INTRODUCTION
Although the economic and environmental benefits obtained through the use of phytase in broiler diets are reported in the literature (Nagata et al., 2011; Santos et al., 2011a,b; Gomide et al., 2012), its catalytic efficiency to make the phytate phosphorus (Pphy) of the feed available is not yet well established, and, according to Angel et al. (2002), this should be done while considering the Pphy concentration in both the feed and the excreta. Among the analytical methods used for the phytate determination, those that utilize colorimetric and refraction principles stand out (Wu et al., 2009). However, these methodologies have not yet been compared with each other for samples of feed and excreta. This comparison is important because the use of an inappropriate analytical methodology might lead to misinterpretations of the phytase efficiency.
The colorimetric method is based on a reaction between the ferric ion (Fe3+) and the sulfosalicylic acid (wade reagent [WR]) with the formation of a complex that can be spectrophotometrically monitored at 500 nm. In the presence of phytate, Fe3+ binds preferentially to Pphy, reducing the color intensity of WR. However, as the Fe3+ does not discriminate between the Pphy or the inorganic P (Pinorg) present in the sample, it is necessary that, before the colorimetric dosing, the sample go through an anion exchange resin to separate Pphy from Pinorg (Latta and Eskin, 1980). After the correlation with a standard curve of phytic acid (IP6), the result obtained is multiplied by 0.282 to express the content of Pphy in the sample because this constant corresponds to the molar ratio of P in the IP6 molecule (Angel et al., 2002). However, it is necessary to assess whether this methodology overestimates the Pphy in samples containing phosphate esters with fewer than six phosphate groups, which is expected in excreta from broilers fed diets with phytase because according to Greiner et al. (2002), the products of the enzyme degradation of IP6 can include different phosphorylation degrees of the myo-inositol (IP5 to IP1). In contrast, the high-performance liquid chromatography with a refractive index detector (HPLC-IR) discriminated the fractions from IP3 to IP6 in rice bran treated with phytase (Cúneo et al., 2000), arousing the interest in evaluating it in experiments with broilers.
Thus, the objectives of this study were to select the most adequate methodology for the determination of the Pphy content in both feed and excreta from broilers, to calculate the supplementation level of phytase in which there is the highest retention coefficient of Pphy and to estimate the P concentration value that can be reduced in the feed formulation when this level of phytase is used.
MATERIALS AND METHODS
Experimental materials and procedures
One metabolism experiment was conducted in Lavras, state of Minas Gerais, Brazil (21°13′52″ S; 44°58′16″W), with broiler chickens in the period from 15 to 28 days of age to compare two analytical methodologies employed to quantify the content of Pphy in both the feeds and excreta from birds and to determine the catalytic efficiency of the phytase supplemented in the diet by utilizing the results from the methodology that was shown to be the most appropriate.
All the experimental procedures adopted were approved by the Committee of Ethics in the Use of Animals (protocol code. 004/11). The experiment was conducted in a completely randomized design, distributed in a (4+1)×2 factorial arrangement, corresponding to 4 diets equally deficient in available P (Pav) supplemented with increasing levels of phytase (0, 750, 1,500, and 2,250 units of phytase activity – FTU – per kg of feed) plus 1 positive control diet without phytase, formulated according to Rostagno et al. (2005). The 6-phytase utilized in this study (EC 3.1.3.26 synthesized by Aspergillus oryzae) was a powder commercial product marketed by DSM Company, with determined activity of 9,980 FTU/g of product, according to the protocol of Engelen et al. (1994). All 5 diets were supplied to male and female broilers for a total of 10 treatments with 3 replications of 4 birds per pen. The use of male and female birds within a cage (mixed housing) could increase the coefficient of variation of the experiment; thus, the sex was considered a factor within the factorial design in order to minimize the variation between repetitions of the same treatment, promoting an experiment with a smaller mean square error.
One hundred and twenty broiler chicks (60 birds of each sex) of the Cobb-500 lineage were acquired at one day of age, sexed, and raised in a conventional shed for broilers up to 14 days of age and received a diet based on corn and soybean meal that was formulated to meet their nutritional requirements (Rostagno et al., 2005). On the 15th day of age, the broilers were weighed individually, separated by weight ranges, and transferred to the cages (experimental unit; with dimensions 50×50×50 cm) in a metabolism room. The experimental units were arranged to the average initial weight of the broilers, which was similar to one another (0.465 g±0.002). The room offered illumination and thermostatically controlled temperature. Each cage was provided with a pressure-type drinker, an individual gutter-type feeder with border, and a tray to collect the excreta. Experimental diets (Table 1) and water were offered ad libitum in the period from 15 to 28 days of age.
The experimental period comprised 13 days, ten days of which were destined for adaptation to the facilities and experimental diets, followed by three days (Rodrigues et al., 2005) of total collection of excreta, per experimental unit. Feed and leftovers were weighed and recorded on the 25th and 28th days of the age of broilers for subsequent calculations of feed intake during that period.
Sample collection and chemical analysis
The excreta were collected daily in the morning, grouped according to the experimental unit in identified plastic bags, and stored in a freezer up to the last day of collection when the excreta were weighed, homogenized, and pre dried in a forced-ventilation oven at 55°C for 72 h. The pre-dried excreta were ground, and the total dry matter content was obtained by considering the loss of moisture during the pre-drying stage and also at 105°C until constant weight (method 934.01 of the AOAC, 2005).
The Pphy content of the excreta was determined by colorimetry according to Frühbeck et al. (1995) with modifications and also by HPLC-IR according to Nappi et al. (2006), with adaptations. Both methodologies were utilized to analyze the extracts obtained in two extraction ratios of mass of sample:volume of extractor solvent (1:20 and 3:20, in g/mL). Parallel to this, three samplings of corn, soybean meal, and the positive and negative control diets were collected, ground, and analyzed in a manner that was similar to the one employed for the excreta. Thus, 1 or 3 g of sample was suspended in 20 mL of 0.66 M HCl followed by mechanical agitation at room temperature during two hours. Afterward, the extract was centrifuged at 14,881×g for 30 min at 15°C, and the supernatant was collected and filtered through Qualy 14 μm pore filter paper. At the 1:20 extraction, the filtered supernatant was diluted in function of the concentration of phytate present in the sample and of the standard curve of IP6 (colorimetric method). The dilutions (v:v in mL) utilized for the corn, soybean meal, and diets were, respectively, 3:25, 1:25, and 2:25. However, for the excreta, the dilution varied from 1:25 to 2:25 in function of the treatment because when a higher level of phytase is supplemented in the diet, there is a lower content of Pphy in the excreta requiring, consequently, a lower dilution level. After the dilution, the pH was adjusted with 1.0 M NaOH to 6.0. In the case of the 3:20 extraction, the filtered solution collected after centrifugation had its pH adjusted to 6.0 without going through the dilution step conducted at the 1:20 extraction.
The sample purification to separate the Pphy from Pinorg was conducted in glass columns (0.7×15 cm) packed with 0.5 g of anion exchange resin (AG 1-X4, 100–200 dry mesh, Bio-Rad) and washed with 15 mL of 0.6 M NaCl and 30 mL of ultrapure water, respectively. After this resin activation process, 10 mL of a sample with pH 6.0 were applied to the column, and the eluate was discarded. Subsequently, 15 mL of 0.6 M NaCl were utilized for the elution of the polyphosphates retained in the resin, and the pH of the eluate was collected and adjusted to 3.0 with 0.1 M HCl because this pH facilitates the link between phosphate and WR (Latta and Eskin, 1980).
In the sequence of the colorimetric method, an aliquot of the eluate that had been collected (3 mL for the 1:20 extraction or 100 to 600 μL for the 3:20 extraction; in these latter cases, ultrapure water was pipetted to maintain a final volume of 3 mL) was mixed with 1 mL of WR (consisting of 0.03% of ferric chloride hexahydrate and 0.3% of sulfosalicylic acid). The tubes were agitated in a vortex, and absorbance was read at 500 nm using ultrapure water to zero the spectrophotometer. The standard curve of IP6 was made by pipetting increasing volumes of the stock solution (200 mg/L) of IP6 (P0109, batch 057k0049, Sigma-Aldrich), so as to obtain concentrations varying from 0 to 150 μg; each tube received 1 mL WR, and the final volume was equivalent to 4 mL. The conversion of the concentration of phytate into Pphy was done considering the molar ratio of the P in the molecule of IP6 of 28.2% (Angel et al., 2002).
The remainder of the eluate that had been collected (15 mL eluted from the anion exchange resin minus the aliquot utilized for the colorimetric determination) was evaporated at 40°C till that, a solid residue was obtained, which was subsequently re-suspended in 2 mL of the mobile phase, centrifuged twice sequentially (15,000×g for 10 min at 4°C), filtered in a GV hydrophilic membrane (0.22 μm pore and 25 mm diameter, Durapore), and stored at 4°C until quantification of the myo-inositol phosphates by HPLC-IR.
The mobile phase consisted of 900 mL of 50 mM formic acid, 900 mL of methanol, 45 mL of 5 mM EDTA, and 54 mL of tetrabutylammonium hydroxide 20%. The pH of the mobile phase was adjusted to 4.3 with sulfuric acid and before being injected in the chromatograph, the mobile phase was vacuum filtered and sonicated (degassed) for 50 min. The analyses were performed in a Shimadzu chromatograph that was equipped with a refractive index detector (RID10Ai) and a C18 CAPCELL PAK column (5 μm×4.6 mm×150 mm) with a mobile phase at a flow of 0.5 mL/min. The temperature of the oven was maintained at 45°C, and the injection volume was 20 μL.
For the identification of phosphorylated fractions, the retention time of the standard compounds myo-inositol hexaphosphate or phytic acid (IP6, code P0109, batch 057k0049); myo-inositol-1,3,4,5,6-pentaphosphate (IP5, code I9261, batch 114K12831); myo-inositol-1,3,4,6-tetraphosphate (IP4, code I9386, batch 60K17321); and myo-inositol-1,4,5-triphosphate (IP3, code. I8761, batch 21H84341), obtained under the same conditions of analysis, were compared with the retention times of the peaks obtained in the samples. The IP5 was utilized as an internal standard for quantification of the phosphorylated fractions (IP3 to IP6) present in the samples using the following equation (Sandberg and Ahderinne, 1986):
In this equation, IPY corresponds to the different fractions of myo-inositol phosphates (IP3, IP4, IP5, or IP6). The conversion of the concentration of IP6 in P was calculated, considering the molar ratio of P in the IP6 molecule, which is 28.2%. For fractions IP5, IP4, and IP3, the molar ratios of 26.7, 24.8, and 22.1%, respectively, were utilized.
The protein content in the extracts (mg of protein per ml of extract) was dosed according to method of Bradford (1976) by utilizing bovine serum albumin as a standard. This analysis was conducted in samples extracts centrifuged and no centrifuged to investigate the hypothesis of that phytate can form complexes with proteins of manner that these phytate-protein complexes can interfere in the phytate quantification.
Balance and coefficient of hydrolysis (or retention) of phytate phosphorus
These parameters were calculated on a dry matter basis, utilizing the Pphy contents determined by the two methodologies evaluated. The Pphy intake (g/bird) was calculated by multiplying the feed intake (g/bird) by the Pphy content (%) in the feed. To calculate the absolute excretion of Pphy (g/bird), the amount of excreta (g/bird) was multiplied by the Pphy content (%) in the excreta. The hydrolysis coefficient of Pphy was calculated by equation:
Statistical analysis
Each cage was considered one experimental unit, and an independent statistical analysis was conducted for each analytical methodology evaluated. The data of the intake, excretion, and coefficient of hydrolysis of Pphy were subjected to analysis of variance (p<.05) by utilizing command General Linear Model of software SAS (2004), and when significant, polynomial regression models were utilized (p<0.05) to evaluate the effect of increasing supplementation of phytase on these parameters. The significance of the effect of the sex of birds was evaluated by the F test itself (p<0.05). In addition, the positive control diet was compared with the other experimental diets by the Dunnett test (p<0.05).
RESULTS AND DISCUSSION
Concentrations of phytate and phytate phosphorus determined in ingredients, diets and excreta
The initial proposal of this research was to compare the two analytical methodologies (colorimetry and HPLC-IR) only at the extraction ratio of 1:20 (mass of sample in g:volume of extractor solvent in mL), as Brito (2008) had already managed to quantify phytate in diets and excreta from broilers by colorimetry at this extraction ratio. However, with the 1:20 extraction, it was not possible to detect all the peaks with regard to the phosphorylated fractions of myo-inositol by the HPLC-IR method (especially for the samples of excreta from broilers fed the diets containing the highest levels of phytase) and, besides, the peaks identified in the chromatogram presented an intensity that was inferior to the limit of quantification of the analyte in the analysis conditions. Therefore, the extract was concentrated so as to enable the analytes of interest to be quantified via HPLC. During this procedure of concentration of the extract, it was observed that the highest possible ratio was of 3:20 because at higher ratios, the sample would become pasty, which is undesirable, as this could compromise the process of solubilization of the analytes.
For the colorimetric method, the extraction at the ratio of 3:20 resulted in the determination of lower concentrations of phytate and, consequently, lower contents of Pphy (Tables 2 and 3), probably due to the interference from the proteins present in this extract. According to Frühbeck et al. (1995), the pH of the diluted aliquot should be adjusted to 6.0 because this value is above of the isoelectric point of most of the vegetable proteins; which means that in theory, these proteins would present negative net charge and there would be electrostatic repulsion with the phytate, thus preventing the formation of complexes between inositol phosphate (IP) and protein. However, it appears that the formation of these phytate-protein complexes occurred, even adjusting the pH to 6.0.
To investigate the formation of these phytate-protein complexes, samples extracted at the ratio of 3:20 were centrifuged, and a great amount of precipitate (pellet) was visually verified, with only 52.07±2.32 μg protein per ml of extract determined in the supernatant (the non-centrifuged extracts resulting from extraction ratios of 1:20 and 3:20 had presented 109.41±1.30 and 800.73±1.71 μg protein/mL, respectively). Moreover, no trace of phytate was detected in the centrifuged supernatant, reinforcing the hypothesis that phytate was complexed with the proteins from the sample, and precipitated after centrifugation due to the high molecular weight of these phytate-protein complexes.
Furthermore, with the 1:20 extraction, the sample would naturally go through the anion exchange resin by the action of gravity; however, with the 3:20 extraction, the sample appeared blurred, and for their passage through the resin, a pressure pump had to be used. Therefore, at the extraction ratio of 3:20, the complexation with proteins results in an underestimation of the phytate content because an portion of the IP-protein complexes probably does not go through resin, and those which might be eluted with the saline solution, cannot be properly quantified due to their inadequate reaction with the WR. In addition to the proteins, phytate can also complex with minerals (Kornegay, 2001), which might interfere in the dosage of phytate; however, as the protein content in the diet is much higher than the mineral content, it is likely that the effect of protein is more pronounced.
When the extract at the rate of 1:20 was dosed by the colorimetric method (Table 2), the contents of Pphy determined for corn and soybean meal in this study were approximately 30% and 44% higher than the mean values presented in the last edition of the Brazilian Tables for Poultry and Swine: 0.217 and 0.437 g Pphy per 100 g DM of corn and soybean meal, respectively (Rostagno et al., 2011); however, the methodology utilized for the determination of these contents was not published. In addition to the methodology, the differences observed can also be due to environmental, genetic, and/or cultivation variations, which can alter the content of Pphy in the seeds and grains. For example, Wadt et al. (2010) collected 94 samples of corn originated from the South, Southeast, and Midwest regions of the Brazil in the summer 2009 harvest, and they verified differences not only within the same region but also between cultivation regions, with Pphy contents determined raging from 1.0 to 2.6 g/kg of corn, which represents a variation of up to 160% in the Pphy contents determined in the samples. Therefore, in this study, the colorimetric methodology at the extraction ratio of 1:20 was shown to be the most adequate method for quantifying the Pphy.
The retention times (in minutes) for standards IP3, IP4, IP5, and IP6 were 6.793, 10.386, 15.113, and 18.791, respectively. The Pphy contents determined in corn, soybean meal, and diets by the HPLC-IR methodology at the extraction ratio of 3:20 were inferior to those determined by the colorimetric method at the extraction ratio of 1:20 (Table 2), which also should be due to the complexation of phytate with other molecules present in the sample, especially proteins. However, this was not observed for the excreta, which can be justified by the profile of esters of myo-inositol phosphate present in the sample (Table 3).
In general, the higher the degree of phosphorylation of myo-inositol, the higher its power of complexation because the greater the number of phosphate groups in the molecule, the greater the number of negative charges resulting from the deprotonation of these phosphate groups (Kornegay, 2001). In this sense, Yu et al. (2012) recently evaluated the effect of the degree of phosphorylation of myo-inositol on the potential of the molecule to complex with the soy protein and bovine β-casein and concluded that, for both proteins, IP6 has a high chelating power which is strongly reduced by the loss of one phosphate group (IP5), becoming practically null in the other phosphorylated fractions (IP4 to IP1). Therefore, due to the fractions with fewer than five phosphate groups in the excreta (Table 3), the interference of the proteins in these samples was lower than in the ingredients and diets.
In the corn, only fraction IP6 was detected; whereas in soybean meal and the diets, the myo-inositol phosphates were distributed between IP5 and IP6, but with predominance of IP6 (Table 2). Consequently, probably there were high rates of complexing of these fractions with other molecules or ions, and once complexed, the physico-chemical properties of these complexes are different from isolated fractions, thus causing alterations in the retention times, impeding these complexes from being identified in the chromatogram, leading to underestimation of the results. This interference by the formation of complexes was lower in the excreta due to the predominance of fractions with low complexing capacity such as IP4 and IP3, which are products from the enzymatic action of phytase on the phytate.
Balance and coefficient of hydrolysis (or retention) of phytate phosphorus
Although it is possible to make comparisons between the methodologies to quantify Pphy based on the contents determined, these data are inappropriate to evaluate the phytase efficiency to make Pphy available for broilers. Therefore, it is necessary to take into account not only the Pphy content determined in the diets and excreta, but also the feed intake and the amount of excreta produced in the collection period. As previously discussed, the methodologies of colorimetry and HPLC-IR at the extraction ratio of 3:20 presented problems in the determination of the Pphy content, which is reflected in inadequate balance and retention of Pphy (Table 4).
In this regard, the colorimetric methodology with the extraction ratio of 1:20 was shown to be the most adequate, and, therefore, presented the most reliable results on the balance and retention of Pphy by the broilers (Table 4). There was no interaction (p>0.05) between the levels of phytase and sex for intake, excretion, or the coefficient of hydrolysis of Pphy. In addition, the birds sex affected (p<0.01) only the intake of Pphy, so that the males consumed 12% more Pphy than did the females. Therefore, it is possible to consider that the mean values obtained to calculate the regression equations for the excretion and the retention coefficient were originated from six replicates (three for each sex). The increase in the phytase level in the diet did not affect (p>0.05) intake, but linearly reduced (p<0.01) the Pphy excretion up to 57.75%, which represents good results, as it demonstrates the possibility of producing broilers with less P deposition in the environment using phytase in the diet.
In the diets without supplemented phytase, there was good use of the Pphy present in the diet, which was similar to the observations by Han et al. (2009), who attributed this result to the action of the endogenous phytase that can be synthesized by cells of the intestinal epithelium of broilers (Applegate et al., 2003). Besides, the decrease of Pav from 4.26 to 2.13 g/kg of the diet increased the coefficient of hydrolysis of the Pphy from 43.37% to 69.67%, which was similar to the results reported by Onyango et al. (2006), who evaluated the effect of two levels of P in the diet (without phytase supplementation) on the activity of phytase present in the intestinal mucosa (duodenum) of broilers, in the period from 8 to 20 days of age, and observed that the activity of this enzyme rose from 29.46 to 43.25 nmols of inorgP/mg of protein per min when the concentration of total P in the diet was reduced from 7.0 to 3.6 g/kg of diet. These results suggest that there is an influence of dietary P levels on the activity and efficacy of intestinal phytase, although the mechanisms are not yet fully understood (Bedford and Partridge, 2010). One possible biochemical explication for these results is based in the mechanism of enzymatic inhibition in the presence of the product from catalysis of manner that the increase in the Pinorg concentration in the diet causes a down-regulation in the activity of the intestinal phytase (Onyango et al., 2006).
The increase in the inclusion of phytase in the diet increased (p<0.01) the retention of Pphy, corroborating with the research of Han et al. (2009), who also verified increasing improvement in the Pphy retention in function of increase in the phytase level in the diet. The greatest retention coefficient was estimated at 87.85% when the diet contained 1,950 FTU/kg of diet (Coefficient of retention of the Pphy = −0.00000492x2+0.01919x+69.14; R2 = 0.97). Thus, as the mean content of Pphy determined in the diet was 3.25 g Pphy/kg of the diet in natural matter (considering the mean determined value of 3.78 g Pphy/kg of diet in DM and the content of moisture of 13.88%±0.45), it is possible to reduce the P content in the diet in 87.85% (or 2.86 g of P per kg) by adding 1,950 FTU/kg of the diet.
IMPLICATIONS
The extraction ratio and methodology of dosage utilized to determine the Pphy content in both the diets and excreta from broilers interfere directly with the results of Pphy retention. The combination of the extraction ratio of 1:20 (mass of sample in g:volume of extractor solvent in mL) and colorimetric methodology is the one that yielded the best results. The broiler sex did not affect the Pphy utilization, in the period from 15 to 28 days of age. Supplementation of 1,950 FTU/kg corresponds to a reduction of 2.86 g in P/kg of the diet.
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