ABSTRACT

A technique was developed to produce crude microbial phytase (CMPhy) enzyme form
Aspergillus niger, with the objective to enhance phytate phosphorus (pP) availability in corn
and soybean meal based broiler diets. The results of in vitro experiments showed that CMPhy
enzyme was most active at pH 2.0 and 5.0, as it results in the release of about 79.0% to
88.0% inorganic phosphorus (iP) as a percentage of total phosphorus (tP) from ground corn
and soybean meal. In vitro pP hydrolysis was 87.14% in ground corn at 40C and 5.0-5.5 pH
after an incubation period of 8 hrs. with a ratio of 1:1 (CMPhy enzyme:substrate). The pP
hydrolysis in soybean meal was 81.25% by applying the same conditions as for ground corn
except an incubation period of 10 hrs. When CMPhy enzyme was added to low-P diets for
broilers the availability of P increased (63.35%) as compared to the diet with normal-P
contents (43.31%) supplied by dicalcium phosphate. Similarly P excretion was reduced to
20.04% units by CMPhy enzyme addition to low-P diet than normal-P diet. Microbial
phytase addition to low-P diets, without affecting the performance reduced the P excretion in
the dropping thus minimising the P pollution hazards.
INTRODUCTION
Phytic acid can bound the free P and forms the compound phytate that reduces the
availability of phosphorus (P) for monogastric animals. From two-thirds of the P in cereal
grains and oil seed meals poultry can utilise only one-third of the P because they lack the
phytase enzyme necessary to hydrolyse pP. Due to this reason protein, phosphorus and other
minerals bound to phytic acid are excreted as such in the faeces of the birds. The excess of P
and nitrogen excretion will not only causes a great threat to our environment but also results
in an increase in feed cost due to supplementation of feeds with inorganic phosphorus salts.
The dire need to find an economical source of P for poultry rations prompted researchers to
make available pP in the body of birds. This study was thus conducted with the objectives to
produce phytase enzyme from a fungal species Aspergillus niger and to determine its efficacy
for in vitro and in vivo dephosphorylation of corn and soybean meal

MATERIALS AND METHODS

Enzyme Production and Assay
The enzyme phytase was produced from a fungus Aspergillus niger. Which was obtained
from National Institute of Biotechnology and Genetic Engineering (NIBGE), Faisalabad,
Pakistan. The phytase was produced through 10 days fermentation at 28C on corn starch
based medium containing (g/l): corn starch 91, glucose.1H2O 38, KNO3 12.0, FeSO4.7H2O
0.20, KCl 0.60, MgSO4.7H2O 0.60. The pH was maintained at 4.5 by using 1N H2SO4, or
1N NaOH. After 10 days fermentation, the broth was subjected to a series of filtration by
using cotton cloth and finally filter paper, until a clear filtrate was obtained. This filtrate in
liquid form was then used as the crude phytase enzyme. The assay for phytase activity was

carried out by using the method by Simons et al. (1990) and the amount of free phosphate
was determined by method of Fiske and Subbarow (1925). One unit of phytase is the activity
that liberates 1mmol phosphate from phytic acid in 1 min at pH 5.5 and 40C. Activity of the
enzyme was found to be 1.075 phytase units (PU) per min per ml of the crude culture filtrate
at pH 5.5 and 40C.
In-vitro and in-vivo Enzyme Efficacy
The in vitro enzyme efficacy was measured after the optimum conditions viz.
enzyme:substrate ratio, incubation time, temperature and pH, were determined for the
optimum release of free phosphate from ground corn (GC) and soybean meal (SBM). After
determining these conditions the GC and SBM were treated with predetermined level of
crude phytase enzyme for the degradation of pP. After drying the GC and SBM in the oven,
they were used in three experimental broiler rations designated as control, N+Phyt and
L+Phyt. Control and N+Phyt diets contained the untreated and treated GC and SBM,
respectively, and were prepared according to the National Research Council (NRC,1994)
requirement for available P (non-phytate P, nP). Low-P plus phytase diet was prepared to
contain the enzyme treated GC and SBM, with the content of nP (0.36%), 20% less than the
NRC (1994) recommendations.
The three experimental diets were fed to ninety day-old Hubbard broiler chicks randomly
divided into nine experimental units of ten chicks each. Each diet was given to three
experimental units of ten chicks each. Droppings collected per pen during the last three days
of the fourth week (i.e., days 26, 27 and 28), were thoroughly mixed and weighed.
Representative samples were dried in the oven at 60C and ground to pass through a 1-mm
sieve (Yi et al., 1996). These ground samples and feed samples were used for the analysis of
P (AOAC, 1984) and Ca (Richard, 1954). All calculations were expressed on a DM basis.
Feed consumption by the birds was recorded on a pen basis at weekly intervals and daily for
the last 3 days of week 4 (i.e., days 26-28). Birds per pen were weighed at weekly intervals
and the data was analysed by using analysis of variance technique with completely
randomised design. Treatment means were compared by using Duncan's multiple range test
(Steel and Torrie, 1980).
RESULTS AND DISCUSSION
Enzyme Production and Assay
Enzyme phytase production was measured in terms of phytase activity, which was found to
be 0.248, 0.279, 0.333, 0.384, 0.698, 0.885, 1.075 and 1.083, Phytase Units/ml/min. after 4,
5, 6, 7, 8, 9, 10, and 11 days of fermentation, respectively. A linear increase in phytase
production was found with the days of fermentation. Maximum phytase activity (1.086) was
obtained after 11 days of fermentation; however, the phytase activity was non-significant
(P<0.05) after 10, and 11th day of fermentation Simons et al. (1990). Based on these

observations, a time period of 10 days fermentation was used for the production of phytase
enzyme.
Phytase activity was found to be 1.075 Phytase Units/ml in 1 min at 40C and pH
5.5, by using phytic acid as substrate. Results of the present study are in line with Shieh and
Ware (1968), who reported phytase activity (mmoles P/min) of extracellular phytase
produced by different strains of A. niger (except soil isolates) in the crude culture filtrate
from 0.268 to 1.129 PU/ml/min, at 37.5C, using calcium phytate as substrate. However, the
phytase activity is found to be lower as compared to the results Nelson et al. (1971), who
reported an activity of 950 PU/gm in the acetone dried enzyme preparation from A. ficuum
NRRL3135. This high phytase activity (Nelson et al., 1971) may be due to the use of
different variety of Aspergillus for enzyme production. The most possible reason of high
phytase activity is due to the physical nature of the enzyme, because enzyme preparation
become concentrated as the crude culture filtrate get dried.
in vitro dephosphorylation of corn and soybean meal
The summary of conditions for optimum release of iP has been given in table 1.
These optimised conditions were then applied for maximum release of iP from GC and SBM
used in experimental rations and the results obtained have been give in table 2.
The results (table 2) showed that before phytase enzyme treatment, total phosphorus (tP),
inorganic phosphorus (iP) and phytate phosphorus (pP) contents were 0.28, 0.08, 0.21 and
0.50, 0.20, 0.32% in GC and SBM, respectively. The degradation of pP after phytase enzyme
treatment was 87.14 and 81.25% in GC and SBM, respectively. The results of optimised
conditions and pP hydrolysis by phytase enzyme substantiate the findings of Han and Wilfred
(1988) and Sandberg et al. (1996). Han and Wilfred (1988) reported that phytase from A.
ficuum hydrolyzed about 85% pP in soybean meal at 50C and pH 4-5.5. Sandberg et al.
(1996) reported that phytase from A. niger showed pH optima at pH 2.0 and 6.0. They further
reported that phytase activity occurred at all pH values between 1.0 and 7.5.
In Vivo Dephosphorylation Of Corn And Soybean Meal
Birds' performance
The effects of phytase supplementation on the performance of birds at the end of the
experiment (28 days) are given in table 3. Compared to the control diet, the normal-P phytase
(1.075 PU/gm) treated diet significantly (P<0.05) increased the BW gain by 2.65%. The gain
of the chicks fed on the low-P plus phytase diet was comparable to that obtained on the
control diet (Table 3), which contained higher levels (1.1 vs 0.6%) of dicalcium phosphate (a
source of inorganic P) to satisfy the birds requirement of non-phytate-P. Feed intake followed
a similar pattern to that of BW gains (Table 3). Added phytase in both normal-P and low-P
diets increased the feed intake yet it was even higher (by 5.37%) with the normal-P diet. Feed
intake was found to be significantly (P<0.05) higher with he normal-P plus phytase diet at 28

days, than with the other two diets. It becomes evident that improved gains due to
supplemental phytase were primarily because of the increased feed intake (Table 3). Thus
dietary phytase supplementation did not affect the feed conversion ratio for either treatment.
However, FCR was better with the normal-P plus phytase (1.34) and low-P plus phytase
(1.33) diets than with the control diet (1.36), yet the differences were statistically nonsignificant. The results revealed that supplementation of broiler diets with phytase at 1.075
PU/g of feed can reduce the tP and nP requirements by 15 and 20% respectively, from the
value recommend by NRC (1994), without having any adverse effect on BW gain (Table 3).
Similar improvements in BW gain of broiler chicks with phytase supplementation of diets
have been reported by Simons et al. (1990). The improvement in growth performance of
chicks fed on phytase treated diets may be attributed to the release of mineral from the
phytate mineral complex and the utilisation of inositol by the bird (Simons et al. 1990) or
increased starch digestibility. The same can also be due to the increased availability of
proteins, because phytate also complexes with proteins, making them less available.
Terjemahan
Produksi enzim dan Assay
Enzim phytase dihasilkan dari jamur Aspergillus niger. Yang diperoleh dari National Institute of
Bioteknologi dan Rekayasa Genetika (NIBGE), Faisalabad, Pakistan. Phytase yang dihasilkan melalui
fermentasi 10 hari pada suhu 28 C pada media pati jagung berbasis mengandung (g / l): pati jagung
91, glucose.1H2O 38, KNO3 12,0, FeSO4.7H2O 0.20, KCl 0.60, MgSO4.7H2O 0.60. PH
dipertahankan pada 4,5 dengan menggunakan 1N H2SO4, atau 1N NaOH. Setelah fermentasi 10 hari,
kaldu menjadi sasaran serangkaian penyaringan dengan menggunakan kain katun dan akhirnya
menyaring kertas, sampai filtrat yang jelas diperoleh. Filtrat ini dalam bentuk cair kemudian
digunakan sebagai enzim phytase mentah. Assay untuk kegiatan phytase dilakukan dengan
menggunakan metode oleh Simons et al. (1990) dan jumlah gratis fosfat ditentukan dengan metode
Fiske dan Subbarow (1925). Satu unit phytase adalah kegiatan yang membebaskan 1mmol fosfat dari
asam fitat dalam 1 menit pada pH 5,5 dan 40 C. Aktivitas enzim yang ditemukan menjadi 1.075 unit
phytase (PU) per menit per ml filtrat kultur mentah pada pH 5,5 dan 40 C.

In-vitro dan in-vivo Enzyme Efikasi

Kemanjuran enzim in vitro diukur setelah optimum kondisi yaitu. enzim: rasio substrat,
waktu inkubasi, suhu dan pH, ditentukan untuk rilis optimal gratis fosfat dari tanah jagung
(GC) dan bungkil kedelai (SBM). Setelah menentukan kondisi ini GC dan SBM diperlakukan
dengan tingkat yang ditentukan sebelumnya enzim phytase mentah untuk degradasi pP.
Setelah mengeringkan GC dan MBS di oven, mereka digunakan dalam tiga ransum broiler
eksperimental ditetapkan sebagai kontrol, N + Phyt dan L + Phyt. Kontrol dan N + Phyt diet
berisi GC diobati dan dirawat dan MBS, masing-masing, dan dibuat menurut National
Research Council (NRC, 1994) persyaratan untuk P tersedia (non-fitat P, nP). Low-P
ditambah diet phytase siap untuk mengandung enzim diperlakukan GC dan SBM, dengan
kandungan nP (0,36%), 20% kurang dari NRC (1994) rekomendasi.

Produksi enzim dan Assay

Produksi phytase enzim diukur dalam hal aktivitas phytase, yang ditemukan menjadi 0,248, 0,279,
0,333, 0,384, 0,698, 0,885, 1,075 dan 1,083, Fitase Unit / ml / menit. setelah 4, 5, 6, 7, 8, 9, 10, dan 11
hari fermentasi, masing-masing. Peningkatan linear dalam produksi phytase ditemukan dengan hari
fermentasi. Aktivitas phytase maksimum (1,086) diperoleh setelah 11 hari fermentasi; Namun,
aktivitas phytase yang tidak bermakna (P <0,05) setelah 10, dan hari ke-11 fermentasi Simons et al.
(1990). Berdasarkan pengamatan ini, jangka waktu fermentasi 10 hari digunakan untuk produksi
enzim phytase
Kegiatan fitase ditemukan menjadi 1.075 Fitase Unit / ml dalam 1 menit pada 40 C dan pH 5,5,
dengan menggunakan asam fitat sebagai substrat. Hasil penelitian ini sejalan dengan Shieh dan Ware
(1968), yang melaporkan aktivitas phytase (mmol P / min) dari phytase ekstraseluler yang dihasilkan
oleh strain yang berbeda dari A. niger (kecuali tanah isolat) dalam filtrat kultur mentah dari 0,268 ke
1,129 PU / ml / menit, pada 37,5 C, dengan menggunakan kalsium fitat sebagai substrat. Namun,
aktivitas phytase ditemukan lebih rendah dibandingkan dengan hasil Nelson et al. (1971), yang
melaporkan kegiatan dari 950 PU / gm di aseton kering persiapan enzim dari A. vakum NRRL 3135.
Kegiatan phytase tinggi ini (Nelson et al., 1971) mungkin karena penggunaan varietas yang berbeda
dari Aspergillus untuk produksi enzim. Yang paling mungkin alasan aktivitas phytase tinggi karena
sifat fisik enzim, karena persiapan enzim menjadi terkonsentrasi sebagai filtrat kultur mentah
mendapatkan kering.