Phytic Acid and Phytase: Implications for Protein Utilization by Poultry A. J. Cowieson,*1 T. Acamovic,*2 and M. R. Bedford† *Avian Science Research Centre, Scottish Agricultural College, Ayr Campus, Ayr, KA6 5HW, UK; and †Zymetrics Inc., Chestnut House, Beckhampton, Marlborough, Wiltshire, SN8 1QJ, UK ABSTRACT The effect of the ingestion of myo-inositol acids, N, and DM of casein compared with birds fed hexaphosphate (IP6) and phytase (EC 3.1.3.26) on the di- casein alone. Supplementation of the mixture of casein < gestibility of casein was investigated using growing and IP6 with phytase improved (P 0.05) the digestibility broiler chickens. A total of 64 female Ross broilers were coefficients of amino acids compared with birds fed on used in a precision feeding study. One group of 8 birds casein and IP6 with no supplemental phytase. The excre- < was fed a solution of glucose to estimate endogenous tion of endogenous minerals was increased (P 0.05) < losses. Seven groups, each of 8 birds, were fed either by the ingestion of IP6 and reduced (P 0.05) by the casein, casein + 1,000 units of phytase activity (FTU), supplementation of IP6 with phytase. In the absence of exogenous phytase, the recovery of phytate-P in excreta casein + 2,000 FTU, casein + 0.5 g of IP , casein + 0.5 g 6 was approximately 80%. However, the recovery of phy- of IP + 1,000 FTU, casein+1gofIP, or casein +1gofIP 6 6 6 tate-P was significantly reduced by the addition of exoge- + 1,000 FTU. The excretion of DM, amino acids, nitrogen, nous phytase to the IP6/casein mixture. It can be con- minerals, and phytate-phosphorus was determined over cluded that the ingestion of IP6 reduces the digestibility a 48-h period and nutrient digestibility coefficients were coefficients of amino acids and the metabolizability of calculated. Casein was found to be highly digestible, with nitrogen of casein. This is likely to be mediated partially true coefficients of DM, N, and amino acid digestibility through increased endogenous losses. However, the addi- of between 0.85 and 1.0. However, the ingestion of IP6 tion of phytase can partially ameliorate the detrimental < reduced (P 0.05) the digestibility coefficients of amino effects of IP6 on protein utilization. Key words: inositol phosphate, phytate, phytase, broilers, precision feeding 2006 Poultry Science 85:878–885 INTRODUCTION (Maenz et al., 1999; Maenz, 2001), forming insoluble salts at intestinal pH, reducing the availability of these miner- A variable but large proportion of the phosphorus (P) als for absorption (Lonnerdal et al., 1999; Selle et al., 2000; in plant material is in the form of phytate-P (myo-inositol Sandberg, 2002). Furthermore, the availability of dietary hexakis phosphate; Haug and Lantzsch, 1983; Eeckhout amino acids and energy is reduced with the presence of and De Paepe, 1994; Harland and Morris, 1995; Harland IP6 in the ration (Ravindran et al., 2000; Selle et al., 2000). and Narula, 1999; Sandberg, 2002). Phytate-P is largely Although exogenous phytase can improve the retention unavailable for utilization by poultry due to a lack of of dietary P, the addition of exogenous phytases to poul- effective endogenous phytase, the enzyme responsible try diets improves performance parameters other than for the hydrolysis of phytate. Inositol hexaphosphate is those associated with an improvement in P utilization relatively reactive having 12 dissociable protons with pKa (Kemme et al., 1999; Ravindran et al., 2000, 2001; Selle et values that range from about 1.5 to 10 (Costello et al., al., 2000). The interaction between IP6 and protein is 1976). The reactivity of inositol phosphate isomers is largely dependent on pH, with binary protein-IP6 com- highly dependent on the conformation and configuration plexes being formed at low pH and ternary protein-IP6- of the molecule and the pH of its environment (Costello mineral complexes formed as pH increases toward neu- et al., 1976; Cheryan, 1980). Fully phosphorylated myo- trality (Selle et al., 2000; Maenz, 2001; Adeola and Sands, inositol (IP6) is a potent chelator of many mineral ions 2003). The complexation of minerals by phytate is likely to reduce their participation as cofactors in enzymes, es- pecially under suboptimal supplies. Knuckles et al. (1989) 2006 Poultry Science Association, Inc. demonstrated in vitro that esters of inositol phosphate Received July 22, 2005. inhibited the digestion of casein by pepsin, the extent of Accepted September 8, 2005. inhibition being a function of the degree of phosphoryla- 1Current address: Danisco Animal Nutrition, PO Box 777, Marlbor- ough, Wiltshire, SN8 1XN, UK. tion of the inositol ring. This inhibition may be a function 2Corresponding author: [email protected] of reduced enzyme activity or an interaction between 878 PHYTIC ACID, PHYTASE, AND PROTEIN UTILIZATION 879 inositol phosphate and protein. However, the interaction IP6, casein + 0.5 g of IP6 + 1,000 FTU, casein + 1 g of IP6, between amino acids and proteins, and IP6 in the gastroin- and casein + 1 g of IP6 + 1,000 FTU. Each treatment group testinal tract of poultry is not well understood and re- consisted of 8 replicate birds and a further 8 birds were quires further examination. fed a solution of glucose to estimate endogenous losses. The addition of exogenous phytases to the diets of After feeding, the birds were placed in individual metabo- poultry has been shown to improve weight gain, mineral lism cages and excreta were collected quantitatively over retention, ME, and amino acid digestibility (Ravindran a 48-h period. The total excretion of DM, N, amino acids, et al., 1999; Newkirk and Classen, 2001; Murai et al., 2002; phytate-P, and minerals were determined and, when ap- Rutherfurd et al., 2002; Augspurger et al., 2003; Cowieson propriate, digestibility coefficients were calculated. and Adeola, 2005). Phytases also reduce the excretion of phytate-P, particularly in diets that have been formulated Laboratory Analyses to have a low available P concentration (Zhang et al., 1999; Ahmad et al., 2000; Edens et al., 2000; Selle et al., Following collection, the excreta were freeze dried, 2000). The improvements in amino acid utilization associ- weighed, and milled to pass a 1-mm aperture. The total ated with the addition of exogenous phytases to poultry quantity of DM excreted was recorded and the content diets are likely to be mediated through both a reduced of N in the excreta was determined using a LECO FP- endogenous amino acid loss as well as an improvement 2000 Analyzer (LECO, St. Joseph, MI). Amino acid con- in the retention of dietary amino acids (Cowieson et al., centrations in the excreta were determined by HPLC fol- 2004). Previous work from this laboratory has demon- lowing acid hydrolysis. The HPLC system comprised a strated, in model systems using growing broiler chickens, Dionex ASI-100 autosampler fitted with a Dionex P580 that ingestion of IP increases the excretion of endogenous 6 pump and a Dionex RF-2000 detector (Sunnyvale, CA). compounds including amino acids (Cowieson et al., 2004). The flow rate was 1 mL/min and the column used was Because of the complexity of components in diets, it is a Spherisorb ODS2 (150 × 4.6 mm fitted with a Waters impossible to characterize with clarity what supplemental guard cartridge (Alltech Assoc., Camforth, UK). The min- dietary phytase affects to produce the improvements or eral content of the excreta, after digestion with nitric acid changes seen. The effects of both phytate and phytases in a microwave digestion unit, was determined using an can be studied in a model system in which variation is Optima 4300 DV Dual View ICP-OE spectrometer (Per- minimized. The purpose of the study reported here was to investigate the effects of supplemental exogenous phytase kin-Elmer, Beaconsfield, UK). The concentration of phy- tate-P in the excreta was determined colorimetrically us- and IP6 on the utilization of protein and amino acids from casein using a precision feeding procedure. ing the method of Haug and Lantzsch (1983). MATERIALS AND METHODS Statistical Analysis and Digestibility Coefficient Calculation Bird Management Data were analyzed statistically using ANOVA (Gens- Sixty-four female Ross broilers were reared under stan- tat, release 5, IACR, Rothamstead, UK) to determine the dard commercial conditions until they reached the target main effects of IP6 and phytase. Tukey’s LSD was used BW of approximately 2.5 kg. During the growing phase, to detect significant differences between means. the birds were fed a standard broiler ration (calculated True digestibility coefficients of DM, N, amino acids, provision was 13.3 MJ of ME/kg and 210 g of CP/kg). and S were calculated using the formula [Nin − (Nexc − The precision feeding study was run according to the Nexcg)]/Nin, where Nin is the quantity of nutrient in- method of McNab and Blair (1988), as modified by Ferraz gested, Nexc is the quantity of nutrient excreted, and de Oliveira et al. (1994). Briefly, when the birds reached Nexcg is the mean quantity of nutrient excreted by the 8 the target weight (at approximately 42 d) they were birds fed the glucose solution. The apparent digestibility placed in a pen with a raised slatted floor and feed was coefficient of phytate-P was calculated using the formula withdrawn for a period of 48 h. Water was provided ad (pPin − pPexc)/pPin, where pPin is the quantity of phytate- libitum via suspended nipple drinker lines throughout P ingested and pPexc is the quantity of phytate-P excreted. the experimental period. After the 48-h period of feed deprivation, the birds were precision-fed a suspension of 5 g of casein in 50 mL of distilled water with and without Animal Ethics IP6 [aqueous solution of phytic acid (cat no.