METABOLISM AND NUTRITION. Influence of Microbial Phytase on Apparent Ileal Amino Acid Digestibility of Feedstuffs for Broilers

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1 METABOLISM AND NUTRITION Influence of Microbial Phytase on Apparent Ileal Amino Acid Digestibility of Feedstuffs for Broilers V. RAVINDRAN,1 S. CABAHUG, G. RAVINDRAN, and W. L. BRYDEN Department of Animal Science, The University of Sydney, Camden, NSW 2570, Australia ABSTRACT The influence of microbial phytase on the ileal amino acid digestibilities in three cereals (corn, sorghum, and wheat), four oilseed meals (soybean meal, canola meal, cottonseed meal, and sunflower meal) and two cereal by-products (wheat middlings and rice polishings) was determined using 5-wk-old broilers. Supplementation of microbial phytase (1,200 FTU/kg) improved (P < to 0.10) the digestibilities of protein and amino acids in all feedstuffs, but the magnitude of response varied depending on the feedstuff and the amino acid considered. Mean digestibility of the 15 amino acids in the feedstuffs without and with phytase were: corn, 78.0 and 80.4%; sorghum, 74.7 and 79.4%; wheat, 77.7 and 84.6%; soybean meal, 82.2 and 85.5%; canola meal, 78.7 and 80.7%; cottonseed meal, 70.8 and 74.2%; sunflower meal, 76.7 and 80.2%; wheat middlings, 70.8 and 73.4%; and rice polishings 62.1 and 66.9%, respectively. When individual amino acids were considered, the increments in digestibility were relatively higher for threonine and valine. This effect was consistent across all feedstuffs. The observed variations in response among feedstuffs were influenced by the inherent protein digestibility, but not by dietary phytic acid concentration. No correlations were determined between the dietary concentrations of phytic acid and phytase responses in terms of protein digestibility (r = 0.20; P > 0.31) and mean amino acid digestibility (r = 0.12; P > 0.51); however a significant negative correlation was observed between inherent protein digestibility and phytase responses in protein digestibility (r = 0.42; P < 0.03). It appears that solubilities of phytate salts and protein, and their influence on the degree of phytateprotein complexing in different feedstuffs, may be more relevant than total phytic acid concentrations. Interestingly, dietary phytic acid concentrations were negatively correlated with inherent protein (r = 0.81; P < 0.001) and mean amino acid (r = 0.85; P < 0.001) digestibilities of the feedstuffs evaluated in this study. (Key words: microbial phytase, amino acid digestibility, feedstuffs, phytic acid, broiler) 1999 Poultry Science 78: INTRODUCTION Until recently, phytic acid (PA) was considered primarily as a factor limiting P availability from plantderived feedstuffs; however, current evidence shows that the deleterious effects of PA in the nutrition of simple-stomached animals go beyond just limiting P availability (Ravindran et al., 1995). In its native state, phytate is also complexed with various cations, protein, lipids (Cosgrove, 1966), and starch (Thompson and Yoon, 1984). The association between phytate and proteins begins in seeds during ripening, when phytate accumulates primarily in the protein-rich aleurone layers of monocotyledonous seeds and in the protein bodies of dicotyledonous seeds (Cosgrove, 1980). The Received for publication July 22, Accepted for publication December 8, To whom correspondence should be addressed: V.Ravindran@ massey.ac.nz Present address: Monogastric Research Centre, Institute for Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand. interaction between PA and proteins is believed to be of an ionic type and is dependent on ph (Anderson, 1985). At low ph, PA forms electrostatic linkages with the basic arginine, lysine, and histidine residues, resulting in insoluble complexes. As the ph approaches the isoelectric point, the charge on the protein is neutralized and the PA is no longer bound and becomes soluble. In this soluble state, PA complexes with protein because of the presence of divalent cations. These cations, usually calcium, magnesium, or zinc, act as a bridge between negatively charged protein carboxyl groups and the phytate (Anderson, 1985). In vitro studies have shown that phytate-protein complexes are insoluble and less subject to attack by proteolytic enzymes than the same protein alone (Barre et al., 1954; Ravindran et al., 1995). The reduced solubility of proteins as a result of such complexing can adversely affect certain functional properties of proteins that are Abbreviation Key: AA = amino acid; AIA = acid insoluble ash; PA = phytic acid. 699

2 700 dependent on their hydration and solubility. This aspect and implications in food systems have been reviewed (Cheryan, 1980). The relevance of phytate-protein complexes in poultry and swine nutrition, however, has not received attention until recently. By releasing these phytate-bound nutrients and improving their utilization, dietary supplementation of microbial phytase would be expected to have protein/ amino acid (AA) effects in poultry and swine. The influence of microbial phytase on protein/aa digestion and utilization is a topic of current interest, as evidenced by an upsurge in recent research reports (Kornegay, 1996; Yi et al., 1996; Biehl and Baker, 1997; Sebastian et al., 1997; Ravindran et al., 1998a). All these studies demonstrate generally positive effects of supplemental phytase on protein/aa digestibility and utilization in broilers. Clearly, the protein/aa effect of microbial phytase needs to be quantified to enable its inclusion in least cost diet formulations. The inherent digestibility of protein and the PA concentration in feedstuffs are probably the two important factors that determine the magnitude of this effect. If this hypothesis is correct, then a meaningful approach would be to assign protein/aa replacement values to feedstuffs (by determining protein and AA digestibilities without and with microbial phytase) rather than to the enzyme. The objective of the present study was to determine the influence of microbial phytase in broilers on the apparent ileal AA digestibility in four plant protein sources, three cereals, and two cereal by-products. Ingredients MATERIALS AND METHODS The ingredients tested included three cereals (corn, sorghum, and wheat), four plant protein meals (soybean meal, cottonseed meal, canola meal, and sunflower meal), and two cereal by-products (wheat middlings and rice polishings). The contents of crude protein, AA, total and phytate P, and endogenous phytase activity of the feedstuffs were measured as described later and are presented in Table 1. The three ingredient types were evaluated in separate digestibility assays. All assays followed similar experimental procedures, the only difference being the composition of assay diets. Birds Day-old male broiler chicks (Inghams TM 70 strain) were obtained from a local hatchery and raised in battery 2Dextrose monohydrate; Starch Australasia Ltd., Tamworth, NSW 2340, Australia. 3Celite Corp., Lompoc, CA BASF AG, Ludwigshafen, Germany. RAVINDRAN ET AL. brooders. The birds received a commercial broiler starter diet (230 g crude protein/kg) from Days 1 to 20. The birds were transferred to battery cages housed in an environmentally controlled room (22 C) on Day 21 and fed a commercial broiler finisher diet (180 g crude protein/kg). Experimental procedures were approved by the University of Sydney Animal Care and Ethics Committee and complied with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Digestibility Assays On Day 35, birds of uniform body weight were chosen and randomly assigned in groups of six to colony cages. Test ingredients were incorporated as the sole source of dietary protein in assay diets (Table 2). The composition of assay diets varied depending on the ingredient type (Ravindran et al., 1998b). In the case of cereals and cereal by-products, diets contained 918 g of the test feedstuff/kg. In the case of oilseed meals, assay diets were based on dextrose2 and test feedstuff. The proportions of dextrose and the test feedstuff were varied in each diet to obtain 180 to 200 g crude protein/kg. All diets contained per kilogram 17 to 19 g dicalcium phosphate and 10 to 13 g limestone. No attempt was made to balance the dietary concentrations of calcium and P. Celite,3 a source of acidinsoluble ash (AIA), was added at 20 g/kg level to all diets as an indigestible marker. Each diet, in mash form, (without or with 1200 FTU/kg Natuphos 4 phytase) was offered for at libitum consumption to three pens (five or six birds per pen) from 35 to 42 d of age. One unit of phytase (FTU) is defined as the quantity of enzyme that releases 1 mmol of inorganic P/ min from mol/l sodium phytate at ph 5.5 at 37 C. The dosage of 1,200 FTU/kg employed in our study was twice the level recommended by the manufacturer for use in broiler diets. This procedure was used to ensure the expression of protein/aa effects of microbial phytase. Water was available at all times. On Day 42, all birds were euthanatized by intracardial injection of sodium pentobarbitone, and the contents of the lower half of the ileum were collected by gentle flushing with distilled water into plastic containers. The ileum was defined as that portion of the small intestine extending from the vitelline diverticulum to a point 40 mm proximal to the ileo-cecal junction. Ileal digesta of birds within a pen were pooled, resulting in three composite samples per dietary treatment. The digesta were frozen immediately after collection and subsequently lyophilized. Lyophilized digesta samples were ground to pass through a 0.5-mm sieve and stored in airtight containers at 4 C for chemical analyses. Chemical Analyses Amino acid concentrations in ingredient, diet, and ileal digesta samples were determined by HPLC. Sample preparation for HPLC analysis involved hydrolysis of

3 MICROBIAL PHYTASE AND AMINO ACID DIGESTIBILITY 701 TABLE 1. Analyzed contents 1 of crude protein, amino acids, total phosphorus, phytate phosphorus, and phytic acid, and endogenous phytase activity 2 in the feedstuffs tested in the present study Soybean Canola Cottonseed Sunflower Wheat Rice Parameter Corn Sorghum Wheat meal meal meal meal middlings polishings (g/kg) Crude protein (N 6.25) Indispensable amino acids Arginine Histidine Isoleucine Leucine Lysine Phenylalanine Threonine Valine Dispensable amino acids Alanine Aspartic acid Glycine Glutamic acid Serine Tyrosine Total phosphorus Phytate phosphorus Phytic acid Phytase activity, FTU/kg 10 < <10 <10 <10 2, Mean of duplicate analyses for amino acids and, mean of triplicate analyses for nitrogen, phosphorus, phytate phosphorus, and phytase activity. 2One unit of phytase (FTU) is defined as the quantity of enzyme that releases 1 mmol of inorganic phosphorus/min from mol/l sodium phytate at ph 5.5 at 37 C. 3Calculated assuming a P content of 282 g/kg in phytic acid molecule (Cosgrove, 1966). samples according to the procedures described by Siriwan et al. (1993). Briefly, the samples were hydrolyzed under nitrogen with 8 M HCl containing phenol (3 g/l) for 16 h at 120 C. DL-Norleucine was added to the hydrolysate as an internal standard. Each hydrolysate was then diluted and adjusted to ph 2.2 to 2.3, the same as the AA standard.5 The hydrolysates were passed through a 0.22-mm Nylon 66 membrane filter.6 Aliquots of the hydrolysates were then subjected to ion-exchange column chromatography using a Shimadzu AA analysis system.7 Underivatized amino acids were eluted by a gradient of ph 3.20 sodium citrate eluent to ph sodium citrate eluent at a flow rate of 0.4 ml/min and a column temperature of 60 C. O-phthaldialdehyde8 was used for postcolumn derivatization of AA. Chicken egg white lysozyme9 was used as an analytical control to monitor the reproducibility and accuracy of the AA determinations. Nitrogen content was determined by the method of Sweeney (1989) using a FP-428 nitrogen determinator.10 The AIA contents of diet and ileal digesta samples were measured after ashing the samples and treating the ash with boiling 4 M HCl (Siriwan et al., 1993). Contents of 5Standard H, Pierce Chemicals Co., Rockford, IL Alltech, Baulkham Hills, NSW 2153, Australia. 7Shimadzu Corp., Kyoto, Japan. 8Sigma Chemicals Co., St. Louis, MO Seikagaku Co., Chuo-ku, Tokyo, Japan. 10LECO Corp., St. Joseph, MI total and phytate P and endogenous phytase activity in the ingredients were determined as previously described (Selle et al., 1996). Calculations Apparent ileal protein and AA digestibilities were calculated, using AIA as the indigestible marker, as shown below. Celite was added to diets to increase the AIA fraction and to improve the precision of the measurement. Apparent AA digestibility (percentage) (AA/AIA) d (AA/AIA) i = 100 (AA/AIA) d where (AA/AIA) d = ratio of AA to AIA in diet; and (AA/ AIA) i = ratio of AA to AIA in ileal digesta. Statistical Analysis Data from each assay (ingredient type) were separately analyzed using the General Linear Models procedure with the model containing ingredient, phytase, ingredient by phytase interaction, and replicate. Differences were considered significant at P < 0.05, although probability values up to P < 0.10 are shown in Table 5 because the data suggest a trend. To determine whether the response (percentage improvement) in protein and average AA digestibilities to added phytase was influenced by dietary

4 702 RAVINDRAN ET AL. TABLE 2. Composition of diets (air dry basis) for amino acid digestibility assays Soybean Canola Cottonseed Sunflower Wheat Rice Component Corn Wheat Sorghum meal meal meal meal middlings polishings Test ingredient Glucose Vegetable oil Celite Dicalcium phosphate Limestone Choline chloride Salt Vitamin-trace mineral premix Supplied the following amounts per kilogram of diet: vitamin A (trans-retinol), 8,280 IU; cholecalciferol, 4,920 IU; vitamin E (dl-a-tocopheryl acetate), 28 IU; menadione, 2.8 mg; thiamine, 2.1 mg; riboflavin, 11.2 mg; calcium pantothenate, 21 mg; niacin, 42 mg; pyridoxine, 7 mg; folic acid, 2.8 mg; cyanocobalamin, mg; biotin, 0.14 mg; Mn, 105 mg; Zn, 70 mg; Cu, 7 mg; Mo, 2.2 mg; Co, 0.42 mg; I, 1.4 mg; Fe, 28 mg; Se, mg; choline chloride, 420 mg; and ethoxyquin, 175 mg. (g/kg) PA concentration and inherent digestibility of feedstuffs, linear regression analyses were performed and correlations were computed. The MINITAB (1996) version 11 program was used for the statistical analysis. RESULTS The phytate P contents ranged from 1.60 g/kg in wheat to 15.5 g/kg in rice polishings (Table 1). Phytatebound P accounted for 70 to 80% of the total P in cereals and oilseed meals. In the case of wheat middlings and rice polishings, over 90% of the total P was in the form of phytate P. Of the feedstuffs tested, only wheat middlings (2,500 FTU/kg) and wheat (340 FTU/kg) had significant phytase activity. The influence of microbial phytase on the apparent ileal protein and AA digestibilities of the feedstuffs are presented in Tables 3 to 5. Because methionine may be degraded to some extent when acid hydrolysis is carried out in the presence of carbohydrates (Blackburn, 1978), and because performic acid oxidation was not performed before analysis, values for methionine are not presented. Addition of microbial phytase improved the digestibilities of protein and AA in all feedstuffs, but the TABLE 3. Apparent ileal digestibility of amino acids in corn, sorghum and wheat as influenced by the addition of Natuphos phytase 1 Corn Sorghum Wheat Phytase addition SEM 2 (%) Crude protein Indispensable amino acids Arginine Histidine Isoleucine Leucine 3, Lysine Phenylalanine Threonine Valine Dispensable amino acids Alanine 3, Aspartic acid Glycine Glutamic acid 3, Serine Tyrosine 3, Overall mean Values are means of three observations. Main effect of ingredient was significant (P < to 0.05) for all amino acids except aspartic acid and lysine. = without phytase; + = with phytase. 2Pooled standard error of mean. 3Main effect of phytase (P < 0.01). 4Ingredient by phytase interaction (P < 0.05).

5 MICROBIAL PHYTASE AND AMINO ACID DIGESTIBILITY 703 TABLE 4. Apparent ileal digestibility of amino acids in soybean meal, canola meal, cottonseed meal, and sunflower meal as influenced by the addition of Natuphos phytase 1 Soybean meal Canola meal Cottonseed meal Sunflower meal Phytase addition SEM 2 (%) Crude protein Indispensable amino acids Arginine Histidine Isoleucine Leucine Lysine Phenylalanine Threonine Valine Dispensable amino acids Alanine Aspartic acid Glycine Glutamic acid Serine Tyrosine Overall mean Values are means of three observations. Main effect of ingredient was significant (P < to 0.05) for all amino acids, except for arginine and glutamic acid. = without phytase; + = with phytase. 2Pooled SEM. 3Main effect of phytase (P < 0.01). 4Main effect of phytase (P < 0.05). magnitude of response varied depending on the feedstuff and the AA considered. Main effects (P < 0.01) of phytase were observed for the digestibilities of protein and AA in cereals (Table 3). However, the magnitude of responses in the digestibilities of leucine, alanine, and glutamic acid were much smaller in corn than in wheat and sorghum, resulting in an ingredient by phytase interaction (P < 0.05). The improvements in AA digestibility were greatest in wheat (5.3 to 10.4 percentage units), followed by sorghum (2.7 to 5.5 percentage units), and lowest in corn (0.9 to 4.4 percentage units). The percentage improvements in the mean digestibility of the 15 AA in corn, sorghum, and wheat were 3.1, 6.3, and 8.9, respectively. Protein and AA digestibilities in oilseed meals were similarly improved (P < 0.05 to 0.01) by phytase addition (Table 4). The percentage improvements in the mean digestibility of the 15 amino acids in soybean meal, canola meal, cottonseed meal, and sunflower meal were 4.0, 2.5, 4.8, and 4.6, respectively. Although the magnitude of improvement in AA digestibility was consistently smaller in canola meal, the ingredient by phytase interaction was not significant (P > 0.10) for any of the AA. Improved (P < 0.10 to 0.01) digestibilities of protein and AA were also obtained for wheat middlings and rice polishings when microbial phytase was added (Table 5). The percentage improvements in the mean digestibility of the 15 AA in wheat middlings and rice polishings were 3.7 and 7.7, respectively. When the effect of phytase addition on individual AA were considered, the increments in digestibility were relatively higher for threonine. This effect was consistent across all feedstuffs. When averaged across all feedstuffs, the percentage increments in the different indispensable AA were: arginine, 3.7; histidine, 5.1; isoleucine, 4.8; leucine, 4.5; lysine, 4.5; phenylalanine, 4.7; threonine, 7.9 and valine, 5.4. The corresponding values for the different dispensable AA were: alanine, 4.8; aspartic acid, 6.0; glycine, 6.1; glutamic acid, 3.4; serine, 6.0 and tyrosine, 5.6. Correlations between dietary PA levels and inherent protein digestibility and phytase responses in protein and digestibility mean AA digestibility are presented in Table 6. No correlations were observed between the dietary concentrations of PA and phytase responses in terms of protein digestibility (r = 0.20; P > 0.31) and mean AA digestibility (r = 0.12; P > 0.51). There were significant negative correlations between inherent protein digestibility and phytase responses in protein digestibility (r = 0.42; P < 0.03) and between inherent mean AA digestibility and phytase responses in mean AA digestibility (r = 0.51; P < 0.01). Dietary PA concentrations were negatively correlated with inherent protein digestibility (r = 0.81; P < 0.001) and inherent mean AA digestibility (r = 0.85; P < 0.001) of the feedstuffs. DISCUSSION The present study demonstrated the beneficial effects of microbial phytase (1,200 FTU/kg) on the apparent ileal digestibility of protein and AA for broilers in a

6 704 RAVINDRAN ET AL. TABLE 5. Apparent ileal digestibility of amino acids in wheat middlings and rice polishings as influenced by the addition of Natuphos phytase 1 Wheat middlings Rice polishings Phytase addition + + SEM 2 (%) Crude protein Indispensable amino acids Arginine Histidine Isoleucine Leucine Lysine Phenylalanine Threonine Valine Dispensable amino acids Alanine Aspartic acid Glycine Glutamic acid Serine Tyrosine Overall mean Values are means of three observations. Main effect of ingredient was significant (P < to 0.05) for all amino acids, except histidine. = without phytase; + = with phytase. 2Pooled SEM. 3Main effect of phytase (P < 0.01). 4Main effect of phytase (P < 0.05). 5Main effect of phytase (P < 0.10). range of feedstuffs. These results are in general agreement with the findings of several recent studies (Kornegay, 1996; Yi et al., 1996; Sebastian et al., 1997; Ravindran et al., 1998a). It is noteworthy, however, in the studies reported by Sebastian et al. (1997), supplemental phytase was found to improve ileal AA digestibility in female broilers but not in males. The reasons for this apparent sex effect are unclear, but the results of the present study, along with others (Kornegay, 1996; Ravindran et al., 1998a) in which male broilers were used, contradict these findings. TABLE 6. Correlation coefficients between dietary phytic acid and inherent protein/amino acid digestibility and phytase responses in protein digestibility and mean amino acid digestibility of feedstuffs in Tables 3 to 5 Correlation r Dietary phytic acid vs inherent protein digestibility 0.81*** Dietary phytic acid vs inherent mean AA digestibility 0.85*** Dietary phytic acid vs phytase responses in protein digestibility 0.20 Dietary phytic acid vs phytase responses in mean AA digestibility 0.12 Inherent protein digestibility vs phytase responses in protein digestibility 0.42* Inherent mean AA digestibility vs phytase responses in mean AA digestibility 0.51** *P < **P < ***P < One of the aims of the current study was to test the hypothesis that the magnitude of responses in protein/ AA digestibility would be determined by the dietary PA concentration and inherent protein/aa digestibility of feedstuffs. It was anticipated that the responses would be greater in feedstuffs that contain high levels of PA. There was, however, no significant correlation between the percentage improvements in protein/average AA digestibility and dietary total PA concentrations. It would appear that the responses to supplemental phytase may be influenced by more subtle structural or chemical properties of both the PA and protein rather than by the total concentration of PA. It is probable that these structural and chemical properties determine the degree of phytate-protein binding, which, in turn, may influence the protein/aa responses to phytase addition. The properties that contribute to a protein s ability to bind PA include its structure, solubility, and the available AA residues. The ability of PA to complex with protein, on the other hand, is determined largely by its solubility, which, in turn, is dependent on the degree of its complexing with minerals (Abdul-Kadir, 1980). The significant negative correlations between inherent protein/aa digestibility and phytase responses in protein/aa digestibility indicate that feedstuffs that are poorly digested may respond better to phytase addition than those with higher inherent digestibility. Interestingly, the inherent protein and AA digestibilities were

7 MICROBIAL PHYTASE AND AMINO ACID DIGESTIBILITY 705 influenced by dietary PA concentrations. Dietary PA concentration explained 66 and 68% of the variation in inherent protein and AA digestibilities, respectively, among the feedstuffs evaluated in the present study. Overall, our results confirm the protein/aa effect of microbial phytase in plant feedstuffs for broilers. The basis of these responses is becoming increasingly evident and is clearly related to the capacity of PA to bind protein/aa and to the ability of the enzyme to release these bound nutrients by hydrolyzing PA. One or more of the following complexes may be relevant in this context. 1) the presence of native phytate-protein complexes in plant feedstuffs, 2) the de novo formation of complexes between solubilized phytate and solubilized proteins/free AA in the ph range within the gut environment (Jongbloed et al., 1997; Rutherfurd et al., 1997), and 3) the inhibition/reduction in enzyme activity by the formation of phytate-enzymic protein complexes (see review, Ravindran et al., 1995). Inhibition may also result from the chelation of calcium ions, which are essential for the activity of trypsin and a-amylase, or possibly to an interaction with the substrates for these enzymes (Liener, 1989). The observed improvements in apparent digestibility of AA with microbial phytase may also reflect, at least in part, reduced endogenous AA losses resulting from the amelioration of the antinutritive effects of PA. Although specific studies relating to phytic acid are lacking, evidence with other antinutritional factors (such as trypsin inhibitors, nonstarch polysaccharides) indicate that these components can significantly increase gut endogenous nitrogen flows in simple-stomached animals (Barth et al., 1993; Angkanaporn et al., 1994). The relatively large effect of microbial phytase on threonine digestibility in the present study lends support to this premise. Endogenous secretions in chickens are known to contain relatively high concentrations of threonine (Siriwan et al., 1994; Angkanaporn et al., 1994). The improvements in protein/aa digestibility with added phytase were greatest for wheat. Because the phytate in wheat is largely located in the aleurone layer, it may be speculated that this increment results, at least in part, from the disruption of cell wall by microbial phytase, in a manner similar to that of exogenous xylanase enzymes (Petterson and Aman, 1989). This cell wall disruption would cause a better diffusion or enhanced contact between digestive enzymes, substrates, and digestion end-products. In summary, phytase supplementation increased apparent ileal digestibilities of protein and AA in a variety of feedstuffs. The magnitude of response varied among feedstuffs and among different amino acids within a feedstuff. The percentage improvements were greater in wheat, sorghum, and rice polishings, and lowest in canola meal. The observed variations in response among feedstuffs were affected by the inherent protein/aa digestibility of the feedstuffs, but were not related to dietary PA concentration. It would appear that the solubilities of phytate salts and protein, and their effects on the degree of phytate-protein binding in different feedstuffs, may be more important in determining the responses to dietary phytase addition. ACKNOWLEDGMENTS The study was supported by the Poultry Research Foundation of the University of Sydney. The able assistance of Lap Im Hew during amino acid analyses is acknowledged. REFERENCES Abdul-Kadir, R. B., The Effect of Phytate Content on the Nutritional Quality of Soy and Wheat Bran Proteins. Ph.D. thesis, University of Nebraska, Lincoln, NE. Angkanaporn, K., M. Choct, W. L. Bryden, E. F. Annison, and G. Annison, Effect of wheat pentosans on endogenous amino acid losses in chickens. J. Sci. Food Agric. 66: Anderson, P. A., Interactions between proteins and constituents that affect protein quality. Pages in: Digestibility and Amino Acid Availability in Cereals and Oilseeds. G. W. Finley and D. T. Hopkins, ed. American Association of Cereal Chemists, St. Paul, MN. Barre, R., J. E. Curtois, and G. Wormser, Etude de la structure de l acide phytique au moyen de ses courbes de titration et de la conductivite se ses solutions. Bull. Soc. Chim. Biol. 36: Barth, C. A., B. Lunding, M. Schmitz, and H. Hagemeister, Soybean trypsin inhibitor(s) reduce absorption of exogenous and increase loss of endogenous protein in miniature pigs. J. Nutr. 123: Biehl, R. R., and D. H. Baker, Microbial phytase improves amino acid utilisation in young chicks fed diets based on soybean meal, but not in diets based on peanut meal. Poultry Sci. 76: Blackburn, S., Amino Acid Determination: Methods and Techniques. Marcel Dekker, New York, NY. Cheryan, M., Phytic acid interactions in food systems. CRC Crit. Rev. Food Sci. 13: Cosgrove, D. J., The chemistry and biochemistry of inositol polyphosphates. Rev. Pure Appl. Chem. 16: Cosgrove, D. J., Inositol Phosphates: Their Chemistry, Biochemistry and Physiology. Elsevier Science Publishing Co., New York, NY. Jongbloed, A. W., L. de Jonge, P. A. Kemme, Z. Mroz, and A. K. Kies, Non-mineral related effects of phytase in pig diets. Pages in: Proceedings Sixth BASF Forum on Animal Nutrition, Ludwigshafen, Germany. Kornegay, E. T., Effects of Natuphos phytase on protein and amino acid digestibility and nitrogen retention of poultry. Pages in: Phytase in Animal Nutrition and Waste Management. M. B. Coelho and E. T. Kornegay, ed. BASF Corp., Mount Olive, NJ. Liener, I. E., Antinutritional factors in legume seeds: State of the art. Pages 6 11 in: Recent Advances of Research in Antinutritional Factors in Legume Seeds. J. Huisman, T.F.B.van der Poel, and I. E. Liener, ed. Pudoc, Wageningen, The Netherlands.

8 706 RAVINDRAN ET AL. Minitab, MINITAB Reference Manual. PC Version. Release 11. Minitab Inc., State College, PA. Pettersson, D., and P. Aman, Enzyme supplementation of a poultry diet containing rye and wheat. Br. J. Nutr. 62: Ravindran, V., W. L. Bryden, and E. T. Kornegay, Phytates: occurrence, bioavailability and implications in poultry nutrition. Poult. Avian Biol. Rev. 6: Ravindran, V., W. L. Bryden, S. Cabahug, and P. H. Selle, 1998a. Impact of microbial phytase on the digestibility of protein, amino acids and energy in broilers. Pages in: Proceedings of the Maryland Nutrition Conference for Feed Manufacturers, Baltimore, MD. Ravindran, V., L. I. Hew, and W. L. Bryden, 1998b. Digestible Amino Acids in Poultry Feedstuffs, Poultry Research Foundation, The University of Sydney, Camden and Rural Industries Research and Development Corporation, Canberra, Australia. Rutherfurd, S. M., A. C. Edwards, and P. H. Selle, Effect of phytase on lysine-rice pollard complexes. Page 248 in: Manipulating Pig Production VI. P. D. Cranwell, ed. Australasian Pig Science Association, Canberra, Australia. Sebastian, S., S. P. Touchburn, E. R. Chavez, and P. C. Lague, Apparent digestibility of protein and amino acids in broiler chickens fed a corn-soybean diet supplemented with microbial phytase. Poultry Sci. 76: Selle, P. H., V. Ravindran, D. J. Cadogan, A. R. Walker, and W. L. Bryden, The role of microbial phytases in poultry and pig nutrition. Pages in: Proceedings of the Tenth Australian Poultry and Feed Convention, Melbourne, Australia. Siriwan, P., W. L. Bryden, Y. Mollah, and E. F. Annison, Measurement of endogenous amino acid losses in poultry. Br. Poult. Sci. 34: Siriwan, P., W. L. Bryden, and E. F. Annison, Use of guanidinated dietary protein to measure losses of endogenous amino acid in poultry. Br. J. Nutr. 71: Sweeney, R. A., 1989: Generic combustion method for determination of crude protein in feeds: Collaborative study. J. Assoc. Off. Anal. Chem. 72: Thompson, L. U., and J. H. Yoon, Starch digestibility as affected by polyphenols and phytic acid. J. Food Sci. 49: Yi, Z., E. T. Kornegay, and D. M. Denbow, Effect of microbial phytase on nitrogen and amino acid digestibility and nitrogen retention of turkey poults fed corn-soybean meal diets. Poultry Sci. 75: