A METHOD FOR IMPROVING THE NUTRITIONAL VALUE OF ANIMAL FEED

Abstract

The invention relates to the use of at least one bacterial phytase in combination with one or more protease(s) in animal feed for improving nutrient and E ileal digestibility of animal feed, in particular an improved digestibility of Threonine, Proline and Cysteine, the method comprising the step of applying to the animal a feed with an efficient amount of one or more proteolytic enzyme in combination with at least one phytase.

Claims

1. A method for increasing nutrient and E ileal digestibility of animal feed in farm animals, the method comprising the step of applying to the animal a feed with an efficient amount of one or more proteolytic enzyme in combination with at least one phytase.

2. The method of claim 1 for increasing the digestibility of Threonine, Proline and Cysteine available in the protein source of animal feed.

3. The method of claim 1, wherein the animal feed comprises a corn/soybean meal diet.

4. The method of claim 1, wherein a. the phytase is administered in such amounts that the specific activity in the final feed is between 1000 FYT/kg feed and 54000 FYT/kg feed and b. the protease is administered in a dosage of between 10′000 units/kg feed and 30′000 units/kg feed.

5. The method according to claim 1, wherein the phytase is classified as belonging to the EC 3.1.3.26 group.

6. The method according to claim 1, wherein the protease is an acid stable serine proteases obtained or obtainable from the order Actinomycetales.

7. The method according to claim 6, wherein the protease is an acid stable serine protease derived from Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235 (A1918L1), Nocardiopsis prasina DSM 15649 (NN018335L1), Nocardiopsis prasina (previously alba) DSM 14010 (NN18140L1), Nocardiopsis sp. DSM 16424 (NN018704L2), Nocardiopsis alkaliphila DSM 44657 (NN019340L2) and Nocardiopsis lucentensis DSM 44048 (NN019002L2), as well as homologous proteases.

8. Use of one or more proteolytic enzyme in combination with at least one phytase in animal feed for increasing nutrient and E ileal digestibility of animal feed in farm animals.

9. Use according to claim 8 for increasing the digestibility of Threonine, Proline and Cysteine available in the protein source of animal feed.

10. Use according to claim 8, wherein the animal feed comprises a corn/soybean meal diet.

11. Use according to claim 8, wherein a. the phytase is administered in such amounts that the specific activity in the final feed is between 1000 FYT/kg feed and 5000 FYT/kg feed and b. the protease is administered in a dosage of between 10′000 units/kg feed and 30′000 units/kg feed.

12. Use according to claim 9, wherein the phytase is classified as belonging to the EC 3.1.3.26 group.

13. Use according to claim 9, wherein the protease is an acid stable serine proteases obtained or obtainable from the order Actinomycetales.

14. The use according to claim 13, wherein the protease is an acid stable serine protease derived from Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235 (A1918L1), Nocardiopsis prasina DSM 15649 (NN018335L1), Nocardiopsis prasina (previously alba) DSM 14010 (NN18140L1), Nocardiopsis sp. DSM 16424 (NN018704L2), Nocardiopsis alkaliphila DSM 44657 (NN019340L2) and Nocardiopsis lucentensis DSM 44048 (NN019002L2), as well as homologous proteases.

Description

EXAMPLE 1: SPECIFIC ACTIVITY OF PHYTASES

[0071] The specific activity of phytases can be determined on highly purified samples dialysed against 20 mM sodium acetate, pH 5.5. The purity can be checked beforehand on an SDS poly acryl amide gel showing the presence of only one component.

[0072] The protein concentration can be determined by amino acid analysis as follows: An aliquot of the sample is hydrolyzed in 6N HCl, 0.1% phenol for 16 h at 110 C in an evacuated glass tube. The resulting amino acids is quantified using an Applied Biosystems 420A amino acid analysis system operated according to the manufacturer's instructions. From the amounts of the amino acids the total mass—and thus also the concentration—of protein in the hydrolyzed aliquot can be calculated.

[0073] The phytase activity is determined in the units of FYT, and the specific activity is calculated as the phytase activity measured in FYT units per mg phytase variant enzyme protein.

EXAMPLE 2: ANIMAL TRIAL—TOWARD STANDARDIZED AMINO ACID MATRICES FOR EXOGENOUS PHYTASE AND PROTEASE IN CORN/SOY-BASED DIETS FOR BROILERS

[0074] Materials and Methods

[0075] Birds and Diets

[0076] The study procedures were reviewed and approved by the Massey University Animal Ethics Committee and complied with the New Zealand Code of Practice for the Care and Use of Animals for Scientific Purposes.

[0077] A total of 468 male Ross 308 broiler chicks were obtained at 1 d of age from a commercial hatchery and reared in an environmentally-controlled room until d 21. A pre-experimental corn/soy-based broiler starter diet that met all the nutrient requirements of the birds was fed from d1 to 21. This diet was formulated to contain an AME of 3050 kcal/kg, 220 g/kg crude protein, 9 g/kg calcium, 4.5 g/kg available phosphorus and 12.3 g/kg digestible lysine. On d 21, birds were randomly distributed to 78 wire-floored metabolism cages (6 birds per cage) and offered one of 13 experimental diets (Table 1; 6 replicate cages per diet) from d21 to 28 or a nitrogen (N)-free diet (d 25-28). The experimental diets were based on the standardized ileal amino acid digestibility assay protocol (Ravindran et al., 2017) and were based on corn, soybean meal or a mixture of corn and soybean meal (Table 1). Each diet was fed without or with phytase (RONOZYME HiPhos, DSM Nutritional Products, Kaiseraugst, Switzerland, 3000 FYT/kg feed), protease (RONOZYME ProAct, DSM Nutritional Products, Kaiseraugst, Switzerland, 15000 PROT/kg feed) ora combination of phytase and protease (at the same inclusion concentration). One protease unit (PROT) is defined as the amount of enzyme that releases 1 mmol of p-nitroaniline from 1 mM substrate (Suc-Ala-Ala-Pro-Phe-pNA) per minute at pH 9.0 and 37° C. One phytase unit (FYT) is defined as the quantity of enzyme which liberates 1 μmol of inorganic phosphate per minute from 5.0 μmol/l sodium phytate at pH 5.5 and 37° C. Birds that received the N-free diet received the commercial starter diet until d25 (Ravindran et al. 2017). The cages were housed in an environmentally-controlled room. Temperature was maintained at 32° C. on d 1 and gradually reduced to 24° C. by d 21 and further to 21° C. by d28. Temperature modification was achieved by the use of thermostatically controlled fans and electric heaters. The birds received 20 hours fluorescent illumination per day and were allowed free access to diets and water. Birds were checked at least 3 times per day (09.00, 13.00 and 16.00 hrs) and any unusual aspect of bird behavior or condition was recorded. Sick or injured animals were weighed and removed from the study. All diets contained titanium dioxide (TiO2; 5 g/kg) as an indigestible marker.

Measurements

[0078] On d28, all birds from each replicate cage were euthanized by intra-cardial injection of sodium pentobarbitone for ileal digesta collection. The small intestine was immediately exposed and the contents of the distal half of the ileum were collected by gently flushing with distilled water into plastic containers. The ileum was defined as that portion of the small intestine extending from vitelline diverticulum to a point 40 mm proximal to the ileo-caecal junction. Digesta from birds within a cage were pooled, resulting in 6 samples per dietary treatment. The digesta samples were frozen immediately after collection, lyophilized and processed. Samples of digesta and diets were analyzed for or amino acids, including methionine and cysteine.

Chemical Analysis

[0079] Amino acids (including proline) were determined by hydrolyzing the samples with 6 N HCl (containing phenol) for 24 h at 110±2° C. in glass tubes sealed under vacuum. Amino acids were detected on a Waters ion-exchange HPLC system, and the chromatograms were integrated by using dedicated software (Millenium, version 3.05.01, Waters, Millipore, Milford, Mass.) with the amino acids identified and quantified by using a standard amino acid mixture (product no. A2908, Sigma, St. Louis, Mo.). The HPLC system consisted of anion-exchange column, two 510 pumps, a Waters 715 ultra-WISP sample processor, a column heater, a post-column reaction coil heater, a ninhydrin pump, and a dual-wavelength detector. Amino acids were eluted by a gradient of pH 3.3 sodium citrate eluent to pH 9.8 sodium borate eluent at a flow rate of 0.4 mL/min and a column temperature of 60° C. Cysteine and methionine were analyzed as cysteic acid and methionine sulfone, respectively, by oxidation with performic acid for 16 h at 0° C. and neutralization with hydrobromic acid before hydrolysis (Ravindran et al., 2008).

Calculations

[0080] The apparent ileal digestibility (AID) of AA was calculated by the following formula using the titanium marker ratio in the diet and ileal digesta.

AID of AA=((AA/Ti).sub.d−(AA/Ti).sub.i)/(AA/Ti).sub.d

[0081] Where, (AA/Ti)d=ratio of amino acid and titanium in diet, and (AA/Ti)i=ratio of amino acid and titanium in ileal digesta.

[0082] The basal endogenous AA (EAA) flow at the terminal ileum was calculated as grams lost per kilogram of DM intake (DMI; Moughan et al., 1992).

Basal endogenous AA flow (g/kg DMI)=(AA in ileal digesta (g/kg)×Ti.sub.d (g/kg))/Ti.sub.i (g/kg)

[0083] Where, Tid=titanium in diet and Tii=titanium in ileal digesta.

[0084] Apparent digestibility data for N and AA were then converted to standardized digestibility values, using endogenous N and AA values determined from birds fed the N-free diet (Ravindran et al., 2017).

SID=AID+[Basal EAA (g/kg DMI)]/Ing. AA (g/kg DM)

[0085] Where, AID=apparent ileal digestibility of the AA, Basal EAA=basal endogenous AA loss and Ing. AA=concentration of the AA in the ingredient.

[0086] Apparent digestibility values were standardized using the following basal ileal endogenous flow values (g/kg DM intake), determined by feeding N-free diet: N, 1.206; Met, 0.110; Cys, 0.149; Lys, 0.263; Thr, 0.468; Arg, 0.320; Ile, 0.285; Leu, 0.460; Val, 0.380; His, 0.098; Phe, 0.264; Gly, 0.317; Ser, 0.408; Pro, 0.375; Ala, 0.314; Asp, 0.573, Glu, 0.792 and Tyr, 0.261.

Results

[0087] The proximate, mineral and AA composition of the corn and soybean meal (g/kg as received) is presented in Table 2 and all values are in agreement with expectations, including the recovery of titanium dioxide in the feed samples. Phytase and protease activity recovered in the experimental diets is presented in Table 3 and is in line with expectations for these products.

[0088] The flow of endogenous AA, presented in the methodology section above, in the terminal ileum of broilers that received the N-free diet are presented in FIG. 1. The mean flow of endogenous AA was 0.34 g/kg DM intake. The flow of endogenous Asp, Glu, Thr, Ser, Leu, Pro and Val were higher than this mean value and other AA (notably Met and His where the endogenous flow was only 0.11 g/kg DM intake) were lower. The flow of endogenous AA in the present experiment (FIG. 4) correlated positively (P<0.001; r.sup.2=0.97) with the flow of endogenous AA from broilers fed an N-free diet in previous work.

[0089] The AID of AA in corn, SBM, a mixture of corn and SBM and the same without or with phytase, protease or a combination of phytase and protease is presented in Table 4. The AID of all AA other than Met, Cys, Leu and Ala, was higher (P<0.01) in SBM or in the corn/SBM mixture compared with corn. There was no effect (P>0.05) of phytase addition on the AID of AA. However, the effect of protease on the AID of Thr, Pro and Cys was greater when offered in combination with phytase than without, resulting in a significant phytase*protease interaction for those AA. Addition of protease resulted in an increase (P<0.01) in the AID of AA by 3.6%, which ranged from 2.1-2.3% for Arg and Met respectively to 6.0-6.4% for Thr and Cys respectively.

[0090] The SID of AA in corn, SBM, a mixture of corn and SBM and the same without or with phytase, protease or a combination of phytase and protease is presented in Table 5. The SID of AA was not significantly different between diets for Glu, Val, Ile, His and Arg whereas the SID of Cys, Met, Phe, Leu, Ala and Pro was higher (P<0.01) in corn compared with SBM. For N and the other AA the SID was generally higher for SBM than for corn. Similar to the AID observations, the presence of phytase increased the effectiveness of protease on the SID of Thr, Pro and Cys, resulting in a significant interaction for those AA. Protease addition resulted in an increase in the SID of all AA by an average of 3.4%, ranging from 2.0% for Arg to 6.1% for Cys.

[0091] The additivity of AID or SID values and the influence of exogenous enzyme addition on the same is presented in FIGS. 2 and 3. On an AID basis (FIG. 2) the calculated digestibility of the mixture of corn and SBM (based on the determined AID of AA in the individual ingredients and their relative proportions in the mixture i.e. 65% corn and 35% SBM) underestimated the measured digestibility by 7.4%, ranging from 1.0% for Cys to 21.9% for Thr. However, when fed with a combination of phytase and protease this underestimation was reduced (P<0.05) to 6.7%. On a SID basis the calculated digestibility of AA in corn and SBM underestimated the measured digestibility by only 2.9% and the calculated and measured values were not significantly different. Addition of phytase and protease numerically reduced this underestimation to 2.5%, being particularly notable for Thr and Lys.

[0092] The effect of diet (corn, SBM or a mixture of corn and SBM) and enzyme (unsupplemented, phytase or protease) on the AID and SID of AA are presented in Tables 4 and 5. Some variance from experiment to experiment is expected due to the influence of the individual ingredient sources used (both corn and SBM are known to vary in digestibility of macro-nutrients; Leeson et al., 1993; Douglas et al., 2000; Ravindran et al., 2007), slight changes in methodology and in the capacity of the birds to extract nutrients from the feed (Hughes & Choct, 1997). Importantly, the corn/SBM mixture has returned a consistently higher AID of AA than the corn or SBM alone. This observation can be observed especially for Thr, Lys, Asp, Gly and Ser where under-estimation of the true value of the individual ingredients was >10% (FIG. 2), further demonstrating the problem of arithmetic assumptions about the nutritional value of individual ingredients in complex diets.

[0093] Specifically for Thr, Pro and Cys a significant phytase-protease interaction can be observed. This was caused by the effect of protease being substantially greater when offered in combination with phytase than when either enzyme were fed alone.

Standardized Ileal Digestibility of Threonine

[0094] Control (no enzymes): 80.2%

Protease: 81.5%

Phytase: 79.3%

Protease+Phytase: 86.6%

Standardized Ileal Digestibility of Proline

[0095] Control (no enzymes): 87.2%

Protease: 88.6%

Phytase: 87.1%

Protease+Phytase: 91.1%

Standardized Ileal Digestibility of Cysteine

[0096] Control (no enzymes): 80.3%

Protease: 83.2%

Phytase: 80.5%

Protease+Phytase: 87.4%

[0097] The significant interaction between phytase and protease for the AID and SID of Thr, Pro and Cys is intriguing and not easy to explain. In the absence of phytase, the addition of protease increased the SID of Thr, Pro and Cys by 1.6, 1.6 and 3.6% respectively. Phytase alone had no effect on the SID of these AA. However, when protease was added on top of phytase an increase in the SID of Thr (1.6 to 8.0%), Pro (1.6 to 4.5%) and Cys (3.6 to 8.8%) was observed. This statistically-confirmed synergy between phytase and protease is interesting and may be associated with co-operation between these enzymes for access to substrate e.g. protease improving solubility of phytate, or perhaps through physiological effects involving myo-inositol, sodium portioning or, more generally, amino acid absorption and peptide transport.

[0098] It can be concluded that the SID AA system for feed ingredient appraisal results in more precise and predictable outcomes for mixed diets than is the case for AID AA approaches and should be used wherever possible. Furthermore, exogenous protease is an effective tool to promote the digestibility of AA in broilers and may do so to a greater extent than is the case for phytase. The synergistic effects of phytase and protease on the SID of Thr, Cys and Pro warrants further attention, especially considering endogenous protein flow, peptide transport and sodium partitioning.

REFERENCES

[0099] Douglas, M. W., C. M. Parsons and M. R. Bedford 2000. Effect of various soybean meal sources and Avizyme on chick growth performance and ileal digestible energy. J. Appl. Poult. Res. 9: 74-80. [0100] Leeson, S., A. Yersin and L. Volker. 1993. Nutritive value of the 1992 corn crop. J. Appl. Poult. Res. 2: 208-213. [0101] Moughan, P. J. and G. S. Marlies Leenaars 1992. Endogenous amino acid flow in the stomach and intestine of the young growing pig. J. Sci. Food. Agric. 60: 437-442. [0102] Moughan, P. J. and S. M. Rutherfurd. 2012. Gut luminal endogenous protein: implications for the determination of ileal amino acid digestibility in humans. Brit. J. Nutr. 108: 258-263. [0103] Ravindran, V. and W. H. Hendriks. 2004. Endogenous amino acid flows at the terminal ileum of broilers, layers and adult roosters. Anim. Sci. 79: 265-271. [0104] Ravindran, V., L. I. Hew, G. Ravindran and W. L. Bryden. 2004. Endogenous amino acid flow in the avian ileum: quantification using three techniques. Brit. J. Nutr. 92: 217-223. [0105] Ravindran, V., L. I. Hew, G. Ravindran and W. L. Bryden. 2007. Apparent ileal digestibility of amino acids in dietary ingredients for broiler chickens. Anim. Sci. 81: 85-97. [0106] Ravindran, V., O. Adeola, M. Rodehutscord, H. Kluth, J. D. van der Klis, E. van Eerden and A. Helmbrecht. 2017. Determination of ileal digestibility of amino acids in raw materials for broiler chickens—results of collaborative studies and assay recommendations. Anim. Feed Sci. Technol. 225:62-72.

Tables

[0107]

TABLE-US-00001 TABLE 1 Composition.sup.1 (g/kg) of the experimental diets used in the ileal digestibility assay (21 to 28 d of age). Corn SBM Corn/SBM N-free Ingredient diet diet diet diet Corn 930 — 600 — Soybean meal — 410 330 — Wheat starch — 520 — 842 Soybean oil 30 30 30 50 Sodium bicarbonate 2.0 2.0 2.0 2.0 Sodium chloride 2.0 2.0 2.0 2.0 Dicalcium phosphate 19 19 19 19 Limestone 10 10 10 10 Titanium dioxide 5.0 5.0 5.0 5.0 Vitamin premix.sup.1 1.0 1.0 1.0 1.0 Trace mineral premix.sup.2 1.0 1.0 1.0 1.0 Solkafloc (Cellulose) — — — 50 Dipotassium phosphate — — — 12 .sup.1Diets were formulated as per the instructions in Ravindran et al. (2017) to achieve crude protein concentrations of approximately 70 g/kg in the corn diet, 190 g/kg in the SBM diet and 200 g/kg in the corn/SBM mixture. .sup.2Supplied per kilogram of diet: butylated hydroxy toluene, 100 mg; biotin, 0.2 mg; calcium pantothenate, 12.8 mg; cholecalciferol, 60 μg; cyanocobalamin, 0.017 mg; folic acid, 5.2 mg; menadione, 4 mg; niacin, 35 mg; pyridoxine, 10 mg; trans-retinol, 3.33 mg; riboflavin, 12 mg; thiamine, 3.0 mg; dl-α-tocopheryl acetate, 60 mg; choline chloride, 638 mg; Co, 0.3 mg; Cu, 3.0 mg; Fe, 25 mg; I, 1 mg; Mn, 125 mg; Mo, 0.5 mg; Se, 200 μg; Zn, 60 mg (DSM Nutritional Products, Wagga Wagga, NSW, Australia)

TABLE-US-00002 TABLE 2 Proximate, mineral and amino acid composition of corn and soybean meal (g/kg, as received).sup.1. Corn Soybean meal Dry matter 917 918 Ash 13.1 70.2 Nitrogen 11.5 77.1 Crude Protein (N × 6.25) 71.9 482 Starch 631 17.4 Fat 38.0 18.1 Calcium 0.86 2.90 Total Phosphorus (P) 2.34 6.26 Phytate P 1.84 3.33 Non-phytate P 0.50 2.93 Magnesium 0.92 3.2 Potassium 3.7 26.0 Sodium 0.046 0.13 Iron 0.023 0.10 Chloride 0.47 0.17 Zinc 0.021 0.043 Arginine 3.76 36.75 Histidine 1.95 12.23 Isoleucine 2.30 21.06 Leucine 7.87 35.17 Lysine 2.47 29.82 Methionine 1.54 6.55 Phenylalanine 3.32 23.79 Threonine 2.43 20.17 Valine 3.41 23.21 Alanine 4.82 21.09 Aspartic acid 4.85 59.11 Cysteine 1.57 6.50 Glycine 3.04 21.69 Glutamic acid 11.72 84.41 Proline 6.37 23.77 Serine 3.35 27.21 Tyrosine 2.99 21.63 .sup.1Analyses were done in duplicate (AOAC, 2012).

TABLE-US-00003 TABLE 3 Expected and measured enzyme activities in samples of the experimental diets RONOZYME RONOZYME Phytase Protease Diet HiPhos GT.sup.1 ProAct CT.sup.2 Expected.sup.3 Measured Expected.sup.4 Measured Corn 0 0 0 LOD 0 LOD Corn 0 0 0 LOD 0 LOD Corn 3000 0 >3000 4266 0 LOD Corn 3000 0 >3000 4676 0 LOD SBM 0 15000 0 LOD 15000 16440 SBM 0 15000 0 LOD 15000 18490 SBM 3000 15000 >3000 4970 15000 14330 SBM 3000 15000 >3000 4144 15000 14900 Corn/SBM 0 0 0 LOD 0 LOD Corn/SBM 3000 0 >3000 4139 0 LOD Corn/SBM 0 15000 0 LOD 15000 14150 Corn/SBM 3000 15000 >3000 4679 15000 15980 N-free 0 0 0 LOD 0 LOD All samples were measured in two replicates. LOD—Limit of detection. .sup.1Ronozyme ® HiPhos GT inclusion rate was 0.3 g/kg .sup.2Ronozyme ® ProAct CT inclusion rate was 0.2 g/kg. .sup.3Expected enzyme activity due to addition of Ronozyme ® HiPhos. .sup.4Expected enzyme activity due to addition of Ronozyme ® ProAct CT.

TABLE-US-00004 TABLE 4 Effect of exogenous enzymes on the apparent ileal amino acid digestibility (%) of corn, soybean meal (SBM) or a mixture of corn and SBM for growing broiler chickens (measured on d 28) Diet Phy Pro N Asp Thr Ser Glu Pro Gly Ala Val Corn 0 0 72.9 67.9 57.3 72.9 84.7 83.9 68.9 83.0 74.4 Corn 3000 0 72.4 66.5 54.2 73.0 84.7 82.8 69.3 83.2 74.0 Corn 0 15000 73.9 68.8 56.2 73.9 86.1 84.3 71.0 84.4 76.0 Corn 3000 15000 78.7 75.1 66.6 79.0 88.1 87.7 75.9 87.1 80.0 SBM 0 0 82.9 84.3 79.3 85.9 88.4 83.4 81.7 83.7 82.6 SBM 3000 0 83.6 85.2 80.4 86.5 89.3 84.2 82.5 84.6 83.8 SBM 0 15000 85.3 86.7 82.1 87.9 90.0 85.5 83.7 85.3 84.7 SBM 3000 15000 86.4 88.1 83.4 88.8 90.8 86.5 85.0 86.5 85.8 Corn/SBM 0 0 83.2 83.6 79.2 85.5 89.0 84.9 81.5 85.3 83.8 Corn/SBM 3000 0 82.5 82.9 78.5 84.7 88.7 84.7 80.5 84.2 82.8 Corn/SBM 0 15000 85.1 85.1 81.4 87.0 90.0 86.4 83.7 86.7 85.3 Corn/SBM 3000 15000 88.0 88.6 85.2 89.7 92.2 89.5 87.0 89.2 88.4 Pooled SEM 1.64 2.18 2.46 1.65 1.33 1.16 1.93 1.50 1.85 Model P < 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 Main Effects Corn 74.5.sup.a 69.6.sup.a 58.6.sup.a 74.7.sup.a 85.9.sup.a 84.7 71.3.sup.a 84.4 76.1.sup.a SBM 84.6.sup.b 86.1.sup.b 81.2.sup.b 87.3.sup.b 89.7.sup.b 84.9 83.2.sup.b 85.0 84.2.sup.b Corn/SBM 84.7.sup.b 85.0.sup.b 81.1.sup.b 86.7.sup.b 90.0.sup.b 86.4 83.2.sup.b 86.4 85.1.sup.b P < 0.001 0.001 0.001 0.001 0.001 NS 0.001 NS 0.001 0 80.6 79.4 72.6 82.2 88.0 84.7 78.4 84.7 81.1 3000 81.9 81.1 74.7 83.6 89.0 85.9 80.0 85.8 82.5 P < NS NS NS NS NS NS NS NS NS 0 79.6 78.4 71.5 81.4 87.5 84.0 77.4 84.0 80.2 15000 82.9 82.1 75.8 84.4 89.5 86.7 81.1 86.5 83.4 P < 0.01 0.01 0.01 0.01 0.01 0.001 0.01 0.01 0.01 Interaction Terms Diet*Pro- NS NS NS NS NS NS NS NS NS tease P < Diet*Phy- NS NS NS NS NS NS NS NS NS tase P < Phytase*Pro- NS NS 0.05 NS NS 0.05 NS NS NS tease P < Diet*Phy- NS NS NS NS NS NS NS NS NS tase*Pro- tease P < Diet Ile Leu Tyr Phe His Lys Arg Cys Me Corn 74.0 86.3 79.8 81.8 82.9 68.6 82.9 75.6 84 Corn 73.8 86.4 80.4 82.1 82.5 69.5 83.3 74.9 84 Corn 75.8 87.7 81.7 83.6 83.3 71.3 84.5 77.0 85 Corn 79.8 89.5 83.8 86.1 86.1 75.5 86.6 82.4 88.4 SBM 84.4 84.2 88.7 86.1 87.1 87.0 91.0 74.6 86.2 SBM 85.7 85.3 89.3 87.2 87.7 87.8 91.9 75.8 86.9 SBM 86.3 86.1 89.9 88.0 88.6 88.7 92.3 79.3 87.3 SBM 87.4 87.2 90.4 88.8 89.6 89.5 92.7 81.0 88.4 Corn/SBM 85.2 86.5 88.9 87.3 87.5 86.8 91.1 76.0 88.2 Corn/SBM 84.5 85.9 88.0 86.7 86.9 86.5 90.8 75.7 87.2 Corn/SBM 86.6 87.8 89.7 88.5 88.8 88.4 92.2 78.4 89.4 Corn/SBM 89.5 90.4 92.1 91.1 91.3 90.7 93.9 83.7 91.4 Pooled SEM 1.96 1.26 1.48 1.36 1.26 2.81 1.41 1.58 1.65 Model P < 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 Main Effects Corn 75.8.sup.a 87.5 81.4.sup.a 83.4.sup.a 83.7.sup.a 71.2.sup.a 84.3.sup.a 77.5 85.8.sup.a SBM 86.0.sup.b 85.7 89.6.sup.b 87.5.sup.b 88.2.sup.b 88.2.sup.b 92.0.sup.b 77.7 87.2.sup.ab Corn/SBM 86.5.sup.b 87.6 89.7.sup.b 88.4.sup.b 88.6.sup.b 88.1.sup.b 92.0.sup.b 78.5 89.0.sup.b P < 0.001 NS 0.001 0.001 0.001 0.001 0.001 NS 0.05 82.1 86.4 86.5 85.9 86.4 81.8 89.0 76.8 86.9 83.4 87.5 87.4 87.0 87.3 83.2 89.9 78.9 87.8 NS NS NS NS NS NS NS 0.05 NS 81.3 85.8 85.9 85.2 85.7 81.0 88.5 75.4 86.4 84.2 88.1 87.9 87.7 87.9 84.0 90.4 80.3 88 0.05 0.01 0.05 0.01 0.01 0.05 0.05 0.001 0 Interaction Terms Diet*Pro- NS NS NS NS NS NS NS NS NS tease P < Diet*Phy- NS NS NS NS NS NS NS NS NS tase P < Phytase*Pro- NS NS NS NS NS NS NS 0.05 NS tease P < Diet*Phy- NS NS NS NS NS NS NS NS NS tase*Pro- tease P < Values in columns without a common superscript differ significantly (P < 0.05, unless otherwise stated). indicates data missing or illegible when filed

TABLE-US-00005 TABLE 5 Effect of exogenous enzymes on the standardized ileal amino acid digestibility (%) of corn, soybean meal (SBM) or a mixture of corn and SBM for growing broiler chickens (measured on d 28) Diet Phy Pro N Asp Thr Ser Glu Pro Gly Ala Val Corn 0 0 82.5 78.8 74.9 84.1 90.9 89.3 78.4 88.9 84.6 Corn 3000 0 82.0 77.3 71.2 84.2 90.9 88.2 78.9 89.1 84.2 Corn 0 15000 83.5 79.7 73.9 85.0 92.3 89.7 80.6 90.3 86.2 Corn 3000 15000 88.3 85.9 84.2 90.1 94.3 93.1 85.5 93.1 90.2 SBM 0 0 84.4 85.2 81.4 87.3 89.3 84.8 83.0 85.1 84.1 SBM 3000 0 85.0 86.1 82.5 87.9 90.2 85.6 83.8 85.9 85.3 SBM 0 15000 86.7 87.6 84.2 89.2 90.9 87.0 85.1 86.6 86.2 SBM 3000 15000 87.9 89.0 85.5 90.1 91.7 88.0 86.3 87.9 87.3 Corn/SBM 0 0 86.4 85.8 84.1 88.7 91.0 87.6 84.5 88.0 87.2 Corn/SBM 3000 0 85.7 85.1 83.4 87.8 90.6 87.4 83.5 87.0 86.2 Corn/SBM 0 15000 88.3 87.3 86.3 90.2 91.9 89.2 86.7 89.5 88.6 Corn/SBM 3000 15000 91.2 90.8 90.2 92.9 94.1 92.3 90.1 91.9 91.7 Pooled SEM 1.64 2.18 2.46 1.65 1.33 1.16 1.93 1.50 1.84 Model P < 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 Main Effects Corn 84.1.sup.a 80.4.sup.a 76.2.sup.a 85.8.sup.a 92.1 90.1.sup.b 80.8.sup.a 90.4.sup.b 86.3 SBM 86.0.sup.b 87.0.sup.b 83.4.sup.b 88.6.sup.b 90.5 86.4.sup.a 84.5.sup.b 86.4.sup.a 85.7 Corn/SBM 87.9.sup.b 87.2.sup.b 86.0.sup.b 89.9.sup.b 91.9 89.1.sup.b 86.2.sup.b 89.1.sup.b 88.4 P < 0.01 0.001 0.001 0.01 NS 0.001 0.001 0.01 NS 0 85.3 84.0 80.8 87.4 91.0 87.9 83.0 88.1 86.1 3000 86.7 85.7 83.0 88.8 92.0 89.1 84.7 89.2 87.5 P < NS NS NS NS NS 0.05 NS NS NS 0 84.3 83.0 79.7 86.7 90.5 87.2 82.0 87.3 85.3 15000 87.6 86.7 84.1 89.6 92.5 89.9 85.7 89.9 88.4 P < 0.001 0.01 0.01 0.01 0.01 0.001 0.01 0.01 0.01 Interaction Terms Diet*Pro- NS NS NS NS NS NS NS NS NS tease P < Diet*Phy- NS NS NS NS NS NS NS NS NS tase P < Phytase*Pro- NS NS 0.05 NS NS 0.05 NS NS NS tease P < Diet*Phy- NS NS NS NS NS NS NS NS NS tase*Pro- tease P Diet Ile Leu Tyr Phe His Lys Arg Cys Me Corn 85.3 91.6 87.8 89.1 87.5 78.4 90.7 84.3 91 Corn 85.2 91.7 88.4 89.4 87.1 79.2 91.1 83.7 91 Corn 87.2 93.0 89.7 90.9 87.9 81.1 92.3 85.7 91 Corn 91.1 94.9 91.8 93.4 90.7 85.2 94.4 91.1 94.9 SBM 85.7 85.4 89.8 87.1 87.8 87.9 91.8 76.7 87.8 SBM 87.0 86.5 90.5 88.2 88.4 88.6 92.7 77.9 88.5 SBM 87.6 87.3 91.0 89.0 89.3 89.5 93.1 81.4 88.9 SBM 88.6 88.4 91.5 89.8 90.3 90.3 93.5 83.1 90.0 Corn/SBM 88.1 88.9 91.4 89.5 89.1 88.7 93.0 80.1 91.3 Corn/SBM 87.4 88.3 90.5 89.0 88.5 88.5 92.7 79.8 90.3 Corn/SBM 89.5 90.2 92.2 90.8 90.5 90.4 94.1 82.6 92.4 Corn/SBM 92.4 92.8 94.6 93.4 92.9 92.7 95.8 87.9 94.4 Pooled SEM 1.96 1.26 1.48 1.36 1.26 2.81 1.41 1.58 1.65 Model P < 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 Main Effects Corn 87.2 92.8.sup.c 89.4.sup.a 90.7.sup.b 88.3 81.0.sup.a 92.1 86.2.sup.c 92.4.sup.b SBM 87.2 87.0.sup.a 90.7.sup.ab 88.5.sup.a 90.2 89.0.sup.b 92.8 79.8.sup.a 88.8.sup.a Corn/SBM 89.4 90.0.sup.b 92.2.sup.b 90.7.sup.b 89.0 90.1.sup.b 92.1 82.6.sup.b 92.1.sup.b P < NS 0.001 0.05 0.05 NS 0.001 NS 0.001 0.01 87.2 89.4 90.3 89.4 88.7 86.0 82.5 81.8 90.6 88.6 90.4 91.2 90.5 89.7 87.4 93.4 83.9 91.6 NS NS NS NS NS NS NS 0.05 NS 86.5 88.7 89.7 88.7 88.1 85.2 92.0 80.4 90.1 89.4 91.1 91.8 91.2 90.2 88.2 93.9 85.3 92 0.05 0.01 0.05 0.01 0.01 0.05 0.05 0.001 0 Interaction Terms Diet*Pro- NS NS NS NS NS NS NS NS NS tease P < Diet*Phy- NS NS NS NS NS NS NS NS NS tase P < Phytase*Pro- NS NS NS NS NS NS NS 0.05 NS tease P < Diet*Phy- NS NS NS NS NS NS NS NS NS tase*Pro- tease P Values in columns without a common superscript differ significantly (P < 0.05, unless otherwise stated). indicates data missing or illegible when filed