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Poult Sci 2008. 87:689-693. doi:10.3382/ps.2007-00256
© 2008 Poultry Science Association
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METABOLISM AND NUTRITION

The Effect of Several Organic Acids on Phytate Phosphorus Hydrolysis in Broiler Chicks

A. Liem1, G. M. Pesti and H. M. Edwards, Jr.

Department of Poultry Science, University of Georgia, Athens 30602-2772

1 Corresponding author: sidoagung{at}yahoo.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Supplementation of some organic acids to a P-deficient diet has been shown to improve phytate P utilization. Two experiments were conducted from 0 to 16 d in battery brooders to determine the effect of various organic acids supplementation on phytate P utilization. In both experiments, birds were fed P-deficient corn and soybean meal-based diets. In experiment 1, citric acid, malic acid, fumaric acid, and EDTA were supplemented. Experiment 2 had a 2 x 2 factorial design with 2 sources of Met, 2-hydroxy-4-(methylthio) butanoic acid (HMB) and DL-Met, with or without 500 U/kg of phytase. In experiment 1, the addition of citric, malic, and fumaric acids increased percentage of bone ash, but only the effect of citric acid was significant. The addition of citric and malic acids also significantly increased the retention of P and phytate P (P < 0.05). In experiment 2, the addition of phytase to the diet significantly increased 16-d BW gain, feed intake, percentage of bone ash, milligrams of bone ash, phytate P disappearance, and decreased the incidence of P-deficiency rickets. Methionine source did not affect 16-d BW gain, feed intake, feed efficiency, milligrams of bone ash, or P rickets incidence. However, the birds fed HMB had a higher percentage of bone ash and phytate P disappearance compared with the groups fed DL-Met only when phytase was added to the diets. The additions of citric acid and HMB improved phytate P utilization. However, the reason why some organic acids are effective whereas others are not is not apparent.

Key Words: phytate phosphorus utilization • organic acid • broiler chick


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ravindran et al. (1995) listed numerous factors that influenced phytate P utilization in poultry. In addition, more recent studies showed that organic acids can increase phytate P utilization by poultry. Boling et al. (2000) reported that inclusion of citric acid at 0 to 6% to a P-deficient broiler diet linearly increased weight gain, weight, and percentage of tibia ash. Snow et al. (2004) showed that the effect of citric acid on phytate P utilization is additive with the effects of phytase and 1-{alpha} cholecalciferol supplementation. Rafacz-Livingston et al. (2005) reported that sodium gluconate, calcium gluconate, glucono-{delta}-lactone, 2-hydroxy-4-(methylthio) butanoic acid (HMB; Alimet), and citric acid improved phytate P utilization, but fumaric acid and EDTA did not. The studies published previously had used bone ash data as a measure of phytate P utilization. Angel et al. (2001) reported an increase in percentage of bone ash and a decrease in feed consumption when citric acid was added to the diet. Further study (Shellem and Angel, 2002) suggested that part of the effect of citric acid on bone ash might be confounded by its effect on feed consumption and size of birds. However, Snow et al. (2004) reported that citric acid addition to a P-deficient diet improved tibia ash without reducing weight gain or feed intake.

Some compounds called organic acids increase P utilization by chicks fed P-deficient diets, whereas others do not (Rafacz-Livingston et al., 2005). The organic acids that are effective chelate minerals. If the effects of the organic acids are due to their ability to chelate minerals, then EDTA, a strong chelating agent, should be effective as well (Rafacz-Livingston et al., 2005). The experiments conducted here will clarify the effects of organic acids in phytate P utilization by providing phytate P disappearance data.

The objective of the experiment 1 was to investigate the effects of citric acid, malic acid, fumaric acid, and EDTA on phytate P utilization by broilers fed a P-deficient diet. The second experiment investigated the effect of substituting DL-Met with HMB on phytate P utilization in the presence and absence of supplemental phytase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
General Procedure
Two experiments were conducted for 16 d with 1-d-old broiler Cobb x Cobb 500 mixed sex chickens sourced from a commercial hatchery. The mash corn-soybean meal basal diets for both experiments are shown in Table 1Go. The experiments were conducted in electrically heated wire mesh floor battery brooders. Ultraviolet irradiation was eliminated from the chick room by fitting Arm-a-Lite (Arm-a-Lite Thermoplastic Processes, Sterling, NJ) sleeves to all fluorescent fixtures in the room and battery brooders (Edwards et al., 1994). The fluorescent lights were on 24 h each day. The temperature of the room was maintained at 22°C. The chicks were given access to water and mash feed ad libitum.


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  		Table 1. Composition of basal diets, experiments 1 and 2
 
At termination of the experiments, the birds were weighed by pen, and their feed consumption was recorded. They were killed by CO2 asphyxiation and examined at random for P-deficiency rickets by making a longitudinal cut across the right tibia. Chickens with P-deficiency rickets have a normal proliferating pre-hypertrophied zone and lengthened metaphyseal primary spongiosa (Edwards, 1993). Left tibias were removed for dry fat-free bone ash determination according to AOAC (1995).

Feces samples were collected for the last 72 h of the experiments (d 13 morning to d 16 morning). Chromic oxide was added to the diets at 0.1% (Table 1Go). Feed and excreta samples were analyzed for phytate P (Latta and Eskin, 1980) and Cr2O3 (Brisson, 1956). Phytate P retention was calculated using the methods of Edwards and Gillis (1959). Percentage of phytate P retained = 100 [(% Cr2O3 in feed/% Cr2O3 in feces) x (% phytate P in feces/% phytate P in feed) x 100].

Experiment 1
At the start of the experiment, 150 one-day-old mixed sex Cobb x Cobb 500 broiler chicks were randomly allotted to 15 pens, which were assigned 5 treatments, with 3 pens per treatment. The citric acid, malic acid, fumaric acid, and EDTA were added at 3.23, 2.90, 2.90, and 3.65%, respectively, to the basal diet, at the expense of corn. The levels of citric and fumaric acids and EDTA are theoretically the amount of acids needed to chelate all the Ca in a diet containing 1% Ca, taking into account the number of carboxyl groups and molecular weight of the acids. Malic acid was added at 50% more. At termination of the experiment, 2 birds from each pen were randomly selected, and blood samples were obtained by heart puncture for plasma total Ca (Technicon Corporation, 1969) and dialyzable P (Technicon Corporation, 1970). Feces samples were collected for the last 72 h of the experiments (d 12 morning to d 16 morning) and analyzed for Ca (Hill, 1955) and P (O’Neill and Webb, 1970).

Experiment 2
In experiment 2, one-day-old chicks (240) were allocated to 4 treatments, with 6 pens per treatment and 10 birds per pen. The design of experiment 2 is a 2 x 2 factorial design with 2 kinds of Met sources fed with or without 500 U/kg of phytase (Natuphos, BASF Corp., Mt. Olive, NJ). The DL-Met was substituted for the hydroxyl analog of the same amount, taking into account that the HMB is 88% pure. The DL-Met was added at 0.2%, and HMB was added at 0.227%.

Statistical Analysis
The statistical analyses for experiments 1 and 2 were performed using the GLM procedure by SAS Institute (Cary, NC). The experimental unit was the pen mean. Comparisons of the treatment means were performed with Duncan’s multiple range test and orthogonal contrasts.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1
Addition of citric acid, malic acid and fumaric acid did not increase the 16-d BW and gain:feed; however, addition of EDTA decreased 16-d BW and gain:feed (Table 2Go). The additions of all acids increased percentage of bone ash numerically, but only the effect of citric acid was significant. The addition of citric acid, fumaric acid, and EDTA decreased the incidence of P-deficiency rickets significantly, but the addition of malic acid did not. All the birds that had rickets in all treatments, except EDTA, had an average score of 3 (severe). Calcium retention did not differ between the basal diet and any of the groups with added acids. The addition of citric, malic, and fumaric acid increased P retention, but EDTA did not. The phytate P retention was increased by acid addition, but only addition of citric and malic acids produced significant increases at P < 0.05. The plasma Ca level of groups fed the basal diet and citric, malic, and fumaric acids treatments did not differ, but birds fed the diet with EDTA had significantly lower plasma Ca. When compared with the basal diet, fumaric acid and EDTA additions to the diet increased plasma P level, whereas citric and malic acid additions did not increase plasma P levels.


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  		Table 2. The effect of organic acids supplementation on broiler performance, bone ash, P rickets, mineral retentions, and plasma minerals, experiment 11
 
Experiment 2
The addition of phytase to the diet significantly increased 16-d BW gain, feed intake, percentage of bone ash, milligrams of bone ash, phytate P disappearance, and decreased the incidence of P-deficiency rickets (Table 3Go). Methionine source did not affect 16-d BW gain, feed intake, feed efficiency, milligrams of bone ash, and P-deficiency rickets incidence. The birds fed HMB had higher percentage of bone ash and phytate P disappearance compared with the groups fed DL-Met only when phytase was added to the diet. This was indicated by the orthogonal contrast analysis.


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  		Table 3. The effect of Met source and phytase on BW gain (BWG), feed intake, gain:feed, percentage of bone ash, milligrams of bone ash, phytate P disappearance, and P rickets incidence, experiment 21
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The effects of organic acids on phytate P utilization might result from a change to the pH of the gastrointestinal tract to a pH more favorable for phytase to hydrolyze phytate. Citric, malic, fumaric acids, EDTA, and HMB are weak acids. All but HMB have more than 1 – log10 acid dissociation constant and functional carboxyl group. Even when added at same levels to the diets, the acids would probably affect GI tract pH differently. The effect of organic acids might not be due to pH reduction only. Boling et al. (2000) suggested that citric acid improved phytate P utilization by competitively chelating Ca, reducing the formation of insoluble Ca-phytate complexes.

Interpreting the bone ash, phytate P retention, and BW gain data, it seemed that citric acid is the most efficacious of the acids studied in improving phytate P utilization, followed by malic and fumaric acids. These results are consistent with Rafacz-Livingston et al. (2005), who reported that citric acid improved phytate P utilization, whereas fumaric acid and EDTA did not. The levels of citric and fumaric acids fed in our experiment were similar to Rafacz-Livingston et al. (2005), but the levels of EDTA were different. The level of EDTA (3.65%) in this experiment was too high and therefore toxic to the birds. Rafacz-Livingston et al. (2005) reported that EDTA supplementation to a P-deficient diet at 0.05 and 0.1% did not improve performance or tibia ash, but instead it seemed to magnify the P deficiency. The level of EDTA used in our experiment was much higher than the level of EDTA used as chelating agent. For example, EDTA is supplemented at 0.12 to 0.14% in the diet for maximizing Zn absorption. It is also important to realize that EDTA does not always increase absorption of trace minerals. For instance, increasing EDTA levels from 0.06 to 0.24% in the diet actually decreased retention of Co and Fe (Suso and Edwards, 1968).

Although the mechanism for how citric and malic acid and HMB improve phytate P utilization is still not clear, this study clarified that the effect was not mediated by reduction in growth and feed consumption, which was suggested by Shellem and Angel (2002). Although Snow et al. (2004) had already reported that the inclusion of citric acid in a P-deficient diet improved bone ash without reducing growth and feed intake, no work has reported the effect of organic acid addition on phytate P retention. Aside from the growth data that indicated that no reduction in weight was produced from citric, malic acid, and HMB supplementation, this study also demonstrated that the additions of those acids increased phytate P retention by broiler chicks.

Only phytase addition to the diet had a significant effect on BW gain, feed intake, percentage and milligrams of tibia ash, and P-deficiency rickets incidence. These results are similar to previous works on the effects of phytase addition to broiler diet, as reviewed in Ravindran et al. (1995). There was no difference in BW gain between birds fed DL-Met and HMB. The P-deficiency rickets data showed that the birds were very deficient in P, causing the high P-deficiency rickets incidence in all treatments. The birds in this experiment seemed to be more P deficient compared with birds in previous experiments fed very similar diets. The chicks received adequate dietary levels of vitamin D3 and Ca to prevent Ca-deficiency rickets and tibial dyschondroplasia. When organic acids are added to the diet, the magnitude and value of other dietary components to increase phytate P utilization should be changed (Ca and available P, Boling et al., 2000). Feeding HMB as opposed to DL-Met only increased phytate P utilization when phytase was present. 2-Hydroxy-4-(methylthio) butanoic acid is an acid which later is transaminated to Met in the body of birds. Adding HMB to the diet might have lowered the pH of the digestive tract, creating a more favorable pH for the phytase to work. In experiment 2, the HMB improved phytate P retention and bone ash only when phytase was present. This might be due to the low (practical) level of the acid, 0.20% as opposed to citric acid level of 3.23% in experiment 1 and 1.14% in the study of Rafacz-Livingston et al. (2005). Therefore, the magnitude of the effect of HMB was only significant when it was magnified by the presence of phytase. The results of experiment 2 suggest that supplementing HMB in place of DL-Met in practical diets would be beneficial to phytate P utilization as well as a source of Met activity. This needs clarified in longer-term studies.

Received for publication June 18, 2007. Accepted for publication December 24, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angel, R., A. S. Dhandu, T. J. Applegate, and M. Chrisman. 2001. Phosphorus sparing effect of phytase, 25-hydroxycholecalciferol, and citric acid when fed to broiler chicks. Poult. Sci. 80(Suppl. 1):133–134. (Abstr.)

AOAC. 1995. Pages 57–58 in Official Methods of Analysis. 16th ed. Vol. II. Assoc. Off. Anal. Chem., Washington, DC.

Boling, S. D., D. M. Webel, I. Mavromichalis, C. M. Parsons, and D. H. Baker. 2000. The effects of citric acid on phytate-phosphorus utilization in young chicks and pigs. J. Anim. Sci. 78:682–689.[Abstract/Free Full Text]

Brisson, G. J. 1956. On the routine determination of chromic oxide in feces. Can. J. Agric. Sci. 36:210–211.

Edwards, H. M. 1993. Dietary 1,25-dihydroxycholecalciferol supplementation increases natural phytate phosphorus utilization in chickens. J. Nutr. 123:567–577.[Abstract/Free Full Text]

Edwards, H. M., M. A. Elliot, S. Sooncharernying, and W. M. Britton. 1994. Quantitative requirement for cholecalciferol in the absence of ultraviolet light. Poult. Sci. 73:288–294.[Web of Science][Medline]

Edwards, H. M., and M. B. Gillis. 1959. A chromic oxide balance method for determining phosphate availability. Poult. Sci. 38:569–574.[Web of Science]

Hill, J. B. 1955. Automated fluorometric method for determination of serum calcium. Clin. Chem. 2:122–130.[Medline]

Latta, M., and M. Eskin. 1980. A simple and rapid colorimetric method for phytate determination. J. Agric. Food Chem. 28:1313–1315.[CrossRef][Web of Science]

NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC.

O’Neill, J. V., and R. A. Webb. 1970. Simultaneous determination of nitrogen, phosphorus, and potassium in plant materials by automatic methods. J. Sci. Food Agric. 21:217–219.[CrossRef][Web of Science]

Rafacz-Livingston, K. A., C. M. Parsons, and R. A. Jungk. 2005. The effects of various organic acids on phytate phosphorus utilization in chicks. Poult. Sci. 84:1356–1362.[Abstract/Free Full Text]

Ravindran, V., W. L. Bryden, and E. T. Kornegay. 1995. Phytates: Occurrence, bioavailability and implications in poultry nutrition. Poult. Avian Biol. Rev. 6:125–143.

Shellem, T., and R. Angel. 2002. Is the effect of citric acid on apparent phosphorus availability mediated primarily through feed consumption changes? Poult. Sci. 81(Suppl. 1):94. (Abstr.)

Snow, J. L., D. H. Baker, and C. M. Parsons. 2004. Phytase, citric acid, and 1 {alpha}-hydroxycholecalciferol improve phytate phosphorus utilization in chicks fed a corn-soybean meal diet. Poult. Sci. 83:1187–1192.[Abstract/Free Full Text]

Suso, F. A., and H. M. Edwards Jr. 1968. Influence of various chelating agents on absorption of 60Co, 59Fe, 54Mn and 65Zn by chickens. Poult. Sci. 47:1417–1425.[Web of Science][Medline]

Technicon Corporation. 1969. Technicon Auto Analyzer Methodology, sect. N-31. Techinicon Corp, Tarrytown, NY.

Technicon Corporation. 1970. Technicon Auto Analyzer Methodology, sect. N-4C. Techinicon Corp., Tarrytown, NY.





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