Phytic phosphorus and phytase activity of animal

Feed Ingredients

Susmira Godoy, Claudio Chicco, François Meschy and Fanny Requena

Susmira Godoy. Doctor in Agricultural Sciences, Universidad Central de Venezuela (UCV). Researcher, Instituto Nacional de Investigaciones Agrícolas, INIA-CENIAP, Venezuela. Address: Apartado Postal 2103, Maracay 2105, Venezuela. e-mail: sgodoy@inia.gov.ve

Claudio Chicco. Ph.D. in Biochemical Nutrition, University of Florida, USA. Professor, UCV.

François Meschy. Ph. D. in Biochemistry, Université de Dijon, France. Researcher, Institut National de la Recherche Agronomique (INRA), France.

Fanny Requena. Doctor in Zootechnics. Unuversitá di Perugia, Italy. Researcher, INIA-CENIAP, Venezuela.

Resumen

Para determinar fósforo total y fítico, y la actividad de fitasas endógenas de granos de cereales y oleaginosas y sus subproductos, se evaluaron ingredientes alimenticios utilizados en la producción animal en los trópicos. Los cereales y sus subproductos fueron maíz amarillo, sorgo, trigo duro, tercerilla de arroz, harina de germen y afrecho de maíz desgrasado grueso y fino, afrecho de trigo y pulitura de arroz. Las oleaginosas y sus subproductos fueron semilla y harina de algodón, y harinas de soya, palmiste y coco. El ácido fítico se determinó mediante un método colorimétrico y la actividad fitásica por un procedimiento enzimático-colorimétrico. Además se determinó, por métodos convencionales, la composición química y mineral de los materiales evaluados. El contenido (%) de proteína cruda, extracto etéreo, cenizas y composición mineral fue similar a los valores de las tablas de composición de alimentos. La concentración de P total estuvo entre 0,12 y 1,57% en granos de cereales y sus subproductos, y entre 0,43 y 1,34% en granos de oleaginosas y sus subproductos. La concentración de P fítico en cereales y oleaginosas varió entre 0,08 y 0,49% y en sus subproductos entre 0,24 y 1,13%. Las ecuaciones de regresión entre P total y fítico fueron positivas y significativas en los cereales y subproductos, y en las oleaginosas y subproductos. Las correlaciones fueron significativas entre P fítico y contenido de Mg, K y Ca en los cereales y subproductos. La actividad fitásica (U/kg) fue significativamente mayor para el trigo (1565). Arroz, afrecho de trigo y pulitura de arroz presentaron actividades >100U/kg. Los restantes granos y subproductos se consideran como materiales sin actividad fitásica.

Summary

To determine total and phytic phosphorus and endogenous phytase activity in cereals, oilseeds and by-products, feed ingredients used in animal production in the tropics were evaluated. The cereals and by-products were yellow corn, sorghum, hard wheat, broken rice, wheat bran, fine and gross defatted corn germ and bran, and rice polishing; the oilseeds and by-products were cotton seed and cotton meal, and soy, palm oil and coconut meals. In five samples of each ingredient, phytic acid was measured by a colorimetric procedure and phytase activity with an enzymatic-colorimetric method. In addition, mineral and chemical components were determined by conventional methods. Crude protein, ether extract, ash and mineral content were similar to values reported in feed composition tables. Total P concentration in cereal grains and by-products ranged from 0.12 to 1.57%, and in oilseeds and by-products, from 0.43 to 1.34%. In cereals and oilseeds, phytic acid concentrations varied from 0.08 to 0.49%, and in by-products, from 0.24 to 1.13%. Significant regressions were found between total and phytic P in cereals and by-products, and in oilseeds and by-products. In the former, correlations were also found between phytic P and Mg, K and Ca content. Phytase activity (U/kg) was significantly higher in wheat (1565). Rice, wheat bran and rice polishing had values >100U/kg. The remaining grains and by-products did not show phytase activity.

Resumo

Para determinar o fósforo total e fítico e, a atividade da fitase endógena de grãos de cereais e oleaginosas e seus subprodutos, avaliaram-se ingredientes alimentícios que se utilizam na produção animal nos trópicos. Estes foram; milho amarelo, sorgo, trigo duro e farelo de arroz; dos subprodutos de cereais, a farinha de gérmen e farelo de milho desengordurado grosso e fino, farelo de trigo e polidura de arroz; das oleaginosas e subprodutos, a semente e farinha de algodão, e as farinhas de soja, palmiste e coco. O ácido fítico determinou-se, em cinco amostras, mediante um método colorimétrico e a atividade fitásica por um procedimento enzimático colorimétrico. Além disso, determinou-se, por métodos convencionais, a composição química e mineral dos materiais avaliados. O conteúdo (%) de proteína crua, extrato etéreo, cinzas e composição mineral foi similar aos valores resenhados nas tabelas de composição de alimentos de diferentes países. Os grãos de cereais e subprodutos apresentaram concentrações de fósforo total variando de 0,12 a 1,57%. Nos grãos de oleaginosas e seus subprodutos a concentração de fósforo total variou de 0,43 a 1,34%. A concentração de fósforo fítico (%) em cereais e oleaginosas variou de 0,08 a 0,49 e, para os subprodutos, de 0,24 a 1.13%. As equações de regressão entre o fósforo total e fítico foram positivas e significativas nos cereais e subprodutos e nas oleaginosas e subprodutos. As correlações foram significativas entre fósforo fítico e conteúdo de magnésio, potássio e cálcio, nos cereais e seus subprodutos. A atividade fitásica (U/kg) foi significativamente mais elevada para o trigo (1.565). O arroz, o farelo de trigo e a polidura de arroz, apresentaram atividades maiores de 100 U/kg. Os grãos e subprodutos restantes se consideram como materiais sem atividade fitásica.

KEYWORDS / Cereals / Oilseeds / Phosphorus / Phytase / Phytate /

Received: 06/14/2004. Modified: 12/01/2004. Accepted: 12/02/2004

Introduction

Cereals, oilseeds and their by-products are the main ingredients for poultry, swine and high productive ruminant diets. Total P concentration in cereals (%) ranges from 0.35 to 0.45 and between 0.65 and 1.12 in oilseeds and by-products (Eeckhout and De Paepe, 1994). In these ingredients, P is present mainly in the form of phytates, which represent 50 to 80% of total P (Kirby and Nelson, 1988). In addition, phytate content in cereals, legumes and oilseeds varies, depending upon cultivars and soil types, climatic and irrigation conditions, processing and locations.

Phytates are poorly utilized by non ruminant animals, due to a low activity of phytase, the enzyme that hydrolyzes these compounds in the digestive tract of poultry and swine (Perney et al., 1993). In ruminants, it is generally accepted that the microorganisms present in the fermentative compartments hydrolyze phytates, releasing P for absorption (Raun et al., 1956). However, Ellis and Tillman (1960) suggest that when high phytate diets are fed, total hydrolysis may not occur, in spite of the high phytase activity of the digestive tract of ruminants.

In addition, there is an endogenous phytase activity in cereals, oilseeds and by-products, which varies according to grain and type of by-product. Thus, this activity (Pointillart, 1994; Eeckhout and De Paepe, 1994) is (U/kg) high in wheat (1200), rye (2700) and triticale (1100), intermediate in barley (580) and low in rice (120), corn (12), sorghum (24) soybean (31) and oat (42 ).

To reach a better understanding of P bioavalability in vegetable ingredients used in animal feeding, knowledge of phytic P content and endogenous phytase activity of these materials is required.

Materials and Methods

Phosphorus content and phytase activity was determined in cereals, oilseeds and by-products, which are widely used for animal production in the tropics. In addition, crude protein, ether extract, structural components, ash and minerals were measured by conventional methods (AOAC, 1984).

The cereals and by-products studied were yellow corn, high tannic acid sorghum, hard wheat and broken rice, fine and gross defatted corn germ and bran, wheat bran, and rice polishing; among oilseeds and by-products, cotton seed and cotton meal, and soybean, palm oil and coconut meals. In addition, brewery grains were included.

Five samples from each material were ground and sifted to 1mm particle size for phytic acid extraction. Phytate assays employing Fe analysis were done by a modification of the method of Early and Turk (1944). A sample (2g) was placed in a flask, into which 100ml of 1.2% HCl + 10% Na₂SO₄ were added. The flask was stoppered and shaken for 2h with a mechanical shaker. The extract was vacuum filtered through Nº54 Whatman paper and 10ml of the filtrate were pipetted into a 50ml centrifuge tube. Deionised water (10ml) was added, followed by 12ml of FeCl₃ solution (2g FeCl₃·6H₂O + 16.3ml conc. HCl/l). The contents were stirred, heated for 75min in boiling water, and cooled, covered, for 1h at room temperature. The tube was centrifuged at 1000G for 15min. The supernatant was decanted and discarded, and the pellet was thoroughly washed three times with a solution of 0.6% HCl and 2.5% Na₂SO₄. After each wash the contents were centrifuged at 1000G for 10min and the supernatant discarded. Concentrated HNO3 (10ml) were added to the resulting pellets and the contents transferred quantitatively to a 400ml beaker with several small portions of deionised water. Four drops of concentrated H₂SO₄ were added and the contents were heated approximately 30min on a hot plate until only the H₂SO₄ remained. Approximately 4-5ml of 30% H₂O₂ were added and the mixture was returned to the hot plate at a low heat until bubbling ceased. The residue was dissolved in 15ml 3N HCl and heated for 10-15min. The resulting solution was brought up to 100ml volume, diluted 1:5. Total P was determined by a colorimetric procedure (Thompson and Erdman, 1982).

Endogenous phytase activity was measured by the procedure described by Bitar and Reinhold (1972). Ground material sifted to 1mm particle size (1g) was diluted in buffer tris-HCl, 0.02M; pH 7.5, and centrifuged (20G) during 30min. The samples were incubated at 37ºC during 5, 15 and 30min in acetate buffer solution (0.25M). The material was incubated with (A) or without (B) 5mM of sodium phytate, including a control without sample (C), but with sodium phytate. The incubation was stopped with 3ml of TCA (20%). Samples were centrifuged (120G) during 10min and free P in the supernatant was measured (Fiske and Subbarrow, 1925). Phosphorus released from the phytate molecule from sample A was adjusted by removing P present in sample B (without phytate) and in the blank (C, without sample), and expressed as inorganic P per g/ml/min.

Phytase activity was calculated as follows: Phytase Unit = (P × 1,000) / (weight × time), where P is expressed in µmols, weight in g of sample and time in min of incubation. In this way one unit of phytase is described as the amount of inorganic P freed per min from 5mM of a sodium phytate solution at a rate of 1µmol/min, at a pH of 5.5 and 37ºC.

Standard deviations were determined for each average value, and correlations and regressions between variables were calculated (Steel and Torrie, 1988).

Results and Discussion

Crude protein, ether extract, neutral and acid detergent fiber, and ash values are presented in Table I. Crude protein, ether extract and ash contents are similar to values reported by international feed composition tables (NRC, 1998; FEDNA, 1999; Sauvant et al., 2002). Ether extract values of coconut and palm oil meal presented some differences with referred standards, probably due to different oil extraction methods.

Mineral composition is presented in Table II. These values are similar to those encountered in the literature (NRC, 1998; Sauvant et al., 2002) for Ca, Mg and K. Some variations were found for Fe, Cu, Mn and Zn.

Total P and phytic P contents, and phytase activity values are presented in Table III. Cereal grains had total P concentrations between 0.12 and 0.33%, lower than values registered by the by-products (0.66-1.57%) These results are similar to those reported by Nelson et al. (1968) and Eeckhout and De Paepe (1994). Oil seeds and by-products presented phosphorus values ranging from 0.43 to 1.34%.

Phytic P concentrations were greater in cereals and oil seed by-products (0.24-1.13%) than in grains (0.08-0.49%). Phytic P, as percent of total P, was >55% in all materials, reaching values over 70% for corn, rice polishing, wheat bran and cotton seed (Table III).

Comparing the values of total and phytic P with those reported by other authors may present some difficulties, even using the same analytical methods, and factors such as cultivar type, climatic conditions, processing methods and soil type may explain some of the variations encountered. Hopkins et al. (1989) using the method of Haug and Lantzesh (1983), reported values of phytic P, as percent of total P, similar to those registered in the present study, and also to the ones published by Eeckout and De Paepe (1994). On the other hand, Nelson et al. (1968) found lower values in the same materials.

Wheat phytase activity (U/kg) was significantly higher (1565) than in the other grains and by-products. Several authors have reported wheat values (U/kg) of 1193 (Eeckhout and De Paepe, 1994; Ravindran et al., 1995), 508 (Barrier-Guillot et al., 1996), 700 (Ponitillart, 1994), and 668 (Frapin, 1996; Nys et al., 1996). Wheat is considered a grain with high phytase activity, as indicated by Eeckhout and De Paepe (1994), who suggested that when phytase activity is higher than 100 U/kg, the material should be considered to have phytase activity.

Based upon the criterion proposed by Eeckhout and De Paepe (1994), hard wheat, wheat bran and rice polishing had significant phytase activity. Corn, sorghum, fine and gross defatted corn germ and bran, broken rice, soybean, coconut and palm oil meals did not show endogenous enzyme activity. Brewery grains had high total P content with no phytase activity.

The significance of intrinsic grain phytases in poultry and swine feeding is still a controversial issue. However, certain feedstuffs such as wheat and wheat bran, when added to swine diets have shown to improve dietary phytate P utilization (Usaryan and Balnave, 1995; Han et al., 1997). Some authors (Jongbloed et al., 1992) have postulated that intrinsic phytases are more resistant to proteolysis in the stomach than extrinsic phytases. In addition, cereal phytases, particularly wheat phytases, could be more active at low gastric pH than microbial phytases (Ranhotra and Loewe, 1975).

Regression equations between total P and phytic P were significant (Table IV). Correlation values were 0.85 (P<0.0001), 0.94 (P<0.0001) and 0.63 (P<0.01), respectively, for all ingredients, cereals and their by-products, and oil seeds and their by-products. No significant correlations were found between phytic P and phytase activity. Similar results were reported by Eeckhout and De Paepe (1994).

Among mineral elements, in case of cereals and by-products, significant correlations were found between phytic P and Mg, K and Ca, with values of 0.70 (P<0.001), 0.86 (P<0.0001) and 0.59 (P<0.01), respectively. No correlations were found for oil seeds and by-product components. Several authors have reported the presence of Ca, Mg and K in the molecular structure of phytates (O'Dell et al., 1972; Morris and Ellis, 1976; Ogawa et al., 1977; Erdman, 1979).

Conclusions

It is concluded that the grains and by-products studied have a high phytate content that varies between 40 to 75% of total P. Significant correlations were found between phytates and total P, as well as some minerals. Wheat and rice, and their by-products, have phytase activity higher than 100U/kg. The remaining grains and by-products do not have significant phytase activity.

ACKNOWLEDGMENTS

The authors thank ECOS-NORD (France) and FONACIT (Venezuela) for financial support and scientific assistance.

REFERENCES

1. AOAC (1984) Official methods analysis. 15th Ed. Association of Official Analytical Chemists. Washington, DC, USA. 1018 pp.        [ Links ]

2. Barrier-Guillot B, Casado P, Maupetit P, Jondreville C, Gatel C (1996) Wheat phosphorus availability. 1. In vitro study in broilers and pigs: relationship with endogenous phytasic activity and phytic phosphorus content in wheat. J. Sci. Food Agric. 70: 69-74.        [ Links ]

3. Bitar K, Reinhold H (1972) Phytase and alkaline phosphatase activities in intestinal mucosa of rat, chicken, calf and man. Biochem. Biophys. Acta 268: 442-452.        [ Links ]

4. Early EB, Turk EE (1944) Time and rate of synthesis of phytin in corn grain during the reproductive period. J. Anim. Sci. Agron. 36: 803-808.        [ Links ]

5. Eeckhout W, De Paepe M (1994) Total phosphorus, phytate-phosphorus and phytase activity in plant feedstuffs. Animal Feed Sci. Technol. 47: 19-29.        [ Links ]

6. Ellis LC, Tillman AD (1960) Utilization of phytin phosphorus in wheat bran by sheep. J. Anim. Sci. 50: 606-607.        [ Links ]

7. Erdman JWJr (1979) Oilseed phytates: Nutritional implications. J. Amer. Oil Chem. Soc. 56: 736-741.        [ Links ]

8. FEDNA (1999) Normas FEDNA para la formulación de piensos compuestos. Blas C, Mateos GG, Rebollar PG (Eds.) Fundación Española para el Desarrollo de la Nutrición Animal. Madrid, Spain. 496pp.        [ Links ]

9. Fiske C, Subbarrow E (1925) The colorimetric determination of phosphorus. J. Biol. Chem. 66: 375-384.        [ Links ]

10. Frapin D (1996) Valorisation du phosphore phytyque vegetal chez I‘oiseau: intérét et mode d‘action des phytases végétales et microbienne. Thesis. Ecole Nationale Supérieure Agronomique de Rennes; SRA, INRA, Centre de Tours, France. 133 pp.        [ Links ]

11. Han YM, Yang F, Zhou AG, Miller ER, Ku PK, Hogberg MG, Lei XG (1997) Supplemental phytases of microbial and cereal sources improve dietary phytate phosphorus utilization by pigs from weaning through finishing. J. Animal Sci. 75: 1017-1025.        [ Links ]

12. Haug W, Lantzsch HJ (1983) Sensitive method for the rapid determination of phytate in cereals and cereal products. J. Sci. Food Agric. 34: 1423-1426.        [ Links ]

13. Hopkins J, Ballantyne AJ, Jones JL (1989) Dietary phosphorus for laying hens. In Cole DJ, Haresign W (Eds.) Recent Developments in Poultry Nutrition. Butterworths. London, UK. pp. 231-238.        [ Links ]

14. Jongbloed AW, Mroz Z, Kemme PA (1992) The effect of supplementary Aspergillus niger phytase in diets for pigs on concentration and apparent digestibility of dry matter, total phosphorus, and phytic acid in different sections of the alimentary tract. J. Animal Sci. 70: 1159-1168        [ Links ]

15. Kirby L, Nelson T (1988) Total and phytate phosphorus content of some feed ingredients derived from grains. Nutr. Reports Intl. 37: 277-280.        [ Links ]

16. Morris ER, Ellis R (1976) Isolation of monoferric phytate from wheat bran and its biological value as an iron source to the rat. J. Nutr. 106: 753-760.        [ Links ]

17. NRC (1998) Nutrient Requirements of swine. 9th ed. National Research Council. National Academy Press. Washington, DC, USA. 189 pp.        [ Links ]

18. Nelson TS, Ferrara LW, Storer NL (1968) Phytate phosphorus content of feed ingredients derived from plants. Poultry Sci. 47: 1372-1374.        [ Links ]

19. Nys Y, Frapin D, Pointillart A (1996) Occurrence of phytase in plants, animals and microorganisms. In Coelho MB, ET Kornegay (Eds) Phytase in Animal Nutrition and Waste Management. BASF. Mount Olive, NJ, USA. pp 213-236.        [ Links ]

20. O'Dell BL, DeBoland AR, Koirtyoham SR (1972) Distribution of phytate and nutritionally important elements among the morphological components of cereal grains. J. Agric. Food Chem. 20: 718-721.        [ Links ]

21. Ogawa M, Tanaka K, Kasai Z (1977) Note on the phytin-containing particles isolated from rice scutellum. Cereal Chem. 54: 1029-1034.        [ Links ]

22. Perney KM, Cantor AH, Stranw ML, Herkelman KL (1993) The effect of dietary phytase on growth performance and phosphorus utilization of broiler chicks. Poultry Sci. 72: 2106-2114.        [ Links ]

23. Pointillart A (1994) Phytates, Phytase: leur importance dans l´alimentation des monogastriques. Prod. Anim. 7: 29-39.        [ Links ]

24. Ranhotra GS, Loewe RJ (1975) Effect of wheat phytase on dietary phytic acid. J. Food Sci. 40: 940-949.        [ Links ]

25. Raun A, Cheng E, Burroughs W (1956) Phytate phosphorus hydrolysis and availability to rumen microorganisms. J. Agr. Food Chem. 4: 869-871.        [ Links ]

26. Ravindran V, Bryden WL, Kornegay ET (1995) Phytates: Occurrer bioavailability and implications in poultry nutrition. Poultry Avian Biol. Rev. 6: 125-143.        [ Links ]

27. Sauvant D, Pérez JM, Tran G, (2002) Tables de composition et de valeur nutritive. In Sauvant D, Pérez JM, Tran G (Eds.). Tables de composition et de valeur nutritive des matieres premieres destinees aux animaux d‘elevage. Paris. France. Pp. 73-289.        [ Links ]

28. Steel RGD, Torrie JH (1988) Principles and Procedures of Statistics. A Biometrics Approach. 2nd ed. McGraw-Hill. New York, USA. 622 pp.        [ Links ]

29. Thompson DB, Erdman JW (1982) Phytic acid determination in soybeans. J. Food Sci. 47: 513-516.        [ Links ]

30. Usayran N, Balnave D (1995) Phosphorus requirements of laying hens fed on wheat-based diets. Br. Poult. Sci. 36: 285-301.         [ Links ]