J. Anim Sci.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Adeola, O.
Right arrow Articles by Schulze, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Adeola, O.
Right arrow Articles by Schulze, H.
J. Anim. Sci. 2004. 82:2657-2666
© 2004 American Society of Animal Science

ANIMAL NUTRITION

The efficacy of an Escherichia coli-derived phytase preparation1

O. Adeola*,2, J. S. Sands*, P. H. Simmins{dagger} and H. Schulze{dagger}

* Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-2054, and and {dagger} Danisco Animal Nutrition, Marlborough SN8 1XN, U.K.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Literature Cited
 
Five experiments were conducted to evaluate the effect of an Escherichia coli-derived phytase on phytate-P use and growth performance by young pigs. The first experiment involved time course, pH dependence, and phytase activity studies to investigate the in vitro release of P from corn, soybean meal, and an inorganic P-unsupplemented corn-soybean meal negative control diet. In Exp. 2, which was designed to determine the efficacy of the E. coli-derived vs. fungal phytase-added diets at 0, 250, 500, 750, 1,000, or 1,250 FTU/kg (as-fed basis; one phytase unit or FTU is defined as the quantity of enzyme required to liberate 1 µmol of inorganic P/min, at pH 5.5, from an excess of 15 µM sodium phytate at 37°C) and a positive control diet, eight individually penned 10-kg pigs per diet (12 diets, 96 pigs) were used in a 28-d growth study. The third experiment was a 10-d nutrient balance study involving six 13-kg pigs per diet (four diets, 24 pigs) in individual metabolism crates. In Exp. 4, eight pens (four pigs per pen) of 19-kg pigs per treatment were used in a 42-d growth performance study to examine the effect of adding the E. coli-derived phytase to corn-soybean diets at 0, 500, or 1,000 FTU/kg (as-fed basis) and a positive control (four diets, 128 pigs). In Exp. 5, six 19-kg pigs per treatment were used in a 10-d nutrient balance study to investigate the effects of the E. coli-derived phytase added to diets at 0, 250, 500, 750, or 1,000 FTU/kg (as-fed basis) and a positive control diet (six diets, 36 pigs). The in vitro study showed that the E. coli-derived phytase has an optimal activity and pH range of 2 to 4.5. Inorganic phosphate release was greatest for soybean meal, least for corn, and intermediate for the negative control diet. Dietary supplementation with graded amounts of E. coli-derived phytase resulted in linear increases (P < 0.05) in weight gain, feed efficiency, and plasma Ca and P concentrations in 10-kg pigs in Exp. 2. Phytase also increased P digestibility and retention in the 13-kg pigs in Exp. 3. In Exp. 4, dietary supplementation with E. coli-derived phytase resulted in linear increases (P < 0.05) in weight gain and feed efficiency of 19-kg pigs. Supplementation of the diets of 19-kg pigs with the E. coli-derived phytase also improved Ca and P digestibility and retention in Exp. 5. In the current study, the new E. coli-derived phytase was efficacious in hydrolyzing phytate-P, both in vitro and in vivo, in young pigs.

Key Words: Growth Performance • Nutrient Balance • Phosphorus • Phytase • Pigs


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Literature Cited
 
A significant portion of the P in mature cereal grains and oilseeds is present as phytate-bound P. Because monogastric animals have little intestinal phytase, use of P in cereal grains and oilseed meals is limited. Consequently, phytate not assimilated by animals is excreted in manure and contributes to P-related environmental pollution problems (Adeola, 1999Go). Furthermore, phytic acid has strong chelating potential in the gut and complexes Ca and Zn, thereby decreasing their bioavailability (Thomson and Yoon, 1984Go; Nolan et al., 1987Go). Adding microbial phytase to diets has been demonstrated to increase phytate hydrolysis and the availability of P and other minerals that may be chelated by phytic acid (Lei et al., 1993bGo; Adeola et al., 1995Go).

The 3- and 6-phytases derived from Aspergillus niger and Peniophora lycii initiate dephosphorylation from positions 3 and 6, respectively, on the myo-inositol ring (Wodzinski and Ullah, 1996Go). A 6-phytase cloned from Escherichia coli obtained from pig intestine was shown to be effective in releasing phytate-bound P in chicks and pigs (Leeson et al., 2000Go; Augspurger et al., 2003Go). The E. coli phytase (EP) exhibited a single pH optimum range (2.5 to 3.5), which is different from the two pH optima of 2.5 and 5.5 for the fungal 3-phytase Natuphos (Rodriguez et al., 1999aGo). Previous in vitro studies showed that EP is more resistant to inactivation in the digestive tract than are other commercially available phytase (Igbasan et al., 2000Go). Any new phytase preparation requires rigorous evaluation of its efficacy in hydrolyzing phytic acid. We hypothesized that supplementation of low-nonphytate-P diets with the new phytase improves phytate P digestibility and growth performance. In the current research, in vitro and in vivo studies were designed to investigate the efficacy of a phytase preparation, whose encoding genes originated from E. coli (Grenier et al., 1993) and were expressed in Bacillus subtilis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Literature Cited
 
In Vitro Studies (Exp. 1)
The first experiment involved three in vitro studies examining the release of P in corn, soybean meal, and complete diet (Table 1Go) from added EP. The three in vitro studies consisted of time course, pH dependence, and phytase activity. Commercial buffers 2-[N-morpholino]ethanesulfonic acid, HEPES, and Tris, and sodium phytate were purchased from Sigma Chemical Co. (St. Louis, MO). A 50-mM concentration of each buffer was prepared and adjusted to the required pH with HCl, NaOH, or Tris. Corn and soybean meal (same batch used in mixing the negative control diet) or negative control diet (Table 1Go) were ground to pass a 1-mm screen.


View this table:
[in this window]
[in a new window]
  		Table 1. Composition of diets, as-fed basis
 
Time Course Study.
Six replicates (approximately 100 mg) each of corn, soybean meal, or a negative control diet were weighed into incubation tubes. Buffer was added to samples in the incubation tubes and vortexed. To initiate the reaction, phytase enzyme stock solution was added to give 500 FTU/kg of sample. One phytase unit (FTU) is defined as the quantity of enzyme required to liberate 1 µmol of inorganic P (iP)/min, at pH 5.5, from an excess of 15 µM sodium phytate at 37°C (International Union of Biochemistry, 1979Go). The samples were incubated in a shaking water bath maintained at 40°C for 0, 0.5, 1, 2, 4, 12, or 24 h. At the specified times, a stop solution (2.4 M HCl) placed on ice bath was added to give a concentration of 1 M HCl. For time zero, the stop solution was added before initiating with phytase. The contents of the incubation tube were centrifuged at 5,000 x g for 10 min at 4°C. The supernatant was analyzed for inorganic phosphate as described in the chemical analysis section.

pH Dependence Study.
Six replicates (approximately 100 mg) each of corn, soybean meal, or the negative control diet were weighed into incubation tubes. Buffers with pH 1.5 to 7 were brought up to temperature by placing in a 40°C water bath, added to samples in the incubation tubes, and vortexed. Phytase solution was added to give 500 FTU/kg of sample and incubated in a shaking water bath maintained at 40°C for 1 h. The reaction was stopped and samples were processed as described above.

Phytase Activity Study.
Six replicates (approximately 100 mg) each of corn, soybean meal, the negative control diet and the positive diet were weighed into incubation tubes. Buffer (pH 4.5 or 5.5) was brought up to temperature by placing in a water bath at 40°C, added to samples in the incubation tubes, and vortexed. Phytase solution was added to give 0, 125, 250, 500, 750, 1,000, 2,000, or 4,000 FTU/kg of sample. Sample incubation and processing were conducted as described above.

Growth Performance of 10-kg Pigs (Exp. 2)
The experiment was conducted to determine the effect of EP in diets with no added iP when fed to 10-kg pigs for 28 d. A fungal phytase (FP) was included for comparison. Twelve diets including a positive control with no added phytase but adequate in Ca and P levels (Table 1Go); a negative control with no added iP (NC); NC plus (as-fed basis) 250, 500, 750, 1,000, or 1,250 FTU of EP/kg; and NC plus 250, 500, 750, 1,000, or 1,250 FTU of FP/kg were used. All diets were corn-soybean meal-based and formulated to meet or exceed recommendations for the 10-kg pig (NRC, 1998Go), with the exception of dietary P in the negative control diet. There were 96 individually penned pigs (48 barrows and 48 gilts) with an average initial weight of 10 kg in eight blocks (initial weight was used as the basis for blocking) in a randomized complete block design.

Pigs were provided ad libitum access to feed and water for 28 d using protocols and facilities similar to those described by Adeola et al. (1995)Go. Feed was withheld for 16 h before taking initial pig weights on d 1. On d 28, feed was weighed and withdrawn for approximately 16 h for an overnight fast. On d 29, pigs were weighed, given 500 g of their respective diets, and bled approximately 2 h after feeding. Blood samples for all pigs were taken from the anterior vena cava into heparinized Vacutainer tubes and placed in ice buckets. Plasma was separated by centrifugation at 2,000 x g for 15 min at 5°C and stored at –18°C until analyzed for P, Ca, and urea N.

Energy and Nutrient Balance of 10-kg Pigs (Exp. 3)
Based on the results of Exp. 2, four diets from those used in Exp. 2 were selected for an energy and nutrient retention study. The diets selected were a positive control, NC, NC plus 750 FTU of EP/kg, and NC plus 750 FTU FP/kg (as-fed basis). Pigs were fed a 22% CP, 1.15% lysine diet until they attained a weight of 13 kg. Twenty-four 13-kg barrows were selected and assigned to the four diets, resulting in six pigs per treatment in a randomized complete block design and used in a nutrient balance study consisting of a 5-d adjustment and 5-d collection. Pigs were housed in stainless steel metabolism crates (0.83 m x 0.71 m) that allowed for separate collection of feces and urine using protocols described by Adeola and Bajjalieh (1997)Go.

Growth Performance of 20-kg Pigs (Exp. 4)
Growth performance study was conducted to examine the effect of adding the EP in diets with no added iP when fed to 19-kg pigs. Four diets, including a positive control with no added phytase but adequate in Ca and P levels (Table 1Go), a NC, NC plus 500 or 1,000 FTU of EP/kg (as-fed basis) were used. All diets were corn-soybean meal-based and formulated to meet or exceed recommendations for the 20-kg pig (NRC, 1998Go), with the exception of dietary P in the NC diet. A total of 128 pigs (64 barrows and 64 gilts) with an average initial weight of 19 kg was assigned to the four treatments, resulting in 32 pigs per treatment. Four replicate pens of barrows and four replicate pens of gilts consisting of four pigs per pen were used in a randomized complete block design. Pigs were housed in slatted-floor pens (1.68 m x 3.05 m) equipped with nipple drinkers. The pens were located in an environmentally regulated building maintained at 23 ± 2°C with a 12-h light (0700 to 1900) cycle. Body weight was recorded every 2 wk and ad libitum access to feed and water was provided for 42 d.

Energy and Nutrient Balance of 20-kg Pigs (Exp. 5)
An energy and nutrient digestibility study was designed to examine the effect of dietary EP in pigs with an initial weight of 20 kg. Six dietary treatments, including a positive control with no added phytase but adequate in Ca and P levels (Table 1Go); NC; and NC plus 250, 500, 750, or 1,000 FTU of EP/kg (as-fed basis), were used. Thirty-six barrows were assigned to the six diets in randomized complete block design. The study was conducted as described above in Exp. 3.

Chemical Analysis
Inorganic phosphate analysis in the in vitro study was conducted by using Sigma kit No. 670 (Sigma Diagnostics, St. Louis, MO). Using appropriate dilutions, aliquots of the supernatant were plated on microtiter plates in triplicate. Inorganic phosphate in the samples was complexed with ammonium molybdate, incubated at 60°C for 10 min, and allowed to cool for 30 min. Absorbance of the reduced complex was read at 660 nm.

Fecal samples were thawed, mixed thoroughly, and 20% subsamples were dried at 55°C for 72 h. Fecal subsamples and diets were air-equilibrated and ground through 1-mm screen in a Wiley mill. Urine samples were thawed and strained through glass wool to remove particulate matter, and 300-mL samples were weighed into aluminum pans and dried in a forced-air oven at 55°C for 72 h. Dry weights were recorded immediately after removal from oven; samples were placed in Whirl-Pak bags (Nasco, Ft. Atkinson, WI) and frozen at –18°C. Samples were taken out of the freezer immediately before energy determination with a Parr 1261 adiabatic calorimeter (Parr Instrument Co., Moline, IL). Nitrogen analysis of diets, feces, and liquid urine samples was carried out by combustion method using a FP 2000 nitrogen analyzer (Leco Corp., St. Joseph, MN). Diets, feces, and urine were analyzed for Ca and P, and diets were analyzed for nonphytate P as described by Sands et al. (2001Go, 2003)Go. Enzymatic activity of phytase was determined by the method of Engelen et al. (1994)Go.

Statistical Analyses
Data were analyzed as a randomized complete block design using the GLM procedures of SAS (SAS Inst., Inc., Cary, NC). Each incubation tube served as the experimental unit in the in vitro studies, and pen served as the experimental unit in the growth performance and nutrient balance studies. In the in vitro studies, nonlinear regression analyses of iP release against incubation time or phytase activity were performed using FigP software (BioSoft, Cambridge, U.K.). Differences between ingredient/diet means within each time point or pH, and differences between pH means within each ingredient/diet were separated using protected LSD. For the growth performance and nutrient balance studies, linear and quadratic contrasts, as well as contrasts of positive and negative control diets, were used to separate means as appropriate. An alpha level of 0.05 was considered statistically significant.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Literature Cited
 
The respective analyzed total and nonphytate P of the samples used in the in vitro studies were (g/kg): corn, 2.6 and 0.9; soybean meal, 6.9 and 3.5; and the negative control diet, 3.4 and 1.4. For Exp. 2 and 3, analyzed phytase activity in diets formulated to contain 250, 500, 750, 1,000, or 1,250 FTU of EP/kg were 198, 560, 696, 1,003, or 1,396 FTU/kg, respectively; corresponding values for diets containing FP were 244, 496, 804, 986, or 1,249 FTU/kg. Diets formulated to contain 250, 500, 750, or 1,000 FTU of EP/kg in Exp. 4 and 5 analyzed at 241, 519, 778, or 1,024 FTU/kg, respectively.

In Vitro Studies (Exp. 1)
The time course of phytate hydrolysis in corn, soybean meal, and NC samples incubated with 500 FTU EP/kg at pH 4.5 is presented in Figure 1Go. The release of iP responded curvilinearly to incubation time and was fitted to a nonlinear model. At all time points, iP release from soybean meal was greater (P < 0.01) than from the NC diet or corn, and the release of iP from corn was lower (P < 0.05) than that from the NC diet at all time points after 4 h. The relatively quick phytate hydrolysis in soybean was probably due to both higher concentration of nonphytate P and the site of phytin within the seed. In dicotyledonous seeds, including oilseeds and other grain legume seeds, phytin accumulates in globoid crystals that are evenly dispersed within protein bodies (Erdman, 1979Go). The phytin in soybean meal is closely associated with protein bodies, but is unique in that there seems to be no specific site of localization as it is distributed evenly in the seed. The structure, form, and site of phytin in grains and legume seeds may determine the extent of accessibility to enzymes and interactions with other nutrients (Adeola and Sands, 2003Go). As would be expected from the higher total P in soybean meal (6.9 g/kg), iP release due to phytase addition was greatest in soybean meal, least in corn, and intermediate in the NC diet.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 1. Time course of phytate hydrolysis in corn, soybean meal, or the negative control diet by E. coli-derived phytase. Samples were incubated with 500 phytase units (FTU)/kg at pH 4.5 in a shaking water bath maintained at 40°C for specified times. Analyzed total and nonphytate P of the samples, respectively, were (g/kg): corn, 2.6 and 0.9; soybean meal, 6.9 and 3.5; and the negative control diet, 3.4 and 1.4. Values are means ± SE of six observations. The nonlinear model that converged the data were Y = [(1,200 x X)/(0.914 + X)] + (41 x X), r2 = 0.996 for corn; Y = [(1,396 x X)/(0.201 + X)] + (89 x X), r2 = 0.966 for soybean meal; and Y = [(1,351 x X)/(0.797 + X)] + (49 x X), r2 = 0.984 for the negative control (NC) diet. At all time points, inorganic P (iP) release from soybean meal was greater (P < 0.01) than from the NC diet or corn, whereas iP release from corn was lower (P < 0.05) than from the NC diet at all time points after 4 h.

 
The release of iP, during a 1-h incubation period, responded to the pH of the incubation medium (Figure 2Go). Inorganic P release from soybean meal was greater (P < 0.01) than that from the NC diet or corn; the release of iP from the NC diet and corn were similar. The hydrolysis of phytate in soybean meal was highest (P < 0.01) between pH 2 and 4.5, with a precipitous decrease in iP release between pH 6 and 7. Inorganic phosphate release from corn and NC was highest (P < 0.01) between pH 2 and 4. This was followed by a decline in phytase activity between pH 4 and 6 and a steep decline thereafter. It is possible that substrate (phytate) availability in corn and the NC diet was limiting iP release. The pH dependence of phytase is likely related to the pH optima of the enzyme rather than to inherent characteristics of the ingredient or diet. However, the buffering capacity of the diet may vary depending on the form in which nutrients or additives are added to the diet. The optimal pH of the phytase used in this study is within the range of 4.5 to 5.5. This would explain the rapid drop in P release above pH 6. The pH dependence of phytase may also be related to phytate solubility. Phytate solubility rapidly decreases above pH 5.5 as insoluble salts of phytic acid are formed (Cheryan, 1980Go). Previous in vitro studies using sodium phytate as a substrate demonstrated that EP had a pH optimum of 4.5 (Greiner et al., 1993Go; Igbasan et al., 2000Go) and was mainly active in the upper part of digestive system of pigs (Igbasan et al., 2000Go). Rodriguez et al. (1999b)Go showed that the EP retained only 65% of its original pH 3.5-activity when assayed at pH 5.5. Similar to the observation in the time course study, iP release due to phytase addition was highest in soybean meal (P < 0.01), lowest in corn, and intermediate in NC. Phytase activity dependence of phytate hydrolysis in corn, soybean meal, and NC is depicted in Figure 3Go. The release of iP from corn, soybean meal, or NC showed a curvilinear response to phytase activity.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. The pH dependence of phytase hydrolysis in corn, soybean meal, or the negative control (NC) diet by E. coli-derived phytase. Samples were incubated with 500 phytase units (FTU)/kg at the specified pH in a shaking water bath maintained at 40°C for 1 h. Analyzed total and nonphytate P of the samples, respectively, were (g/kg): corn, 2.6 and 0.9; soybean meal, 6.9 and 3.5; and the negative control diet, 3.4 and 1.4. Values are means ± SE of six observations. At all pH points, inorganic P (iP) release from soybean meal was greater (P < 0.01) than from the NC diet or corn. Release of iP from soybean meal was higher (P < 0.01) between pH 2 and 4.5 than at any other pH. In corn and NC, iP release was higher (P < 0.01) between pH 2 and 4 than at any other pH.

 


View larger version (20K):
[in this window]
[in a new window]
  		Figure 3. The effect of phytase activity at 0, 125, 250, 500, 750, 1,000, 2,000, or 4,000 phytase units (FTU)/kg on hydrolysis of E. coli-derived phytate in corn, soybean meal, the negative and positive control diets. Samples were incubated in a shaking water bath maintained at 40°C for 1 h at pH 4.5. Analyzed total and nonphytate P of the samples, respectively, were (g/kg): corn, 2.6 and 0.9; soybean meal, 6.9 and 3.5; and the negative control (NC) diet, 3.4 and 1.4. Values are means ± SE of six observations. The nonlinear model that converged the data were Y = [(899 x X)/(169 + X)] + (0.094 x X), r2 = 0.945 for corn; Y = [(1,787 x X)/(84 + X)] + (0.114 x X), r2 = 0.919 for soybean meal; and Y = [(1,121 x X)/(117 + X)] + (0.074 x X), r2 = 0.961 for the NC diet. At all phytase activities used, inorganic P release from soybean meal was greater (P < 0.01) than from the NC diet, which was greater (P < 0.01) from corn.

 
Growth Performance of 10-kg Pigs (Exp. 2)
Increasing the dietary nonphytate P level increased (P < 0.01) final weight, weight gain, and G:F of pigs over the 28-d period (Table 2Go; negative vs. positive control). However, feed intake and plasma urea nitrogen were not different between pigs that received the positive and negative control diets. Plasma urea nitrogen did not respond to dietary phytase supplementation and thus was not a valid response criterion for phytase effect. The efficacy of phytase on phytate-P use is shown by the responses in performance and measures of blood P status. The addition of graded levels of either EP or FP to NC diet resulted in linear increases (P < 0.01) in weight gain and G:F (Table 2Go). This observation confirms the well-documented improvement in weight gain of pigs fed phytase-supplemented low-nonphytate-P diets (Cromwell et al., 1993Go; Adeola et al., 1995Go; Augspurger et al., 2003Go). Previous studies (Lei et al., 1993bGo; Han et al., 1997Go; Sands et al., 2001Go) showed that plasma P concentration is a sensitive measure of P status that is highly correlated with bone strength and growth in young pigs. The plasma concentrations of P and Ca concentration of pigs in the current study increased linearly (P < 0.05) with increasing supplementation of EP or FP to the diets (Table 2Go). Adding EP or FP (750 FTU/kg) to the NC diets resulted in a 21 or 22% increase in plasma P concentration of pigs, respectively (P < 0.01). The significant effect of phytase on plasma Ca concentration in weanling pigs is consistent with previous observations (Lei et al., 1993aGo).


View this table:
[in this window]
[in a new window]
  		Table 2. Growth performance and plasma chemistry of pigs fed two different phytase enzymes for 28 d in Experiment 2
 
For all of the response criteria taken in the experiment, there were no treatment contrast differences between EP vs. FP, which is consistent with the results of Igbasan (2001)Go that EP was as effective as Natuphos in enhancing the availability of phytate-P for pigs. Furthermore, the recent study of Augspurger et al. (2003)Go showed that the supplementation of diets with 400 FTU/kg of Natuphos or EP resulted in a similar weight gain response of weanling pigs.

Energy and Nutrient Balance of 13-kg Pigs (Exp. 3)
The DM, energy, N, P, and Ca use data of 13-kg pigs that were offered EP or FP (750 FTU/kg) in a nutrient balance study are presented in Table 3Go. Pigs that received iP in the positive control diet had higher (P < 0.01) P digestibility and retention than those fed the NC diet. Phytase supplementation of the low-nonphytate-P NC diet resulted in increased (P < 0.01) apparent digestibility and retention of P. The improvement in apparent digestibility and retention of P observed in the current study confirms earlier observations reported by Simons et al. (1990)Go and Sands et al. (2001)Go. In pigs that received the low-nonphytate-P NC and phytase-supplemented diets in the current study, apparent retention of P was close to apparent digestibility of P-value (the difference being less than 0.3 percentage units; Table 3Go), an indication that the pigs retained almost all of the P digested.


View this table:
[in this window]
[in a new window]
  		Table 3. Energy and nutrient utilization by pigs fed two different phytase enzymes in Experiment 3
 
Apparent digestibility of Ca was higher (P < 0.05) in pigs that received the positive control diet than in those that received the NC diet (Table 3Go); however, there was no response in Ca digestibility or retention to phytase supplementation. This observed lack of response in Ca digestibility or retention to phytase supplementation differs from the observations reported by Lei et al. (1993a)Go and Nasi (1990)Go, who showed that digestive use of dietary Ca was improved when FP was added to the diets of weanling pigs. Differences in Ca and ingredients used in basal diets could contribute to these observations. As shown in Table 3Go, there was no response in the digestibilities of DM, energy, or N to phytase or iP supplementation of the NC diet. The average DE (4.048 kcal/g) of the positive control diet was not different from that (3.860 kcal/g) of the NC diet in the study. This is not consistent with the suggestion by Graf (1986)Go that phytic acid, phytate residues, and phytin in nonruminant diets might decrease starch digestibility by inhibiting {alpha}-amylase or lipase, and that supplementation of microbial phytase to diets might neutralize these antinutritional effects and increase the digestibility of starch and lipid, thus improving the digestible energy.

Growth Performance of 19-kg Pigs (Exp. 4)
The 42-d growth performance of pigs (four pigs per pen, eight pens per diet) as affected by dietary EP supplementation is shown in Table 4Go. The low-nonphytate-P diet depressed (P < 0.05) growth performance when compared with the positive control diet. Body weights at the end of wk 2, 4, and 6 increased linearly (P < 0.01) in response to phytase supplementation of the low-nonphytate-P NC diet. The addition of phytase to the low-nonphytate-P NC diet increased weight gain (linear effect, P < 0.01), feed intake (linear effect, P < 0.05), and G:F (linear effect, P < 0.01). Weight gain and G:F were increased by 29 and 17%, respectively, as dietary phytase level increased from 0 to 1,000 FTU/kg. The positive effect of EP on feed intake, weight gain, and feed efficiency are close to the observations reported by Jongbloed et al. (1994)Go with Natuphos phytase in growing pigs.


View this table:
[in this window]
[in a new window]
  		Table 4. Growth performance by pigs fed phytase enzyme for 42 d in Experiment 4
 
Energy and Nutrient Balance of 19-kg Pigs (Exp. 5)
As presented in Table 5Go, the addition of EP or iP to the diets had no effect on DM, energy, or nitrogen digestibilities. Recent results reported by Oryschak et al. (2002)Go showed that supplementation of diet with Natuphos phytase significantly increased N retention by reducing urinary N excretion and not by increasing N digestibility. In the current study however, N retention was not affected by the addition of phytase. The wide divergence of these N use values seemed to be related, in part, to the complex nature of phytate hydrolysis in the digestive system of pigs. Although Jongbloed et al. (1996)Go reported that the addition of Natuphos phytase to the diets of growing pigs enhanced the digestibility of DM, such a positive effect in growing pigs was not observed in the current study using the EP. Pigs fed the positive control diet digested Ca and P approximately nine percentage units better than those fed the NC diet (P < 0.01), but Traylor et al. (2001)Go did not detect any changes in the apparent digestibility of Ca between the corn-soybean basal diet and the diet containing Natuphos phytase (1,500 FTU/kg) fed to growing pigs. There were linear increases in digestibilities and retentions in Ca and P (P < 0.01) when phytase supplementation of the low-nonphytate-P NC diet increased from 0 to 1,000 FTU/kg. The lowest P retention (41.53%), observed in the NC diet, was probably due to the low available P intake in the diet. Adding phytase at 750 FTU/kg to the diets of 19-kg pigs resulted in a relative increase of 36% in P digestibility, which is numerically greater than the 31% increase in the 13-kg pigs used in Exp. 3. Perhaps this is due in part to underlying physiological differences, such as digestive juice secretion, rate of passage, and control of digesta pH, between younger and older pigs. The study of Kemme et al. (1997)Go also showed that the efficacy of phytase supplementation in generating digestible P was less in pigs between 30 and 38 kg BW than in pigs between 40 and 100 kg BW. It appears that there exists a tendency toward an increase of phytase efficacy and P digestibility in pigs with increasing BW.


View this table:
[in this window]
[in a new window]
  		Table 5. Energy and nutrient utilization by pigs fed phytase enzyme in Experiment 5
 
General Discussion
Phosphorus as a nutrient has posed challenges for nonruminant nutritionists due to the inefficiency of P use in cereal-based diets, expense of P supplementation, and potential environmental pollution effects of P in animal wastes. The advent of cost-effective, commercial-scale production of phytase enzyme has presented the monogastric nutritionist with an effective tool to improve P digestibility and use, minimize P excretion, and allow prudent use of P supplements in diet formulation. In the present research, the efficacy of an EP was evaluated using a three-prong approach: in vitro phytate hydrolysis, in vivo digestibility in weanling and growing pigs, and growth performance in weanling and growing pigs. The results indeed demonstrate that the EP, under controlled in vitro conditions, is effective in hydrolyzing phytin in grains and oilseed meal. Furthermore, the EP is also active in the gastrointestinal tract of pigs, as evidenced from the increased P digestibility in the phytase-supplemented low-nonphytate P diets. Presumably, the exogenous phytase-induced increase in P digestibility resulted in improved growth performance of weanling and growing pigs.

During a 24-h incubation with 500 FTU of the EP/kg, 81 and 72% of the total P in corn (2.6 g/kg; analyzed) and the NC diet (3.4 g/kg; analyzed) were released (Figure 1Go). The corresponding value for soybean meal (6.9 g/kg; analyzed) was 55%. In a study on in vitro hydrolysis of soybean meal phytin by 100 to 900 FTU of an EP, Rodriguez et al. (1999a)Go reported that between 1.5 and 8 µmol of iP was released. This translates to a release of between 233 and 1,240 mg of iP/kg of soybean meal or between 3 and 18% of total P in soybean meal (based on analyzed total P of 6.9 g/kg). The duration of incubation, which could not be deciphered from the study, might be responsible for the difference between the current study and Rodriguez et al. (1999a)Go. In the current study, the release of iP, even after correction for nonphytate P in the samples, is a clear manifestation that the EP dephosphorylated the native myo-inositol hexakis dihydrogen phosphate in the feed samples under the in vitro conditions used in the current study. Phytic acid associates with K+ and Mg2+ and, to a lesser extent, Ca2+, to form phytin in plants (Adeola and Sands, 2003Go). The size of globoid phytin crystals depends, to a large extent, on the ratio of divalent cations to K+ and ratios with higher divalent cations favor the formation of large insoluble crystals. Furthermore, the site of phytin in the seed fraction varies between different grains, grains and legumes, and between legumes (Maga, 1982Go). It would seem that the variable location of phytin between and within the seed, coupled with the differences in structure and form, have further implications on the differences between corn and soybean meal in the proportion of total P released during hydrolysis.

Growth performance of weanling and growing pigs was significantly decreased when pigs were fed the low-nonphytate-P diets. The negative effects were attenuated by the addition of phytase to diets, demonstrating that the added phytase produced the desired effect in releasing iP from dietary phytate P and improving P use in corn-soybean meal diets for pigs. This contention is supported by the observed improvement in P digestibility when diets were supplemented with phytase. Thus, it could be surmised that the phytase added to pig diets in the current studies was active in dephosphorylating feed phytin in the gastrointestinal tract of pigs, and that the released P was absorbed and used to meet the P needs for maintenance and growth. The two phytase enzymes used in Exp. 2 and 3 were equally effective. As discussed by Augspurger et al. (2003)Go, however, interpretive problems affect the comparison of phytase enzymes with different pH optima. Determination of the activity of the two phytase enzymes at pH 5.5 (the optimal pH for FP) may underestimate the activity of EP (optimal pH = 2 to 4.5), resulting in a situation whereby more EP activity than that intended was added to diets. Digestibility of P in the positive control diet was approximately 16 percentage units higher than that in the diets supplemented with phytase in Exp. 3; however, plasma P concentration was lower in pigs fed the positive control diets than in those fed the diets supplemented with phytase in Exp. 2. This could be due to regulatory mechanisms involved with P release from the portal-drained viscera into systemic circulation relative to P requirements of the animal. There was a phytase-induced improvement in Ca digestibility in Exp. 5, but not in Exp. 3. This was due to the relatively lower Ca digestibility in the NC diet in Exp. 5 (71%) vs. Exp. 3 (79%). The relatively lower Ca digestibility in Exp. 5 could be related to the wider Ca:P ratio and the age of pigs used. Addition of phytase did not affect N or energy use in Exp. 3 and 5. Adeola and Sands (2003)Go point out that in the literature, in several instances, a lack of microbial phytase-induced improvement in protein and energy use has been reported. The totality of research on phytase-induced effects on protein and energy use in pigs epitomizes a conflicting base of information. The evidence is incontrovertible that microbial phytase is effective for improving digestive use of plant-derived phytin-P.

In summary, results in these studies demonstrate that the EP in a low-nonphytate-P diet was efficacious in hydrolyzing phytate. Supplementation of low-nonphytate-P diets with the new phytase improved phytate P digestibility and growth performance of pigs. Therefore, adding the EP to the diets can increase P use, decrease the need for inorganic P supplementation of diets, and decrease the excretion of P in swine manure.


   Footnotes
 
1 Journal paper No. 17286 of the Purdue Univ. Agric. Res. Programs. The authors thank the staff of Purdue Univ. Swine Research Unit for care of experimental animals, and C. Thomas for technical assistance. Back

2 Correspondence: 915 W. State St. (phone: 765-494-4848; fax: 765-494-9346; e-mail: ladeola{at}purdue.edu).

Received for publication January 6, 2004. Accepted for publication May 25, 2004.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Literature Cited
 


Adeola, O. 1999. Nutrient management procedures to enhance environmental conditions: An introduction. J. Anim. Sci. 77:427–429.[Abstract/Free Full Text]

Adeola, O., and N. L. Bajjalieh. 1997. Energy concentration of high-oil corn varieties for pigs. J. Anim. Sci. 75:430–436.[Abstract/Free Full Text]

Adeola, O., B. V. Lawrence, A. L. Sutton, and T. R. Cline. 1995. Phytase-induced changes in mineral utilization in zinc-supplemented diets for pigs. J. Anim. Sci. 73:3384–3391.[Abstract]

Adeola, O., and J. S. Sands. 2003. Does supplemental dietary microbial phytase improve amino acid utilization? A perspective that it does not. J. Anim. Sci. 81(E Suppl. 2):E78–E85.[Abstract/Free Full Text]

Augspurger, N. R., D. M. Webel, X. G. Lei, and D. H. Baker. 2003. Efficacy of an E. coli phytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs. J. Anim. Sci. 81:474–483.[Abstract/Free Full Text]

Cheryan, M. 1980. Phytic acid interactions in food systems. CRC Crit. Rev. Food Sci. Nutr. 13:297–335.

Cromwell, G. L., R. D. Coffey, G. R. Parker, H. J. Monegue, and J. H. Randolph. 1995. Efficacy of a recombinant-derived phytase in improving the bioavailability of phosphorus in corn-soybean meal diets for pigs. J. Anim. Sci. 73:2000–2008.[Abstract]

Cromwell, G. L., T. S. Stahly, R. D. Coffey, H. J. Monegue, and J. H. Randolph. 1993. Efficacy of phytase in improving the bioavailability of phosphorus in soybean meal and corn-soybean meal diets for pigs. J. Anim. Sci. 71:1831–1840.[Abstract]

Engelen, A. J., F. C. van der Heeft, P. H. G. Randsdorp, and E. L. C. Smit. 1994. Simple and Rapid Determination of Phytase Activity. J. AOAC Int. 77:760–764.[Medline]

Erdman, J. W., Jr. 1979. Oilseed phytates: Nutritional implications. J. Am. Oil Chem. Soc. 56:736–741.

Graf, E. 1986. Chemistry and applications of phytic acid: An overview. Pages 173–194 in Phytic Acid: Chemistry and Applications. E. Graf, ed. Pilatus Press, Minneapolis, MN.

Greiner, R., U. Konietzny, and K. D. Jany. 1993. Purification and characterization of two phytases from Escherichia coli. Arch. Biochem. Biophys. 303:107–113.[Medline]

Han, Y. M., F. Yang, A. G. Zhou, E. R. Miller, P. K. Ku, M. G. Hogberg, and X. G. Lei. 1997. Supplemental phytases of microbial and cereal sources improve dietary phytate phosphorus utilization by pigs from weaning through finishing. J. Anim. Sci. 75:1017–1025.[Abstract/Free Full Text]

Igbasan, F. A., K. Männer, G. Miksch, R. Borriss, A. Farouk, and O. Simon. 2000. Comparative studies on the in vitro properties of phytase from various microbial origins. Arch. Anim. Nutr. 53:353–373.

Igbasan, F. A., O. Simon, G. Miksch, and K. Männer. 2001. The effectiveness of an Escherichia coli phytase in improving phosphorus and calcium bioavailabilities in poultry and young pigs. Arch. Anim. Nutr. 54:117–126.

International Union of Biochemistry. 1979. Enzyme Nomenclature: Recommendations of the Nomenclature Committee of the International Union of Biochemistry. Academic Press: New York, NY.

Jongbloed, A. W., P. A. Kemme, and H. Everts. 1994. Digestible P requirements for pigs. Pages 38–48 in CVB Documentation Report No. 10. Centraal Veevoederbureau, Lelystad, The Netherlands.

Jongbloed, A. W., P. A. Kemme, Z. Mroz, and J. TH. M. van Diepen. 1996. Phytase in swine rations: Impact on nutrition and environment. A review. Pages 44–56 in BASF Technical Symp., Des Moines, IA.

Kemme, P. A., A. W. Jongbloed, Z. Mroz, and A. C. Beynen. 1997. The efficacy of Aspergillus niger phytase in rendering phytate phosphorus available for absorption in pigs is influenced by pig physiological status. J. Anim. Sci. 75:2129–2138.[Abstract/Free Full Text]

Leeson, S., H. Namkung, M. Cottrill, and C. W. Forsberg. 2000. Efficacy of a new bacterial phytase in poultry diets. Can. J. Anim. Sci. 80:527–528.

Lei, X. G., P. K. Ku, E. R. Miller, and M. T. Yokoyama. 1993b. Supplementing corn-soybean meal diets with microbial phytase linearly improves phytate phosphorus utilization by weaning pigs. J. Anim. Sci. 71:3359–3367.[Abstract]

Lei, X. G., P. K. Ku, E. R. Miller, and M. T. Yokoyama. 1993a. Supplementing corn-soybean meal diets with microbial phytase maximizes phytate phosphorus utilization by weaning pigs. J. Anim. Sci. 71:3368–3375.[Abstract]

Maga, J. A. 1982. Phytate: Its chemistry, occurrence, food interactions, nutritional significance and methods of analysis. J. Agric. Food Chem. 30:1–9.

Näsi, M. 1990. Microbial phytase supplementation for improving availability of plant phosphorus in the diet of growing pigs. J. Agri. Sci. Finl. 62:435–443.

Nolan, K. B., P. A. Duffin, and D. J. McWeeny. 1987. Effects of phytate on mineral bioavailability. In vitro studies on Mg, Ca, Fe, Cu, and Zn (also CD) solubilities in the presence of phytate. J. Sci. Food Agric. 40:79–85.

NRC. 1998. Nutrient Requirements of Swine. 10th ed. Natl. Acad. Press, Washington, DC.

Oryschak, M. A., P. H. Simmins, and R. T. Zijlstra. 2002. Effect of dietary particle size and carbohydrase and/or phytase supplementation on nitrogen and phosphorus excretion of grower pigs. Can. J. Anim. Sci. 82:533–540.

Rodriguez, E., Y. Han, and X. G. Lei. 1999a. Cloning, sequencing, and expression of an Escherichia coli phosphatase/phytase gene (AppA2) isolated from pig colon. Biochem. Biophys. Res. Commun. 257:117–123.[Medline]

Rodriguez, E., J. M. Porres, Y. Han, and X. G. Lei. 1999b. Different sensitivity of recombinant Aspergillus niger phytase (r-PhyA) and Escherichia coli pH 2.5 aid phosphatase (r-AppA) to trypsin and pepsin in vitro. Arch. Biochem. Biophys. 365:262–257.[Medline]

Sands, J. S., D. Ragland, C. Baxter, B. C. Joern, T. E. Sauber, and O. Adeola. 2001. Phosphorus bioavailability, growth performance, and nutrient balance in pigs fed high available phosphorus corn and phytase. J. Anim. Sci. 79:2134–2142.[Abstract/Free Full Text]

Sands, J. S., D. Ragland, J. Wilcox, and O. Adeola. 2003. Relative bioavailability of phosphorus and retention of nutrients from low-phytate soybean meal for broiler chicks. Can. J. Anim. Sci. 83:95–100.

Simons, P. C. M., H. A. J. Versteegh, A. W. Jongbloed, P. A. Kemme, P. Stump, K. D. Bos, M. G. E. Wolters, R. F. Beudeker, and G. J. Verschoor. 1990. Improvement of phosphorus availability by microbial phytase in broilers and pigs. Br. J. Nutr. 64:525–540.[Medline]

Thomson, L. U., and J. H. Yoon. 1984. Starch digestibility as affected by poly phenols and phytic acid. J. Food Sci. 49:1228–1229.

Traylor, S. L., G. L. Cromwell, M. D. Lindemann, and D. A. Knabe. 2001. Effect of levels of supplemental phytase on ileal digestibility of amino acids, calcium, and phosphorus in dehulled soybean meal for growing pigs. J. Anim. Sci. 79:2634–2642.[Abstract/Free Full Text]

Wodzinski, R. J., and A. H. J. Ullah. 1996. Phytase. Adv. App. Microbiol. 42:263–303.


This article has been cited by other articles:


Home page
 J ANIM SCIHome page
T. A. Woyengo, J. S. Sands, W. Guenter, and C. M. Nyachoti
Nutrient digestibility and performance responses of growing pigs fed phytase- and xylanase-supplemented wheat-based diets
J Anim Sci, April 1, 2008; 86(4): 848 - 857.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
E. K. D. Nyannor, P. Williams, M. R. Bedford, and O. Adeola
Corn expressing an Escherichia coli-derived phytase gene: A proof-of-concept nutritional study in pigs
J Anim Sci, August 1, 2007; 85(8): 1946 - 1952.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
M. Schulin-Zeuthen, E. Kebreab, W. J. J. Gerrits, S. Lopez, M. Z. Fan, R. S. Dias, and J. France
Meta-analysis of phosphorus balance data from growing pigs
J Anim Sci, August 1, 2007; 85(8): 1953 - 1961.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
T. N. Nortey, J. F. Patience, P. H. Simmins, N. L. Trottier, and R. T. Zijlstra
Effects of individual or combined xylanase and phytase supplementation on energy, amino acid, and phosphorus digestibility and growth performance of grower pigs fed wheat-based diets containing wheat millrun
J Anim Sci, June 1, 2007; 85(6): 1432 - 1443.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
J. A. Jendza, R. N. Dilger, J. S. Sands, and O. Adeola
Efficacy and equivalency of an Escherichia coli-derived phytase for replacing inorganic phosphorus in the diets of broiler chickens and young pigs
J Anim Sci, December 1, 2006; 84(12): 3364 - 3374.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
J. A. Jendza, R. N. Dilger, S. A. Adedokun, J. S. Sands, and O. Adeola
Escherichia coli phytase improves growth performance of starter, grower, and finisher pigs fed phosphorus-deficient diets
J Anim Sci, August 1, 2005; 83(8): 1882 - 1889.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Adeola, O.
Right arrow Articles by Schulze, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Adeola, O.
Right arrow Articles by Schulze, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS