IN VITRO ENZYME ASSAY OF COMMERCIAL FEED ENZYMES*
A.Bharathidhasan1, D.Chandrasekaran2, A.Natarajan3, R.Ravi4, K.Viswanathan5 and S.Ezhilvalavan6
Tamilnadu Veterinary and Animal Sciences University,
Madhavaram Milk Colony, Chennai-600051.
ABSTRACT
A study was conducted to assess the potency of different commercial feed enzyme preparations
available in the market by in vitro. The cellulase activity of the commercial feed enzyme
preparations was ranged from 0.41 IU/g to 145.84 IU/g in Dinitro salicylic acid method. In
Glucose oxidase-peroxidase method, the cellulase activity was ranged from 0.26 IU/g to
20.63 IU/g at 42o C for 2 hours incubation and from 0.6 IU/g to 52.21 IU/g at 50o C for 30
minutes incubation. The xylanase, pectinase, protese, amylase and phytase activities of the
commercial feed enzyme preparations ranged between 35.09-241.35 IU/g, 76.96-249.30
IU/g, 26.05-254 IU/g, 25.25-904.87 IU/g and 0.9-115.84 IU/g respectively. The in vitro
evaluation of an enzyme A with sunflower meal (SFM) showed that the increasing the level
of enzyme addition from 0 to 15.0 mg/10 g of SFM enhanced the release of reducing sugars,
glucose and inorganic phosphorus.
Key words: Enzyme activity, cellulase, xylanase, pectinase and phytase activity
INTRODUCTION the digestibility of the NSP, utilization of these
components to the desired level in the
The major part of poultry feed contains birds.Nowadays various commercial feed enzyme
considerable amount of Non-starch polysaccharides preparations are available in the market. These feed
(NSP) and phytates. These components reduce the enzymes are full of crude enzyme preparations.
energy utilization, protein digestion and decrease Hence, the present study was carried out to assess
the absorption of other nutrients. The ingestion of the actual potency of various feed enzymes.
soluble NSP like b-glucans increased the digesta
viscosity (Choct and Annison, 1992) and depressed MATERIALSAND METHODS
the growth rate (White et al., 1983) in broiler chicken.
Thirteen commercial feed enzyme (Enzyme
Phytate or phytic acid is a naturally occurring organic
A to L) preparations marketed by different
complex found in plants and 60-80 % of phosphorus
companies were collected and evaluated interms of
found in the cereal grains, oilseeds as phytic acid
cellulase, xylanase, pectinase, protease, amylase
(Simons and Versteegh, 1990). Phytate forms stable
and phytase activities by in vitro technique
complex with minerals like Ca, Zn, Cu etc (Erdman,
1979), complex with protein (Cheryan, 1980) in 1. Cellulase, Xylanase, Pectinase Assay
poultry gut and there by reducing their utilization. a.Use of Dinitro Salicylic acid method (DNSA) for
Hence, supplementation of enzymes will enhance determination of reducing sugars
*Part of M.V.Sc thesis of the first author submitted to Tamilnadu Veterinary and Animal Sciences
University, Chennai-51. 1Asst. Professor, University Research Farm, 2Professor and Head, Dept of
Animal Nutrition, 3Associate Professor, 4Professor and Head, AFAQCL, 5 Dean, VCRI, Namakkal, 6Asst.
Professor, DEE.
20 Tamilnadu J. Veterinary & Animal Sciences 5 (1) 20-24, Januaryr - February 2009
Bharathidhasan et.al.,
10 g crude enzyme sample was taken in 4. Assay of in vitro Enzyme -A activity with
100 ml of 2 % calcium chloride solution and mixed sunflower meal (SFM)
thoroughly for one hour in a mechanical shaker, The Enzyme- A that showed the highest
centrifuged at 2500 rpm for 10 minutes and the potency in vitro evaluation using specific substrate
supernatant enzyme extract was diluted at 1:10 ratio was selected and evaluated with sunflower meal
with 0.2 M acetate buffer and used for further assay. (SFM) as substrate. Five levels of enzyme 0, 2.5, 5.0,
The substrate carboxy methyl cellulose (CMC) or 10.0, 15.0 mg/10 g of SFM was taken in 100 ml beaker
xylan or pectin was prepared by adding 1g in 100 ml with 40 ml of 0.2 M acetate buffer and was incubated
of 0.2 M acetate buffer (pH-5.0) and dissolved by at 42o C for 2 hours with periodical shaking. After
mixing it in a mechanical shaker for minimum period incubation the content was mixed well and allowed
of 12 hours, centrifuged and the supernatant was to stand for 10 minutes, then the contents was
used for further assay. 0.1 ml of enzyme extract was centrifuged and the supernatant was used for
incubated with 0.9 ml of substrate (CMC or xylan or glucose estimation using GOD-POD method (Tiets,
pectin) at 50o C for 30 minutes. After incubations, 1976) reducing sugars (Miller, 1959) and inorganic
the reducing sugars were estimated by phosphorus by Fiske and Subba Rao method
Dinitrosalicylic acid method (Miller, 1959). One IU (Varley, 1965).
of cellulase or xylanase or pectinase activity is
defined as the amount of enzyme that liberates 1 RESULTS AND DISCUSSION
mM of reducing sugars per minute under the assay
condition. In enzyme assay, the cellulase, xylanase,
b.Specific test for glucose(GOD-POD method) pectinase, protease, amylase and phytase activity
In the DNSA method along with reducing of various commercial enzyme preparation are
sugars, glucose, aldehyde groups, disaccharide presented in Table 1 and cellulase activity – specific
such as cellobiose, xylobiose and dextrin etc are test for glucose (GOD-POD method) in Table 2.
being released which are not fully utilized by the
birds. So, specific test for glucose was done using The cellulase activity (IU/g) ranged from minimum
glucose oxidase peroxidase method (GOD-POD). of 0.41 IU/g in enzyme L to maximum of 145.84 IU/g
After incubation of 0.1ml of 10% enzyme with 0.9 ml in Enzyme A by DNSA method. In GOD-POD
of 1 % substrate at 42o C for 2 hours (to simulate method the cellulase activity with respect to release
the condition in poultry gut system) or 50o C for 30 of glucose in vitro was also highest 20.43 IU/g in
minutes, 20 ml was taken and used for estimation of 42 o C for 2 hours and 52.21 IU/g in 50o C for 30
glucose (Tiets, 1976). minutes in enzyme-A, where as other commercial
2. Protease and Amylase assay enzyme preparations had a very low activity 0.26
Protease and amylase activities were IU/g in enzyme B to 2.20 IU/g in enzyme D at 42o C
estimated by using the method of Kunitz (1947) and for 2 hours and 0.60 IU/g in enzyme B to 7.22 IU/g in
Smith and Roe (1949) respectively. enzyme D at 50o C for 30 minutes. The xylanase
3. Phytase assay activity (IU/g) was estimated to maximum of 241.35
The phytase activity was calculated based in Enzyme A and the minimum value of 35.09 in
on the amount of phosphorus liberated per minute enzyme J. In the pectinase activity (IU/g), Enzyme
under assay condition (Heinonen and Lahti.,1981) B, L and E recorded 249.30, 248.28 and 245.09 IU/g
.One unit of phytase activity is defined as the respectively where as other enzyme recorded lower
amount of enzyme that liberates 1mMol of inorganic activity.
phosphorus per minute under assay condition.
Tamilnadu J. Veterinary & Animal Sciences 5 (1) 20-24, Januaryr - February 2009 21
In vitro enzyme assay of commercial feed enzymes
The highest protease activity was observed in an increase in vitro release of phosphate from
enzyme K and D (254 and 242.72 IU/g) where as phytate in maize and soyabean meal incubated with
other enzyme had lower protease activity. The microbial phytase.
highest amylase (IU/g) activity was noticed in From this study it was found that the
enzyme –E (904.87) followed by enzyme-K (792.4), potency of various commercial feed enzymes were
enzyme–A (777.72) and enzyme G (703.63). The estimated. It is a useful indicator to identify the
phytase activity (IU/g) in phytase specific enzyme quality of crude enzyme preparations used in poultry
preparation was higher in enzyme B1-115.84. ration. Also, it might be very helpful when
However, 32.95 IU/g phytase activity was recorded formulating the least cost ration contains feed
in enzyme –A when compared to other enzyme ingredients with high levels of NSP and phytate
preparations which showed little phytase activity phosphorus
(0.90 to 12.86 IU/g).
REFERENCES
The variation in enzyme activity could be
mainly due to the crude enzymes of different Cheryan,M..(1980). Phytic acid interactions in food
commercial preparations and the environmental systems. CRC. Critical Reviews in Food Sci.
factors including temperature, pH, relative humidity and Nutr.,13 : 297-302.
etc may also influence the activities of feed enzymes.
Choct, M. and Annison,G.(1992).The inhibition of
ASSAY OF INVITRO ENZYME ACTIVITY WITH nutrient digestion by wheat pentosans. Br. J.
SFM Anim. Nutr., 67 : 123-132.
The mean value of glucose (mg %),
reducing sugars (mg %) and inorganic phosphorus Erdman, J.W.Jr.(1979). Oilseed phytates nutritional
(%) as influenced by various levels of enzyme – A implications. Journal of American Oil Chemists
in SFM are presented in Table 3. The linear increase Society., 56: 736-741.
in the amount of glucose released viz. 4.91, 6.69,
Heinonen Jukka K. and Reijo Lahti,J.(1981). A new
12.07 and 15.03 % in 2.5, 5.0, 10.0, 15.0 mg/10 g of
convenient calorimetric determination of
SFM respectively over control. The amount of
inorganic orthophosphate and its application
reducing sugar was 1365.62 mg % in control which
to the assay of inorganic pyrophosphatase.
increased by 2.45, 4.44, 5.67 and 7.08 mg % by
Analytical Biochemistry., 113: 313-317.
addition of enzyme –A at 2.5, 5.0, 10.0 and 15 mg/10
g of SFM respectively. Kunitz,M .(1947). Crystalline soyabean trypsin
inhibitor II. General properties. Journal of
The results agree with earlier findings of
General Physiology., 30: 291-300.
Slominski and Campbell (1990) who observed an
increased hydrolysis of polysaccharides of canola Miller, Gail Lorenz.(1959). Use of dinitrosalicylic acid
meal when enzyme added at 1 % level. reagent for determination of reducing sugars.
Analytical Chemistry., 31: 426-428.
The amount of inorganic P (%) released in
control was 0.052 which increased to 0.056, 0.066, Simons, P.C.M. and Versteegh, H.A.J.(1990).
0.074 and 0.082 % by addition of 2.5, 5.0, 10.0, 15.0 Improvement of phosphorus availability by
mg/10 g of SFM of enzyme A respectively and microbial phytase in broiler and pigs. Br. J.
increase over the control was 7.14, 21.21, 29.73 and Nutr., 64: 525-540.
36.59 % respectively. A similar finding was reported
by Simons and Versteegh (1990) who observed that
22 Tamilnadu J. Veterinary & Animal Sciences 5 (1) 20-24, Januaryr - February 2009
Bharathidhasan et.al.,
Slominski, Bogden A. and Cloyd Campbell,D.(1990). Varley, H.(1965). Practical Clinical Biochemistry. 4th
Non starch polysaccharide of canola meal, edn. pp 158-467.Arnold-Heinemann
quantification, digestibility in poultry and Publishers (India) Pvt Ltd, NewDelhi.
potential benefit of dietary enzyme
supplementation, J.Sci. Food Agric., 53: 175- White, W. B., Bird, H.R., Sunde,M.L and
184. Marlett,A.(1983). Viscosity of b-D-glucan as
a factor in the enzymatic improvement of
Smith B.W. and Joseph H. Roe, 1949. A photometric barley for chicks. Poult. Sci., 62: 853-862.
method for the determination of a-amylase in
blood and urine, with use of the starch-
iodine color. J.Biological Chem., 179: 53-59.
Tiets, N.W.(1976). In Clinical Guide to Laboratory
Tests, W.B. Saunders Co., Philadelphia page:
238.
Table 1
Cellulase, xylanase, pectinase, protease, amylase and phytase activity (IU/gm) of commercial enzyme
preparations
Enzymes C ellulase Xylanase Pe ctin as e Proteas e Amylase Phytase
A 145.84 241.35 9 8.10 74.0 0 777.72 32.95
B 67.26 132.40 249.30 45.2 5 140.75 1.52
C 19.60 92.04 163.63 26.0 5 67.30 0.90
D 29.64 101.18 8 4.92 242.7 2 137.90 4.56
E 89.44 181.36 245.09 82.2 9 904.87 1.54
F 34.28 100.00 137.87 50.2 6 25.25 1.18
G 31.99 132.00 140.12 88.8 6 703.63 *
H 6.46 40.70 115.25 191.1 0 78.50 *
I 20.07 129.08 7 9.22 38.0 0 60.51 *
J 1.35 35.09 6 8.88 94.1 0 67.15 *
K 5.56 85.69 7 6.96 254.0 0 792.40 12.86
L 0.41 94.67 248.28 96.0
* The OD value was equal to the 0
blank 85.00 *
B1** - - -
** Phytase specific enzyme - - 115.84
Tamilnadu J. Veterinary & Animal Sciences 5 (1) 20-24, Januaryr - February 2009 23
In vitro enzyme assay of commercial feed enzymes
Table 2
Cellulase activity-specific test for glucose (GOD-POD method)
The OD value was equal to the blank
Table 3
Assay of in vitro enzyme activity with sunflower meal
Glucose (mg %) Reducing sugar
Level of enzyme/10 (mg %) Inorganic
g of SFM (Specific for glucose phosphorus (%)
test) (DNSA method)
0 mg 138.90 ± 38.20 1365.62 ± 9.07 0.052 ± 0.017
2.5mg 146.07 ± 39.31 1399.85 ± 6.11 0.056 ± 0.019
5 mg 148.86 ± 39.04 1429.01 ± 16.13 0.066 ± 0.02
10 mg 157.96 ± 36.94 1447.74 ± 6.91 0.074 ± 0.025
15 mg 163.46 ± 37.12 1469.61 ± 14.24 0.082 ± 0.028
Mean of five observations
24 Tamilnadu J. Veterinary & Animal Sciences 5 (1) 20-24, Januaryr - February 2009
