Interaction of Dietary Phytase and Glutamic Acid. on Bone Ash Response in Chicken Thigh. Atsushi Murai1), Kazumi Kital1, 2), Shoichi Tsurutal1,3)

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1 Journal of Poultry Science, 38: , 2001 Interaction of Dietary Phytase and Glutamic Acid on Bone Ash Response in Chicken Thigh Atsushi Murai1), Kazumi Kital1, 2), Shoichi Tsurutal1,3) and Jun-ichi Okumural1) 1)Laboratory of Animal Nutrition, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya The optimum ph of phytase originated from Aspergillus niger is approximately 5.5. In the present study, we tried to modify the intraluminal ph by dietary acidic amino acid to maximize phytase activity. Single Comb White Leghorn male chicks at 7 days of age were fed the experimental diets for 10 days. The experiment was 2 ~2 ~2 factorial arrangement of treatments with two dietary amino acid sources (L-glutamine or L- glutamic acid at 50g/kg diet), two levels of dietary phytase (0 or 1,000U/kg diet) and two levels of dietary available P supplementation (0 or 6g/kg diet). The glutamic acid supplementation significantly reduced ph in the crop (5.4 }0.2; n=7) compared with the glutamine supplementation (6.0 }0.1; n=7). There was a significant interaction between dietary amino acid source and phytase level on bone ash response in the thigh. Dietary phytase significantly increased bone ash content in chicks fed diets containing glutamic acid but not in the glutamine group. These results suggest that the modification of ph in the crop could activate phytase in chickens. Key words: chicken, phytase, glutamic acid, bone ash content Introduction Dietary phytase improves the availability of phytate P in non-ruminant animals (see Reviews of Liu et al., 1998; Van Der Klis and Versteegh,1999; Waldroup, 1999). Until now, many kinds of phytase derived from fungi, bacteria as well as plant have been developed as dietary supplements for animals. Most of isolated phytases are active with the range of ph (Liu et al., 1998). The microbial phytase derived from Aspergillus niger has optimum ph around 5.5. In chickens, Takemasa et al. (1996) showed that dietary phytase might work mainly in the crop of chicken. Therefore, the ph in crop is one of the important factors to control the activity of dietary phytase. One of the strategies for drawing the maximum potentiality of phytase is the modification of the intraluminal ph by dietary supplements. In the Received: July 27, 2000 Accepted: October 30, 2000 Correspondence should be addressed to: A. Murai, Laboratory of Animal Nutrition, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya Tel: , Fax: atsushi@agr.nagoya-u.ac.jp Present address is: 2) Laboratory of Grassland Science, University Farm, Graduate School of Bioagricultural Sciences, Nagoya University, Togo, Aichi ; 3)Owari-Higashi Agricultural Extension Office, Nagoya

2 Murai et al.: Bone Ash Response by Phytase 147 present study, therefore, we examined the combination effects of dietary acidic amino acid and microbial phytase derived from Aspergillus niger on gut ph and bone ash response in growing chickens. We report here the improvement of bone ash response by dietary phytase due to glutamic acid. Materials and Methods Day-old Single Comb White Leghorn male chicks were fed a commercial diet (Prespo(R), CP 21.5%, ME 12.1kJ/g diet, Marubeni Feed Co. Ltd., Tokyo) for 7 days prior to the experimental diets. The chicks were housed in stainless-steel metabolism cages in a constant temperature (28 }1 Ž) with artificial continuous lighting. Composition of the experimental diet is shown in Table 1. The experimental groups were arranged by factorial design (2 ~2 ~2) having two dietary amino acid sources (Lglutamine or L-glutamic acid at 50g/kg diet), two levels of microbial phytase originated from Aspergillus niger (0 or 1,000U/kg diet; Kyowa Hakko Kogyo Co. Ltd., Tokyo) and two levels of dietary available P supplementation (0 or 6g/kg diet). On day 7, chicks were distributed into 8 groups, and were given the experimental diets and Table 1. Composition of the experimental diets (g/kg diet) 1Contained 20.7g CaHPO4 E2H2 n, 14.8g CaCO3, 10g KH2PO4, 3g KCl, 6g NaCl, 3g MgSO4, 0.5g FeSO4 E7H2O, 0.35g MnSO4 E5H2O, 2.6mg KI, 40mg CuSO4 E5H2O, 62mg ZnO,1.7mg Na2MoO4 E2H2O, 0.4mg Na2SeO3 and 0.93mg CoCl2 E6H2O. 2CaHPO 4 E2H2O and KH2PO4 were substituted for the same amount of CaCO3 and KCl, respectively. The composition of others was the same as described above. 3Contained 15mg calcium pantothenate, 6mg riboflavin, 4mg pyridoxine hydrochloride, 40mg nicotinic acid, 1.5mg folic acid, 0.2mg biotin, 0.02mg cyanocobalamin, 3mg thiamin hydrochloride, 10IU DL-ƒ -tocopheryl acetate, 1,700IU retinyl acetate, 200ICU vitamin D3, 0.5mg vitamin K3, and 1.930g glucose.

3 148 J. Poult. Sci., 38 (2) distilled water (set ph at 7.0) ad libitum for 10 days. There was no significant difference on initial body weight among the treatments (the mean initial body weight of this experiment was 79.2g). On the final day, all chicks were euthanatized by decapitation. The intraluminal ph in the crop, proventriculus, gizzard and intestine was measured by using 4 kinds of ph test papers (UNIV, ph 1-11; BCG, ph ; MR, ph ; BTB, ph ; Toyo Roshi Co. Ltd., Japan). Blood samples were collected from the jugular vein. Plasma P concentration was measured by using commercially available kit (P-test Wako, Wako Pure Chemical Co. Ltd., Osaka). The thighbone was excised to measure the ash content. Statistical analysis. Three-way analysis of variance was applied to evaluate the effects of dietary amino acid sources, dietary phytase and dietary available P levels. Differences between means were assessed by Student's t-test. Results Dietary amino acid source and phytase level had no influence on body weight, food efficiency and thighbone weight when evaluated as a main effect (Table 2). The lack of dietary available P decreased body weight, food efficiency and thighbone weight. There were significant interactions between dietary available P and phytase levels on food intake and plasma P concentration. In chicks given dietary available P-free diet, phytase supplementation increased food intake, so that there was a tendency of improvement of growth performance. The phytase supplementation also increased Table 2. Effects of dietary amino acid source, avaalable P and phytase supplementation on body weight, food intake, food efficiency, thigh bone weight and plasma P concentration in chicks fed experimental diets for 10 days 1 Data were analyzed by use of three-way ANOVA. AP=main effect of available P level; PHY=main effect of phytase level; AP X PHY=interaction between AP and PHY; P<0.05. Values are means }SEM. Number of birds in each treatment is 6 or 7.

4 Murai et al.: Bone Ash Response by Phytase 149 plasma P concentration only in chicks fed dietary available P-free diet. The lack of dietary available P significantly decreased thighbone ash content (Fig. 1, left). The main effect of dietary available P level is statistically independent of those of dietary fatty acid source and phytase levels. On the other hand, there was a significant interaction between dietary amino acid source and phytase level on bone ash response. Therefore, the comparison has been done by the difference of dietary fatty acid source and phytase level. Dietary phytase significantly increased bone ash content in the glutamic acid group but not in the glutamine group (Fig.1; right). The addition of glutamic acid to the experimental diet significantly reduced ph in the crop compared with the glutamine when evaluated as a main effect (Table 3). There were no significant changes in ph in the proventriculus, gizzard and intestine by dietary glutamic acid supplementation. Dietary available P and phytase supplementation did not influence on ph in any places of gastrointestinal tracts. Fig.1. Effects of dietary amino acid source, available P and phytase supplementation on thighbone ash content in chicks fed experimental diets for 10 days. Data were analyzed by three-way ANOVA: ANOVA Probability AA (main effects of dietary amino acid source) NS (not significant) AP (main effects of dietary available P level) P<0.001 PHY (main effect of dietary phytase level) P<0.01 AA ~AP (interaction between AA and AP) NS AP ~PHY (interaction between AP and PHY) NS AA ~PHY (interaction between AA and PHY) P<0.01 AA ~AP ~PHY (interaction between AA, AP and PHY) NS Since there was a significant interaction between AA and PHY, the comparison of means between dietary amino acid source and phytase level was described in the right part of figure. a,b; Significantly different at P< 0.05 within the right part of figure. Values are means }SEM. Number of birds in each treatment is 6 or 7.

5 150 J. Poult. Sci., 38 (2) Table 3. Effects of dietary amino acid source, avialable P and phytase supplementation on ph in gastrointestinal tracts in chicks fed experimental diets for 10 days 1Data were analyzed by use of three -way ANOVA. AA=main effect of amino acid source; P<0.05 NS; not significant. a, b; Means with different superscripts are significantly different; P<0 Values are means }SEM. Number of birds in each treatment is 6 or Discussion The intraluminal ph of gastrointestinal tract is varied depending on the part of gut segments and animal species. Some birds including chicken posses the crop in the middle of esophagus for the storage of food. In chickens, mucous glands only exist near the entrance of the crop, so that the intraluminal ph of the crop is relatively basic compared with the ph in proventriculus and gizzard (Klashing, 1998). In the present study, the glutamic acid supplementation to the diets changed intraluminal ph of the crop to more acidic condition (ph5.4) compared with the glutamine supplementation (ph6.0). In addition, the phytase used here has optimum ph around 5.5. These data showed that the intraluminal condition of the crop approached to the optimum ph for microbial phytase by glutamic acid supplementation. Certainly, the combination of dietary glutamic acid and phytase synergistically increased bone ash content in the present study. It is well known that phytase supplementation improves the availability of dietary P in the body (Simons and Versteegh, 1990; Perney et al., 1993; Broz et al., 1994; Takemasa et al., 1996; Furuse et al., 1997). Takemasa et al. (1996) suggested that dietary phytase might work mainly in the crop of chicken. These reports support that the modification of ph in the crop might improve the potentiality of microbial phytase in chickens. However, the synergistic effect was not observed on other parameters including thigh bone weight and plasma P concentration. Thus, there is another possibility that synergistic bone

6 Murai et al.: Bone Ash Response by Phytase 151 ash response by glutamic acid and phytase might be caused by other unknown mechanism. The determination of combined effect of phytase and other acidic compounds might be expected to resolve this possibility. There were no changes in intraluminal ph of other parts of gut. The intraluminal ph of proventriculus and gizzard keeps acidic condition between ph 3-4 because of H+ secretion from the proventriculus. Therefore, the acidification by glutamic acid might not be effective in these parts and other down stream parts including intestine. also implied that the crop is a unique organ as its intraluminal dietary components. In conclusion, the present study shows that the combination This ph can be modified by of dietary phytase and glutamic acid improves the availability of dietary P in chicks. This indicates that the selection of proper phytase having an optimum ph around the intraluminal ph condition might be important to maximize its activity. Acknowledgement We thank the Japan Poultry Science Association for travel support to present the results of this study at the XXI World's Poultry Congress held in Montreal, 20-24th August, References Canada, on Broz J, Oldale P, Perrin-Voltz AH, Rychen G, Schulze J and Nunes CS. Effects of supplemental phytase on performance and phosphorus utilization in broiler chickens fed a low phosphorus diet without addition of inorganic phosphates. British Poultry Science, 35: Furuse M, Nakajima S-I, Nakagawa J, Okumura M and Okumura J. Effects of dietary guar gum on the performance of growing broilers given diets containing phytase. Japanese Poultry Science, 34: Klasing KC. Comparative avian nutrition. CAB International. Oxon Liu B-L, Rafiq A, Tzeng YM and Rob A. The induction and characterization of phytase and beyond. Enzyme Microbiology and Technology, 22: Perney KM, Cantor AH, Straw ML and Herkeleman KL. The effect of dietary phytase on growth performance and phosphorus utilization of broiler chicks. Poultry Science, 72: Simons PCM and Versteegh HAJ. Improvement of phosphorus availability by microbial phytase in broiler and pigs. British Journal of Nutrition, 64: Takemasa M, Murakami H and Yamazaki M. Reduction of phosphorus excretion of chicks by addition of yeast phytase. Japanese Poultry Science, 33: Van Der Klis JD and Versteegh HAJ. Phosphorus Nutrition of Poultry. In: Recent Advances in Animal Nutrition 1999 (Garnsworthy PC and Wiseman J eds.). pp Nottingham University Press. Nottingham Waldroup PW. Nutritional approaches to reducing phosphorus excretion by poultry. Poultry Science, 78: