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METABOLISM AND NUTRITION |
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* Danisco Animal Nutrition, Marlborough, Wiltshire, SN8 1XN, UK; Institute of Food, Nutrition and Human Health, Massey University, Palmerston North 4442, New Zealand; and Faculty of Veterinary Science, The University of Sydney, Camden, New South Wales 2570, Australia
1 Corresponding author: v.ravindran{at}massey.ac.nz
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: broiler • endogenous amino acid flow • phytic acid • microbial phytase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In feed ingredients, PA exists as a salt of potassium and magnesium, and is relatively unreactive (Ravindran et al., 1995; Lott et al., 2000). However, when the feed is exposed to the low pH conditions in the gastric phase, PA becomes soluble as H+ ions replacing potassium and magnesium (Cosgrove, 1966; Rajendran and Prakash, 1993). Though protonated, PA still carries a net negative charge and can react electrostatically with basic amino acid residues in dietary proteins. The extent of this reaction depends on the concentration and solubility of phytate, ambient pH, the isoelectric point of the protein and its tertiary and quaternary structure (i.e., the degree of steric hindrance between reactive amino acids and phytate). These phytate-protein complexes are variably refractory to digestion by pepsin and solubilization with HCl (Knuckles et al., 1985, 1989; Vaintrub and Bulmaga, 1991), which may lead to an increase in their secretion by the animal (Cowieson and Ravindran, 2007a). On gastric emptying, the distal gut is faced with a luminal challenge to maintain favorable conditions for optimal functioning of the pancreatic and brush border enzyme array, and for a satisfactory ion balance for nutrient transport. Hyper-secretion of mucin and sodium bicarbonate, commensurate with variation in proteolytic and H+ antagonism follows (Munster et al., 1987; Allen and Flemstorm, 2005), increasing the presence of endogenous amino acids and sodium in the lumen and presumably altering maintenance requirements. In addition, the loss of endogenous protein from the ileum has a direct effect on the available energy value of the diet, depending on the amino acid composition of the protein leaving the terminal ileum. This direct energetic cost has been estimated to be as much as 20 kcal/kg of DM intake for every 1 g/kg of dietary phytate-P (Cowieson and Ravindran, 2007b), without including the effects on net energy associated with protein synthesis and turnover. Thus, the ingestion of dietary PA influences endogenous losses indirectly via a reduction in the solubility of dietary protein with a subsequent cascade altering intestinal dynamics via secretive and absorptive mechanisms. Furthermore, the direct interaction of PA with endogenous enzymes, mucin, and the gastrointestinal tract epithelium cannot be ruled out (Cowieson et al., 2004).
A comprehension of mode of action for the extraphosphoric effects of phytase is not only scientifically enlightening but will also assist in explaining the marked variation in published ileal amino acid digestibility responses to supplemental phytase. For example, the amino acid composition of mucin and pepsin is highly correlated with the phytase-induced improvements in ileal amino acid digestibility (Cowieson and Ravindran, 2007a). This is instructive because it explains why the improvements in the digestibility of threonine, serine, and cystine with supplemental phytase is typically superior to that of methionine (endogenous proteins being essentially devoid of methionine). A greater understanding of the physiological consequences of changes in the secretion and resorption of endogenous amino acids and minerals is justified because maintenance requirements, immune function, and microbial host dynamics are expected to be involved.
Several distinct phytase products are currently commercially available and the 3 commonly used phytases are derived either from fungal (Aspergillus niger and Peniophora lycii) or bacterial (Escherichia coli) sources (Selle and Ravindran, 2007). These phytases fall into 2 categories, namely 3-phytases and 6-phytases, depending on the site where the hydrolysis of the PA molecule is initiated. As a result, each phytase has different recommendations regarding digestibility improvements for P, calcium, amino acids, and energy. One possible explanation for the different responses in amino acid digestibility may lie in the different effects of these phytase sources on endogenous amino acid flows. The purpose of the experiment that is reported herein was to assess the effect of PA and 2 different phytase sources (bacterial vs. fungal) on the flow of endogenous amino acids at the terminal ileum of broiler chickens, using the enzyme-hydrolyzed casein (EHC) method. The loss of endogenous protein has considerable energetic consequences for the host. For this reason, the effects of dietary treatments on the energy flow associated with endogenous amino acids were also determined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The PA (myo-inositol hexaphosphate) was added in a purified form (PA, sodium salt; EC No. 238–242–6; Sigma-Aldrich Corporation, St. Louis, MO). The bacterial phytase product (Phyzyme XP) was derived from E. coli and expressed in Schizosaccaromyces pombe (Danisco Animal Nutrition, Marlborough, UK). The fungal phytase product (Natuphos) was derived from A. niger (BASF AG, Ludwigshafen, Germany). The former is a 6-phytase (EC 3.1.3.26 [EC] ), whereas the latter is a 3-phytase (EC 3.1.3.8 [EC] ), and these enzymes commence the dephosphorylation of PA at positions 6 and 3 of myo-inositol, respectively.
Both products contained a minimum phytase activity of 5,000 phytase units (FTU)/g and the addition of 100 g of enzyme/t provided a guaranteed minimum of 500 FTU/kg of diet. One FTU is defined as the quantity of enzyme that releases 1 µmol of inorganic P/min from 0.15 mM sodium phytate at pH 5.5 at 37°C.
Diets
The study was conducted as a 2 x 3 factorial arrangement of treatments to evaluate the effects of 2 concentrations of PA (8.5 or 14.5 g/kg), without or with bacterial or fungal phytase supplementation (0 or 500 FTU/kg of diet), on the endogenous amino acid flow in chickens. In addition, a seventh treatment (without PA and phytase) was included to obtain data on basal endogenous amino acid flow.
A basal diet with 100 g/kg of EHC (Table 1
) as the sole source of N was formulated. Based on this diet, 6 experimental diets were developed. Titanium oxide (3 g/kg) was included in all diets as an inert digesta marker for the calculation of amino acid flows. A casein-based diet was also developed (Table 1), which was similar to the EHC diet except that casein was used in place of EHC and no titanium oxide was included. The phytase products, in powder form on an N-free starch-based carrier, was added and mixed in a vertical mixer. Phytase activity of both phytase products was confirmed at 5,000 ± 50 FTU/g, using the assay procedures of Engelen et al. (2001).
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A total of 300 male broiler (Ross 308) chicks was obtained as day-olds from a commercial hatchery and raised in floor pens. On d 14 posthatching, the birds were individually weighed, and 210 birds of similar BW range were selected and assigned to 42 cages of 5 birds each. The 7 dietary treatments were then randomly assigned to 6 cages each. The cages were housed in an environmentally controlled room with 24-h fluorescent lighting. The experimental procedures were approved by the Animal Ethics Committee of Massey University.
Conduct of the Trial
The birds were fed from d 0 to 14 posthatch a standard corn-soy diet (225 g/kg of CP, 3,020 kcal/kg of AME, 10.2 g/kg of calcium, and 7.8 g/kg of total P) in pellet form. From d 15 to 21, the birds were offered a mash diet, which was made by grinding the standard starter pellets in a hammer-mill to pass through a 3-mm screen. The casein-basal diet (Table 1
) was introduced on d 22. The aim of using this basal diet was to enable the birds to adjust to the change-over to synthetic EHC diets. The casein-basal diet was withdrawn on d 24, and the EHC-based experimental diets were introduced and offered for 4 d. The diets were offered ad libitum, and water was available at all times.
Digesta Collection and Processing
On d 28, the birds were killed and digesta from the lower half of the ileum was collected. Samples from birds within a pen were pooled, homogenized, frozen immediately after collection, and lyophilized (Ravindran et al., 2005). The following procedure was used to separate the endogenous protein fraction (Ravindran et al., 2004). The lyophilized samples were resuspended in deionized water and acidified to pH 3.5 with 9 M H2SO4. The samples were stored overnight at 4°C and then centrifuged at 1,450 x g for 45 min at 0°C. The supernatant was decanted off and retained. The precipitate was washed with 10 mL of deionized water and centrifuged at 1,450 x g for 30 min at 0°C. The second supernatant was added to the first, and the precipitate was stored at –20°C. The combined supernatants were ultrafiltered using a Centriprep-10 ultrafiltering device (molecular weight cut-off filter, 10,000 Da; Ami-con Inc., Beverly, MA) according to the manufacturer’s instructions. The precipitate from the centrifugation step was added to the retentate (>10,000 Da) from the ultrafiltration step, and the material was lyophilized. Diets and digesta were then ground to pass through a 0.5-mm sieve and stored in airtight containers at –4°C for chemical analyses.
Chemical Analyses
The diets and ultra-filtered ileal digesta samples were analyzed for DM, nitrogen, amino acids, and titanium as described below. Dry matter determination was carried out according to standard procedures (AOAC International, 2005, method 930.15). Total N was determined by the combustion method (AOAC International, 2005, method 968.06) using a CNS-2000 carbon, N, and sulfur analyzer (Leco Corporation, St. Joseph, MI). Amino acids were determined by hydrolyzing the samples with HCl (containing phenol) for 24 h at 110 ± 2°C in glass tubes sealed under vacuum, as described by Ravindran et al. (2008a). Amino acids were detected on a Waters ion exchange HPLC system, and the chromatograms were integrated using dedicated software (Maxima 820, Waters, Millipore, Milford, MA) with the amino acids identified and quantified using a standard amino acid solution (Pierce, Rockford, IL). Cystine and methionine were analyzed as cysteic acid and methionine sulphone, respectively, by oxidation with performic acid for 16 h at 0°C and neutralization with hydrobromic acid before hydrolysis. Tryptophan was not determined. Titanium content was measured on an ultraviolet spectrophotometer following the method of Short et al. (1996).
Calculations
The flow of N and individual amino acids at the terminal ileum was calculated, as milligrams lost per ingestion of kilogram of feed DM, using the following formula (Moughan et al., 1992). These values were considered to be the estimates for endogenous flow.
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The amino acid profile of endogenous protein (N x 6.25) was calculated by expressing each amino acid as a percentage of endogenous crude protein.
The gross energy of endogenous proteins lost to the birds was calculated based on their amino acid composition, gross flow, and published gross energy values of amino acids (Boisen and Verstegen, 2000; Table 2).
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Two-way ANOVA was employed to determine the main effects (PA and phytase) and their interaction by using the GLM procedure of SAS Institute (1997) using pen as the experimental unit. The effects of PA concentrations (0, 8.5, and 14.5 g/kg) were compared using a completely randomized design ANOVA. To establish the relative effects of the 2 phytases, nonorthogonal contrasts were carried out. Differences were considered significant at P 
0.05. Multiple regression analyses were carried out using the modeling function of JMP (SAS Institute, 1997). Models were constructed by using the amino acid profiles of mucin and pepsin as constructs to explain the effects of phytate and phytase on the gross flow of the endogenous amino acids.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The comparison of ileal endogenous flow of N and amino acids in birds fed EHC diets containing 0, 8.5, and 14.5 g/kg of PA, with no phytase supplementation, is shown in Tables 4 and 5. Increasing concentrations of PA significantly affected ileal flows of N and amino acids. The flows of N and most amino acids were increased (P < 0.05 to 0.001) when dietary PA concentration was increased from 0 to 8.5 g/kg. The exceptions were the flow of alanine, histidine, and arginine, which were unaffected (P > 0.05). The flow of N and all amino acids in birds fed diets with 14.5 g/kg of PA were higher (P < 0.01 to 0.001) than in those fed diets with 0 and 8.5 g/kg of PA.
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and 5). However, differences between the phytase sources were significant (P < 0.05) only for the flow of glutamic acid, proline, and isoleucine. For these amino acids, the reduction in flow was greater with the bacterial phytase compared with the fungal phytase. Differences between phytase sources were also observed for ileal flow of N (P = 0.06) and total amino acids (P = 0.06). In both cases, the flows were reduced by a greater extent by the bacterial phytase compared with the fungal. Phytic acid x phytase interaction was also observed for glutamic acid (P < 0.05), glycine (P < 0.01), and histidine (P = 0.06). For glutamic acid, the interaction was due to a larger reduction in flow with the bacterial, rather than fungal phytase, at the 14.5 g/kg of dietary PA concentration. For glycine and histidine, the interaction resulted from reductions in ileal flows with phytase supplementation being greater at 14.5 g/kg compared with 8.5 g/kg of PA diet.
The influence of dietary treatments on the amino acid profile of endogenous protein, expressed as g/100 g of protein, is presented in Table 6. The proportion of amino acids, except aspartic acid, threonine, glycine, arginine, and methionine, differed in the endogenous protein of birds fed 8.5 and 14.5 g/kg of PA. The proportion of proline, valine, isoleucine, lysine, and cystine in the endogenous protein were lower (P < 0.05 to 0.001) in birds fed diets with 14.5 g/kg of PA compared with those fed diets with 8.5 g/kg of PA. In contrast, the proportion of serine, leucine, tyrosine, phenylalanine, histidine (P < 0.05 to 0.001), and glutamic acid (P = 0.07) was increased in the endogenous protein of birds fed diets with 14.5 g/kg of PA compared with those fed diets with 8.5 g/kg of PA.
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Differences in the composition of endogenous protein in birds fed diets with 0, 8.5, and 14.5 g/kg of PA were observed for for glycine, alanine, valine, tyrosine, phenylalanine, cystine (P < 0.05), glutamic acid (P = 0.06), isoleucine (P = 0.07), and histidine (P = 0.06). In general, the proportions of these amino acids increased with increasing PA concentrations. The exceptions were those of valine, arginine, cystine, and methionine, which decreased with increasing PA concentrations.
The calculated energetic losses associated with the endogenous protein flows are presented in Table 7. In the absence of supplemental phytase, the ingestion of 14.5 g/kg of PA compared with 8.5 g/kg increased (P < 0.001) the loss of energy as endogenous protein by 23 kcal/kg of DM intake. Supplementation of the diets with phytase reduced (P < 0.01) energy loss by all amino acids, restoring energy loss from endogenous protein in the diet containing 8.5 g/kg of PA to the level of the phytate-free basal diet in the case of the bacterial phytase. The 2 phytase sources differed (P < 0.05) only for energy loss from endogenous glutamic acid, proline, isoleucine, and leucine, and the sum of total amino acids. Phytic acid x phytase interactions (P < 0.05) were established for glutamic acid, glycine, and histidine, reflecting the endogenous flow data presented above.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The beneficial effect of phytase in lowering PA-induced endogenous amino acid flow in the current study was in agreement with previous data (Cowieson et al., 2004; Cowieson and Ravindran, 2007a) and of a similar magnitude (20 to 30% reduction relative to the phytase-free diets). The magnitude of reduction in total endogenous amino acid flow was greater (P = 0.06) for the bacterial phytase compared with the fungal. This finding may suggest that differences in substrate affinity/specificity, proteolytic stability or pH profile between phytases from different sources can influence the capacity to ameliorate the nutritional antagonism associated with PA. The substrate specificity of phytase from E. coli was reported by Greiner et al. (1993) who concluded that E. coli-derived phytases have an exceptionally high specificity for fully phosphorylated myo-inositol ester (IP6) with little or no activity on other phosphate esters. Several other studies (Wyss et al., 1999; Greiner and Farouk, 2007) confirmed these data, demonstrating that the biochemical characteristics of different phytases vary widely, with phytases from E. coli having higher relative activity at gastric pH, greater resistance to intestinal proteases, and 8-fold greater specificity for IP6 than phytase from Aspergillus niger. Given that IP6 is the main protagonist associated with the uptake and transport of minerals (Han et al., 1993) and interaction with dietary protein (Knuckles et al., 1989), it is possible that the superior capacity of the bacterial phytase to reduce endogenous flow of N and amino acids in the current study was due to its high specificity for IP6, whereas other phytases may also preferentially bind to lower molecular weight myo-inositol esters or other phosphate-based compounds. Hu et al. (1996) demonstrated that intestinal phosphatases are capable of degrading the lower molecular weight myo-inositol esters and that IP6 alone escapes significant dephosphorylation from the mucosal phosphatases. It is also of relevance that bacterial phytases have been shown to have a greater resistance to degradation by intestinal proteases than their fungal counterparts. Onyango et al. (2005) presented data showing that an evolved phytase from E. coli had significantly greater residual activity in all segments of the gastrointestinal tract compared with a phytase from P. lycii, concluding that the bacterial phytase may have superior resistance to proteolytic degradation. Similar observations were made by Igbasan et al. (2000) who found that phytases from E. coli have high proteolytic stability. Thus, the tendency for greater capacity of the bacterial phytase compared with that of the fungal phytase to reduce losses in endogenous N and total amino acids in the current study may be associated with the specificity for IP6, a more favorable pH profile, and less susceptibility to degradation by gastric and pancreatic proteases.
The loss of endogenous protein from the terminal ileum of broilers has considerable energetic consequences for the host. Without considering the substantial amount of energy required in the synthesis of the endogenous proteins in the first place, it is possible to calculate the gross energy of the lost proteins based on their amino acid composition and gross flow (Table 7). The data presented in Table 2 show that the gross energy of amino acids ranges from 2,890 kcal/kg for aspartic acid to 6,740 kcal/kg for phenylalanine. This is of relevance when calculating the direct (digestible) caloric consequence of endogenous protein flow at the terminal ileum because endogenous proteins that are rich in phenylalanine, isoleucine, and leucine, compared with those containing higher concentrations of aspartic acid, glycine, and serine, will have a much greater consequence for digestible energy, metabolizable energy, or both. In the current study, the ingestion of 14.5 g/kg of PA compared with 8.5 g/kg increased the endogenous amino acid flow by 66% and gross energy flow associated with those endogenous amino acids by 68%. This discrepancy may be explained by PA-induced changes in the composition of endogenous protein favoring amino acids with a higher caloric density. Increasing the concentration of PA from 8.5 to 14.5 g/kg increased the relative proportion of serine, leucine, tyrosine, phenylalanine, and histidine and decreased the relative proportion of proline, valine, isoleucine, lysine, and cystine in the endogenous protein. Multiple regression analysis shows that these changes are correlated (P < 0.01) with the amino acid profile of pepsin (Table 8) and are further evidence that the ingestion of PA increases the secretion and loss of specific endogenous proteins rather than to stimulate an increase in total endogenous protein production. It is unlikely that the implied increase in the secretion of pepsin is associated with a direct interaction between PA and pepsin because pepsin is essentially devoid of basic amino acid residues (Tang et al., 1973) and so will not readily interact with PA. However, it is possible that PA interacts with dietary protein in the gastric phase of digestion, reducing protein solubility and stimulating an increase in pepsin and HCl as a compensatory mechanism as discussed above. Both phytase sources altered the amino acid composition of the endogenous protein. In the case of threonine, glycine, and cystine, both phytases reduced their relative contribution to the total endogenous protein flow. In the case of glutamic acid and proline, however, the bacterial phytase reduced the relative proportions to a greater extent compared with the fungal phytase, and for methionine the opposite was the case. The exact reasons for these significant differential effects are unclear, but may be related to differences in the functionality of these 2 phytases in the timing or method of dephosphorylation of IP6 and the impact this has on secretory physiology. Regardless of the underlying mechanisms, the present data demonstrate that microbial phytases are effective in reducing endogenous amino acid flow at the terminal ileum of growing chickens through a reduction in the secretion, or alteration in the absorption, of specific endogenous proteins.
The EHC method, described by Moughan et al. (1990), was employed in the current study and this method has been previously used to measure ileal AA losses in chickens (Ravindran and Hendriks, 2004; Ravindran et al., 2004; Cowieson and Ravindran, 2007a). Several assumptions are made when this method is used (Nyachoti et al., 1997b; Ravindran and Bryden, 1999). In this method, the animal is fed a synthetic diet where the sole source of protein is EHC, which consists of small peptides (molecular weight, <5,000 Da) and free amino acids. Digesta from the terminal ileum are collected, centrifuged, and ultrafiltered (molecular weight exclusion limit of 10,000 Da). The precipitate from the centrifugation step and the retentate from the ultrafiltration step (>10,000 Da fraction of digesta) are considered to provide the estimate of the endogenous component of the digesta. However, a potential limitation is that there may be some underestimation of endogenous AA flow due to a loss of low molecular weight endogenous peptides and AA in the ultrafiltrate (Moughan et al., 1998). In the current study, however, the same diet (100 g of EHC/kg of diet) served as the basal formula for all treatments. Thus the effect of any underestimation in endogenous AA flow is inherent to this diet and would not have influenced the comparisons between the treatments employed.
In feed ingredients, PA exists as a complex with various other nutrients, including protein, starch and various minerals (Ravindran et al., 1995). Thus the use of sodium phytate, a purified form, is subject to criticism because it is not representative of practical feeding situations. The alternate option available is to use phytate-rich ingredients, such as rice bran, to manipulate dietary concentrations of PA. However, these phytate-rich ingredients also contain high levels of fiber (Ravindran et al., 2006), which are known to negatively influence endogenous protein losses (Nyachoti et al., 1997b). Conversely, the use of purified PA forms eliminates the confounding effects of diet composition at different concentrations of PA.
It can be concluded that the ingestion of PA increases the flow of endogenous amino acids at the terminal ileum of broiler chickens. This effect may be mediated by an increase in the secretion, or a reduction in digestion, absorption, or both, of endogenous proteins, specifically pepsin and mucin. Exogenous phytases are effective in countering the adverse effects of PA on endogenous nutrient losses, though the magnitude of these effects appears to be different for bacterial and fungal phytases. Further work on the effects of PA and phytase on the secretion and absorption of specific components of endogenous protein will be of interest.
Received for publication February 29, 2008. Accepted for publication June 13, 2008.
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