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Effect of germination on the phytase activity, phytate and total phosphorus contents of

some Nigerian grown cereals

Marshall A. Azeke*, Samuel J. Egielewa, Mary U. Eigbogbo, Godwin I. Ihimire

Department of Biochemistry,

Ambrose Alli University,

P. M. B. 14

Ekpoma, Nigeria

*Corresponding Author, Tel: +234 805 6174857; Fax: +234 1 2637021,

E-mail: m_azeke@yahoo.com

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Abstract

The level of phytase activity, phytate and phosphorus contents of five Nigeria grown cereal

grains were determined. The cereals screened were rice (Oriza sativa), maize ( Zea mays),

millet (Panicum miliaceum), sorghum (Sorghum bicolor) and wheat (Triticum aestivum). The

cereal grains were also germination for 10 days in order to assess the responses of the

aforementioned parameters. Phytase activities were generally high (0.21 0.67 U g-1) in all

samples. Phytate content ranged between 5.58 and 6.23 g/kg-1

while total phosphorus content

ranged between 3.33 and 4.34 g/kg-1

. The level of phytase activity reached its maximal value

after seven (16-fold), six (5-fold), five (7-fold), seven (3-fold) and eight (6-fold) days of

germination for rice, maize, millet, sorghum and wheat respectively. Following this initial

period, phytase activity declined slightly (P < 0.05). The increase in phytase activity during

germination was accompanied by a significant reduction in phytate (P < 0.05) and a small but

significant increase in total phosphorus. The increase in phytase activity and the

accompanying decrease in phytate content could have positive implication for the nutrition of

poultry and ruminants and for the environment.

Keywords: Cereal, phytase, phytate, phosphorus, germination.

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INTRODUCTION

Cereals and legumes form a significant portion of the food supply for humans and other

animals, as they are a major source of carbohydrates, proteins, lipids and minerals (Katina et

al., 2005). The presence of antinutrients such as phytate has been a major limiting factor to

the extensive utilization of these plant resources (Al-Numair et al., 2009). Phytate, myo-

inositol (1,2,3,4,5,6) hexakisphosphate, is present in high concentrations in many food items

derived from plants. It is the major storage form of phosphorus in mature grains and legumes

(Kumar et al., 2010). Its hydrolysis is catalysed by the enzyme phytase (myoinositol

hexaphosphate phosphohydrolase) to inositol and orthophosphate. About 60

90 % of total

phosphorus in plant feedstuffs is bound as phytate phosphorus (Loewus, 2002). Phosphorus,

in this form, is not utilised by human beings, birds or agastric animals because they lack the

sufficient endogenous intestinal digestive enzyme phytase which releases orthophosphate

from the phytate molecule in the gastrointestinal tract (Holm et al., 2002). The unabsorbed

phytate is excreted and contributes to phosphate pollution in water bodies downstream of

agriculturally intensive areas (Sharpley, 1999). Furthermore, phytate works in a broad pH-

region as a highly negatively charged ion and therefore its presence in the diet has a negative

impact on the bioavailability of divalent and trivalent mineral ions such as Zn2+, Fe2+/3+, Ca2+,

Mg2+

, Mn2+

and Cu2+

(Fredlund et al., 2006; Lopez et al., 2002; Lnnerdal, 2002). The need

to maintain sufficient dietary phosphorus levels while reducing phosphorus excretion in

poultry manure has led to an increase in the application of phytase to poultry feed (Hatten et

al., 2001). The phytases (myo-inositol hexakisphosphate phosphohydrolases) are a subfamily

of the high-molecular-weight histidine acid phosphatases. They catalyse the phosphate

monoester hydrolysis of phytate,which results in the stepwise formation of myo-inositol

pentakis-,tetrakis-, tris-, bis-, and monophosphates, as well as the liberation

of inorganic

phosphate (Sathe and Reddy, 2002). The use of phytase reduces phosphorus excretion in

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poultry manure by allowing the birds to utilize more of the phytate phosphorus (Sharpley,

1999). The use of phytase may also free cations and proteases bound in phytate phosphorus

complexes and improve many production parameters and body structure characteristics in

broilers and laying hens, such as body weight, bone ash content, feed consumption, egg

weight, and egg shell quality (Zyla et al., 1999; Hatten et al., 2001). The phytases used for

this purpose are usually of microbial origin and the technology involved in the extraction,

purification and thermo-stabilization of the enzymes is still beyond the reach of developing

countries especially in Africa. High native phytase activities are present in cereals and cereal

by-products, whereas lower activities have been reported for legume seeds (Viveros et al.,

2000; Zimmermann et al., 2002; Steiner et al., 2007). However, no studies have been carried

out on the distribution of phytase activity in cereals grown in Nigeria. The current study was

conducted to assess phytase activities and the contents of phytate and total phosphorus in

samples of different Nigerian grown cereal grains as influenced by sprouting.

MATERIALS AND METHODS

Samples

Seed samples harvested in 2008 were purchased from local farmers in Edo State in Mid-

Western Nigeria. The legumes screened are: rice (Oriza sativa), maize ( Zea mays), millet

(Panicum miliaceum), sorghum (Sorghum bicolor) and wheat (Triticum aestivum).

Surface Sterilization and Germination of seeds

For surface sterilization, seeds were soaked in 0.5% NaOCl for 3 minutes, rinsed very well

with some quantity of sterile water and soaked again in of 0.75% H2O2 for 2 minutes and

again rinsed thoroughly with sterile water. After draining, the grains were spread on plastic

trays overlaid with wet tissue papers. The seeds were then allowed to germinate in disinfected

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dark cupboards for 0 10 days with daily harvestings. The seeds were rinsed once a day with

sterile water, which was removed completely. For the determination of Phytate and total

phosphorus, the harvested samples were oven dried at 60 C for about 18 h, allowed to cool

and then milled. Freshly harvested samples were used for phytase activity determination.

Portions of freshly harvested and the dried, milled samples were used for moisture

determination.

Enzyme preparation

The preparation of the crude enzyme was basically as described by Senna et al. (2006). Fresh

samples of ungerminated and germinated seeds were homogenized in 0.1 M sodium acetate

buffer (pH 5.0). The homogenate was centrifuged at 12,000 g for 5 min. The pellets were

discarded and supernatants were used for enzyme assays, described as total homogenate. All

procedures were carried out at 4 C.

Enzyme activity

Enzyme activity was determined at 40 C. The assay mixture consisted of 350 L of 0.1 M

sodium acetate buffer, pH 5.0 and 100 L of 2 mM sodium phytate. This mixture was pre-

incubated for 10 min at 40C and the enzymatic reactions were started by adding 100 L of

the crude enzyme to the pre-incubated assay mixtures. After incubation at 40 C for 30 min,

the liberated phosphate was measured by using the ammonium molybdate method (Heinonen

and Lahti, 1981). Added to the assay mixture were 1.5 mL of a freshly prepared solution of

acetone/5 N H2SO4/10 mM ammonium molybdate (2:1:1 v/v) and 100 L of 1.0 M citric acid.

Any cloudiness was removed by centrifugation prior to measurement of the absorbance at 355

nm. To calculate the enzyme activity, a calibration curve was produced over the range of 5-

600 nmol of phosphate ( = 8.7 cm2

mol-1

). Activity (units) was expressed as 1 mol of

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phosphate liberated per minute. Blanks were run by adding the ammonium molybdate

solution prior to adding the enzyme to the assay mixture.

Phytate Extraction and Determination

Phytate extraction and determination was essentially according to the method of Wheeler and

Ferrel (1971) with slight modifications. Instead of extraction using 3 % TCA, 1.2 M HCl

containing 10 % Na2SO4 was used because of excessive foaming with TCA (Azeke et al.,

2005).

Total Phosphorus Determination

The method of Jacobs (1999) was applied for the determination of total phosphorus. This

involved digestion of seeds (5 g) with 30 ml conc. HNO3 and 20 ml conc. H2SO4.

Statistics

Significance of difference was tested by analysis of variance and Tukey-Kramer test using the

GraphPad InStat version 2.04a (GraphPad Software Inc, San Diego, California, USA,

www.graphpad.com)

RESULTS AND DISCUSSION

Distribution of phytase activity, phytate and total phosphorus in cereal grains

The phytase activities, phytate and total phosphorus contents of some Nigerian grown cereal

grains are shown in table 1. With the exception of maize and sorghum significant differences

(P < 0.05) were observed in the phytase activities (0.21 0.67 U g-1) of the cereal grains

screened. Steiner et al. (2007) reported a range of 0.4 6.0 U g-1

for oats, barley, triticale, rye

and wheat. They reported 2.8 U g-1