The following fluid bed settings were used: 55 C. inlet air temperature, 5 ml/min spray rate, 1 bar atomizing pressure, 0.8 mm spray nozzle. The rice hull enzyme product was dried after the enzyme spray.

[0062] Formulation 4200 g of rice hulls was loaded in a GEA Aeromatic-Fielder Strea-1 fluid bed granulator; 100 ml of a liquid concentrate phytase formulation containing 20% sucrose was sprayed onto the rice hulls through a top spray two-fluid nozzle. The fluid bed settings were the same as Formulation 3 and were used to form a rice hull enzyme product.

[0063] Formulation 5200 g of wheat flour was loaded in a GEA Aeromatic-Fielder Strea-1 fluid bed granulator; 100 ml of a liquid concentrate phytase formulation containing 20% sucrose and 7% pre-gelatinized starch was sprayed onto the wheat flour through a top spray two-fluid nozzle. The following fluid bed settings were used: 50 C. inlet air temperature, 10-15 ml/min spray rate, 1.5-2 bar atomizing pressure, 0.8 mm spray nozzle. The wheat flour and enzyme product was dried after the enzyme spray to form phytase granules.

[0064] Pelleting Trials and Enzyme Stability Determination were the same as in Example 1. The results are shown in Table 5.

TABLE-US-00005 TABLE 5 Residual Phytase activities (%) in poultry feed after pelleting at different conditioning temperatures Sample # 75 C. 80 C. 85 C. 88 C. 90 C. 93 C. Formulation 3 67% 70% 80% 56% 29% 9% Formulation 4 72% 75% 58% 56% 36% 17% Formulation 5 95% 89% 93% 79% 77% 38%

[0065] Rice hulls were used as carrier in Formulations 3 & 4. The rice hulls were not finely milled and not very absorbent; in addition, no binder was used in these formulations. As a result, no effective granulation was seen in the rice hull enzyme products. The thermal stability for both formulations was relatively low at high conditioning temperatures when compared to Formulation 5. Switching the stabilizer from sodium chloride and sorbitol to sucrose improved thermostability of the phytase. However, when the carrier was switched from rice hulls to finely milled wheat flour in combination with the use of a pre-gelatinized starch as binder as in formulation 5. The wheat flour enzyme product was granulated, and the thermal stability of the phytase extracted from an animal feed pellet improved across all temperatures, especially at 90 C.

Example 3

[0066] Formulation 63 kg of wheat flour was loaded in a Vector VFC-Lab 3 fluid bed granulator; 1.6 kg of liquid concentrate phytase formulation containing 20% sucrose and 0.5% guar gum was sprayed onto the wheat flour through a top spray two-fluid nozzle. The fluid bed settings were 1.2 mm nozzle diameter, 20 psi atomizing pressure, 50-55 C. inlet air temperature, 23-27 C. product temperature, 25-50 g/min pray rate during spray; 70-80 C. inlet air temperature during drying, dried to 45 C. end product temperature to form phytase granules.

[0067] Formulation 73 kg of wheat flour was loaded in a Vector VFC-Lab 3 fluid bed granulator; 1.6 kg of liquid concentrate phytase formulation containing 20% sucrose, 10% maltodextrin and 0.5% guar gum was sprayed onto the wheat flour through a top spray two-fluid nozzle. The fluid bed settings were 1.2 mm nozzle diameter, 20 psi atomizing pressure, 50 C. inlet air temperature, 23-29 C. product temperature, 25-50 g/min pray rate during spray; 70-80 C. inlet air temperature during drying, dried to 45 C. end product temperature to form phytase granules.

[0068] Pelleting Trials and Enzyme Stability Determination were the same as in Example 1. The results are shown in Table 6. All residual activities were normalized to 75 C. stability of the same sample.

TABLE-US-00006 TABLE 6 Residual Phytase activities (%) in poultry feed after pelleting at different conditioning temperatures Sample # 75 C. 88 C. 90 C. 93 C. Formulation 3 100% 31% 9% Formulation 6 100% 92% 35% 5% Formulation 7 100% 76% 40% 4%

[0069] Formulation 3 was the same sample from Example 2, and it was used as a control in this experiment. The thermal stability for formulation 3 is lower here than the value in Example 2, probably due to harsher pelleting conditions in this trial. Both Formulations 6 and 7 resulted in phytase granules that improved thermal stability of the phytase when extracted from a pelleted animal feed.

[0070] Additional Pelleting Trialsto confirm the improvement in thermal stability, the phytase granules produced from the two samples (Formulation 6 and 7) were subjected to additional pelleting trial using a different California Pellet Mill. The duration for steam conditioning was 40 seconds, which is significantly longer than the previous pelleting trials. The results are shown in Table 7. All residual phytase activities were normalized to 75 C. stability of the same sample.

TABLE-US-00007 TABLE 7 Residual Phytase activities (%) in poultry feed after pelleting at different conditioning temperatures. Sample # 75 C. 85 C. 90 C. 92 C. Formulation 6 100% 82% 66% 12% Formulation 7 100% 94% 50% 8%

[0071] The results confirmed that even under prolonged steam conditioning, both phytase granules from Formulations 6 and 7 provided high phytase activity for phytase extracted from animal feed pellets generated at high pelleting temperature.

Example 4

[0072] Formulation 83 kg of wheat flour was loaded in a Vector VFC-Lab 3 fluid bed granulator; 1.6 kg of liquid concentrate phytase formulation containing 20% sucrose, 10% kaolin and 0.5% guar gum was sprayed onto the wheat flour through a top spray two-fluid nozzle. The fluid bed settings were 1.2 mm nozzle diameter, 20 psi atomizing pressure, 50 C. inlet air temperature, 22-30 C. product temperature, 25-50 g/min pray rate during spray; 70-80 C. inlet air temperature during drying, dried to 45 C. end product temperature to form phytase granules.

[0073] Formulation 93 kg of wheat flour was loaded in a Vector VFC-Lab 3 fluid bed granulator; 1.6 kg of liquid concentrate phytase formulation containing 20% sucrose, 10% maltodextrin, 5% kaolin and 0.5% guar gum was sprayed onto the wheat flour through a top spray two-fluid nozzle. The fluid bed settings were the same as in Formulation 8 to form phytase granules.

[0074] Formulation 102 kg of wheat flour was loaded in a Vector VFC-Lab 3 fluid bed granulator; 1 kg of liquid concentrate phytase formulation containing 20% sucrose and 0.5% guar gum was sprayed onto the wheat flour through a top spray two-fluid nozzle; 1 kg of secondary granulation solution was then sprayed containing 10% modified starch, 10% kaolin and 1% glycerol. The fluid bed settings during enzyme spray were the same as in Formulation 8; during secondary granulation solution spray, the spray rate was 17-18 g/min, inlet air temperature was 60 C., and the product temperature was 30-31 C.; during drying, inlet air temperature was 70-80 C. to form phytase granules.

[0075] Pelleting Trials and Enzyme Stability Determination were the same as in Example 1 and the results are shown in Table 8.

TABLE-US-00008 TABLE 8 Residual Phytase activities (%) in poultry feed after pelleting at different conditioning temperatures Sample # 80 C. 85 C. 88 C. 90 C. 93 C. Formulation 8 74% 61% 79% 52% 20% Formulation 9 132% 71% 68% 58% 25% Formulation 10 104% 92% 79% 74% 28%

[0076] The results showed that the inclusion of kaolin in the secondary granulation solution spray led to increased phytase thermal stability; and the use of a secondary granulation solution further enhanced the phytase thermal stability of the phytase extracted from the animal feed pellet.

Example 5

[0077] Formulation 113 kg of wheat flour was loaded in a Vector VFC-Lab 3 fluid bed granulator; 1.3 kg of liquid concentrate phytase formulation containing 20% sucrose and 0.5% guar gum was sprayed onto the wheat flour through a top spray two-fluid nozzle. The fluid bed settings were 1.2 mm nozzle diameter, 20 psi atomizing pressure, 50 C. inlet air temperature, 22-29 C. product temperature, 25-50 g/min pray rate during spray; 70-80 C. inlet air temperature during drying, dried to 45 C. end product temperature to form phytase granules.

[0078] Formulation 124.6 kg of wheat flour was loaded in a 5-kg scale customized fluid bed granulator; 1.25 kg of liquid concentrate phytase formulation containing 20% sucrose and 0.5% guar gum was sprayed onto the wheat flour through a top spray two-fluid nozzle; 1 kg of secondary granulation solution containing 0.5% guar gum only was then sprayed. The fluid bed settings were 60-80 C. inlet air temperature, 28-36 C. product temperature and 40 g/min spray rate to form phytase granules.

[0079] Pelleting Trials and Enzyme Stability Determination were the same as in Example 1 and the results are shown in Table 9.

TABLE-US-00009 TABLE 9 Residual Phytase activities (%) in poultry feed after pelleting at different conditioning temperatures Sample # 75 C. 80 C. 85 C. 88 C. 90 C. 93 C. Formulation 11 93% 111% 76% 113% 57% 38% Formulation 12 108% 108% 107% 129% 91% 73%

[0080] The results showed that both samples had improved phytase thermal stability; and the use of a secondary granulation solution further enhanced the thermal stability of the the phytase extracted from an animal feed pellet generated at high pelleting temperatures.

Example 6

[0081] Most thermally stable feed enzymes currently commercially available rely on thermal protective coating. Some of these coatings have been shown to inadvertently delay the enzyme release into the solution, which lead to reduced enzyme efficacy. Therefore, the time release profile of Phytase activity from the granulated product of the current invention was examined.

[0082] One hundred milligrams of granulated Phytase from Formulation 12 was mixed with 400 ul of 100 mM sodium acetate buffer at pH 5.5. The mixture was agitated for up to 60 minutes. At designated time point, an aliquot was removed and centrifuged. Phytase activity in the clear supernatant was assayed. The results are shown in Table 10.

TABLE-US-00010 TABLE 10 Time Release Profile of Phytase Activity Time (minutes) 2.5 5 7.5 10 15 20 Formulation 12 94% 100% 100% 100% 100% 100%

[0083] The results show that Phytase became soluble in very short amount of time. On the other hand, some commercially available thermal coated Phytase granules displayed significant delay in releasing the enzyme activity into the solution, by as much as 20 minutes (data not shown).