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Effect of Encapsulated Protease Enzyme on the Performance, Carcass, Serum Protein and Retention of Protein and Energy in Broilers

R. Purshotham Naik A. Rajashekher Reddy K. Kondal Reddy J. Jyothi
Vol 7(5), 220-227

Two hundred day old commercial broiler chicks were randomly distributed into 4 treatments with 10 replicates of 5 birds in each replicate, which were reared for a period of 6 weeks under uniform managemental conditions. Encapsulation of protease enzyme was done to prevent the stability of enzyme during pelleting of feed. A corn soya experimental diets were prepared for all the four treatments formulating four diets i.e Control diet, Basal diet (BD) (with 2% less CP compared to control diet), BD+ uncoated protease enzyme and BD+ coated protease enzyme. Blood samples were collected for serum protein estimation. Metabolic trial was conducted for the estimating retention of protein and energy. Supplementation of protease enzyme resulted in better FCR, as compared to control diet. Protease enzyme to low protein BD showed a significance (P<0.05) increase in total serum protein and protein retention when compared to control. Enzyme supplementation improved broiler performance through better FCR, better retention of protein. Coated enzyme showed marginal improvement over uncoated enzyme.

Keywords : Coating Serum Protein Retention Protease


Feeding poultry is a major component that decides the profit from rearing poultry. Corn, soybean meal are the main ingredients used in preparation of poultry feed because of their abundance and economy. Use of dietary enzymes is a common practice for enhancing the bird’s performance through improved digestion, increased nutrient availability and reduced nutrient loss through excreta. Use of exogenous enzymes in pellet diets is a constraint since the enzymes are generally heat labile (Jongbloed et al., 1992). The enzyme activity is reduced or lost during pellet processing involving temperature and steam. The combination of heat and humidity may cause a severe inactivation of the enzymes (Gibson, 1995). Thus, the supplemental carbohydrases, proteases and most of other enzymes must be able to remain active at the feed pelleting temperature. Phytases are known to lose significant amounts of activity at temperature exceeding 70oC similarly carbohydrases and proteases lose activity at temperature exceeding 80oC and 90oC respectively (Gill, 1997, Perez-Portabella et al., 2001).

Coating of the enzyme with a barrier or encapsulation process is one approach to prevent contact of the enzyme with steam, moisture and harsh conditions during storage. The coated or encapsulated products may survive the pelleting process perfectly well and stabilize the enzyme during storage. The goal of encapsulation is to create a microenvironment in which the enzyme should survive during processing, storage and release of enzyme at appropriate sites in the digestive tract. Most of the coating technologies were applied to phytase enzyme due to its high instability and wide application. Recent work on feeding encapsulated phytase, xylanase and cocktail enzymes in corn soya pellet diets to broiler suggested benefits of encapsulation at pellet temperature (Srinath, 2012). In view of the above, present study was conducted to evaluate the supplementation of encapsulated protease enzyme effect on the performance of broilers (FCR, body weight gain, feed intake), livability, carcass parameters, retention of protein and serum protein.

Material and Methods

Birds, Management and Diet

Two hundred day old commercial broiler chicks were randomly distributed into 4 treatments with 10 replicates of 5 birds in each replicate, which were reared for a period of 6 weeks under uniform managemental conditions. On day 1, chicks were wing banded and housed in battery brooders. The brooder temperature was maintained at 35±0.5 0C until 7 days of age and gradually decreased to 27 0C by 21 days of age, after which, chicks were maintained at room temperature (20–27 0C). Birds were immunized for Marek’s disease at hatchery, New Castle disease (ND) at 7th day and 21stday of age with Lasota vaccine and infectious bursal disease at 14thday of age with Georgia strain vaccine. A corn soya starter (0-3wks) and finisher (4-6wks) pelleted diets were formulated (Table 1) for four treatments i.e. control diet, basal diet (BD) (with 2% less CP compared to control diet), BD+ uncoated protease enzyme and BD+ coated protease enzyme. Protease enzyme was added at 6000 IU/kg feed. All the treatments were offered ad-libitum feed and water for 1-42 days.

Table 1: Ingredient (g/kg) and nutrient composition (% dry matter) of basal diet fed to broilers

Ingredients Starter(0-3 wks) Finisher(4-6 wks)
Control Diet Basal Diet Control Diet Basal Diet
Maize 554.08 621.54 628.33 695.71
Soya bean meal 383.04 326.25 303.82 246.89
DORB 0.00 0.00 0 0
Salt 3.99 3.99 4.0 4
Dicalcium phosphate 19.55 19.82 17.12 17.42
Shell grit 4.52 4.79 4.48 4.79
DL-Methionine 2.66 2.39 2.21 1.9
AB2D3K1 0.13 0.13 0.2 0.2
B complex2 0.13 0.13 0.15 0.15
Choline chloride 0.93 0.93 1.0 1.0
Toxin Binder 2.00 2.00 2.0 2.0
Trace mineral mixture3 0.93 0.93 1.0 1.0
Antibiotic(chlortetracycline) 0.53 0.53 0.5 0.5
Cocciodiostat 0.53 0.53 0.5 0.5
L-Lysine 0.53 0.53 0.99 1.34
Vegetable oil 23.81 12.77 33.7 22.61
Nutrient Composition (calculated)*
M.E (kcal/kg) 2900 2900 3100 3100
Protein % 22 20 19 17
Calcium % 0.8 0.8 0.8 0.8
Available phosphorus % 0.45 0.45 0.4 0.4
Lysine % 1.25 1.134 1.097 0.981
Methionine % 0.56 0.506 0.51 0.456
Vitamin premix provided per kg diet: Vitamin A, 20000IU; Vitamin D3, 3000IU; Vitamin E, 10mg; Vitamin K, 2mg; Riboflavin, 25mg; VitaminB1,1mg; Vitamin B6, 2mg; Vitamin B12, 40mcg and Niacin, 15 mg; trace mineral premix provided per kg diet: Cu: 8 mg, Mn: 60 mg, Fe: 80 mg; *Calculated values

Encapsulation of Enzymes (Extrusion Technique)

Coating of enzymes was done by using alginate standard method (Krasaekoopt et al.,2003) Protease enzymes was added into a hydrocolloid solution (alginate) and then the cell suspension was extruded through a syringe needle to form droplets, which free-fall into a setting bath containing hardening solution CaCl2. Concentration of alginate was 1% to form a gel with 0.5M CaCl2. Uniform size and shape beads were obtained with alginate coat over enzymes for use in experimental diets.

Metabolic Trial

A metabolic trial was conducted for three days after 42 days of trial by keeping 2 birds in each replicate. All the birds were fasted to ensure emptying of gastrointestinal tract. Weighed quantity of feed was offered for three days. Litter trays were covered with plastic sheets under each cage cells and were replaced with new plastic sheet very next day. Faecal samples were collected daily carefully by separating feathers, scales and traces of feed. All the faecal samples were oven dried at 60oC for 24 hrs and weighed to record the faecal output on dry matter basis. Representative samples of feed and left over feed were collected and dried at 100±5oC for 8-10 hrs to estimate the dry matter intake. Analysis of crude protein and gross energy of dried excreta as carried as per AOAC (2005).

Slaughter and Serum Parameters

At the end of experiment 2 birds per replicate were slaughtered for carcass parameters like dressing percentage, heart, gizzard, liver and abdominal fat weight. Blood samples were collected in clean sterilized glass tubes for serum collection and the serum was stored in eppendorf tubes at – 200 C for estimation of biochemical constituents i.e. total protein (Reinhold, 1953).

Statistical Analysis

The data were subjected to statistical analysis by applying one way ANOVA as per procedures of Snedecor and Cochran (1980). The differences between the means were tested by significance using Duncan’s multiple range test (Duncan, 1955).

Results and Discussion

Supplementation of uncoated and coated protease enzyme did not show significant difference as compared to control diets during starter, finisher and overall period in terms of body weight gain, feed intake (Table 2) and this was in agreement with Ngxumeshe and Gous (2009) and Reddy et al. (2010) who reported that no significant in body weight gain during entire experimental period, when protease was added to low protein basal diet.

Table 2: Effect of uncoated and coated protease enzyme on body weight gain, feed intake, FCR

0-3 wks 4-6wks 0-6 wks
B.wt gain Feed intake FCR B.wt gain Feed intake FCR B.wt gain Feed intake FCR
Control 617.7 973.1 1.575a 1276a 2431a 1.906c 1894a 3405a 1.798b
Basal diet (BD) 593.8 952.0 1.603b 1182b 2198b 1.860bc 1776b 3150b 1.774b
BD + uncoated protease 596.8 948.9 1.593ab 1194b 2172b 1.818ab 1791b 3121b 1.742a
BD+ coated protease 603.4 957.4 1.588ab 1220b 2181b 1.786a 1824b 3139b 1.721a
SEM 5.358 8.362 0.003 10.153 25.301 0.011 12.644 28.264 0.007
p-value 0.412 0.757 0.019 0.003 0.000 0.000 0.002 0.000 0.000

Values bearing different superscripts within a column are significantly (P<0.05) different

Addition of uncoated and coated protease enzyme in basal diet did not show significant improvement in feed intake and this is in agreement with Cowieson and Ravindran (2008). Dietary addition of uncoated and coated enzymes to basal diets produced comparable FCR with control diet during starter, finisher but significant (P<0.05) improvement in FCR was observed during 0-6 wks when compared to control diet and this was observed same with Persia et al. (2002) working with turkey using multi enzyme complex containing protease who reported no significant difference in BWG but improvements in FCR from 9 to 15 wk age. In support to this Frietas et al. (2011) reported that supplementation of protease had no effect on BWG but improvement in FCR. Inclusion of coated enzyme improved the FCR (P<0.05) compared to control but numerical improvement was observed over uncoated enzyme. The lack of response of protease enzymes on body weight gain and FCR might be due to using good quality feed ingredients which might have favoured the growth of beneficial bacteria and improved gut health (Bedford and Apajalahti, 2000).

The healthy gut responds less to exogenous enzymes than those with higher harmful bacteria. In our study feed intake was not influenced and improvement in FCR was observed and this was observed by Mahagna et al. (1995) where he found that secretions of amylase and protease (Typsin and Chymotrypsin) by the pancreas was reduced when chicks were fed diets supplemented with amylase and protease. When enzyme supplemented to these diets showed improvement in FCR and this may be due to decreased feed intake in low density diets and there by suggesting that the improvement in nutrient utilization brought about by enzyme supplementation completely compensated for reduced protein and energy (Abudabos, 2012). Whereas no difference between coated and uncoated enzymes was found and this might be due to loss of activity of enzyme during pelleting of feed and for coated enzymes it might be due to untimely release of protease from coated particle in the digestive tract of bird. Moreover birds could produce adequate protease enzyme to digest protein as the age increased.

Supplementation of enzymes did not affect mortality and this is in agreement with Irish et al. (1995). Rao et al. (2003) showed that livability was not affected by supplementation of feed enzyme preparation to broiler diet. Supplementation of uncoated and coated protease enzymes did show any significant difference in carcass parameters in terms of dressing percentage, giblet weight percentage and abdominal fat percentage (Table 3) as compared to control and basal diet.

Table 3: Effect of uncoated and coated protease enzyme on dressing yield, giblet weight and fat weight (percentage)

Diets Dressing yield % Heart wt % Gizzard wt % Liver % Fat wt %
Control 71.91 0.438 1.952 1.700 1.739
Basal diet (BD) 71.58 0.457 1.815 1.731 1.641
BD+uncoated protease 72.44 0.421 1.913 1.712 1.686
BD+coated protease 72.64 0.489 1.810 1.698 1.635
SEM 0.1803 0.0146 0.0387 0.0287 0.0219
p-value 0.140 0.403 0.487 0.987 0.316

Values bearing different superscripts within a column are significantly (P<0.05) different

Serum protein level (Table 4) was improved by addition of uncoated and coated protease as compared to basal diet and the results are in agreement with Abudabos (2010 and 2012). Elevated levels of serum protein reflects role of protease on retention of protein.

Table 4: Effect of uncoated and coated protease enzyme on serum protein

Diets Serum Protein (g/dl)
Control 3.395a
Basal diet 3.024c
Basal +uncoated protease 3.198b
Basal +coated protease 3.263ab
SEM 0.0319
p-value 0.000

Values bearing different superscripts within a column are significantly (P<0.05) different

Supplementation of uncoated and coated protease enzyme showed no significant difference on dry matter and retention of energy but enzyme supplementation improved retention of protein (Table 5) compared to basal diet and this may be due improved apparent N digestibility and TME value of SBM (Ghazi et al., 2002; Yu et al., 2007). This was also supported by Marsmann et al. (1997) who found that supplementation of soybeans with proteases and carbohydrases improved the non-starch polysaccharide digestibility of soy bean.

Table 5: Effect of uncoated and coated protease enzyme on retention of protein and energy

Retention %
Diet Dry Matter Crude Protein Energy
Control 66.47 63.63ab 64.10
Basal diet 66.86 61.73b 62.95
Basal +uncoated protease 68.55 64.44a 64.07
Basal +coated protease 67.88 65.18a 64.92
SEM 0.437 0.408 0.327
p-value 0.330 0.009 0.209

Values bearing different superscripts within a column are significantly (P<0.05) different

But contrary to this Samarasinghe et al. (2000) found that conditioning temperature as high as 90oC reduced the N utilization by 4%. Supplementation of protease enzyme had no effect on feed intake, BWG but significantly improved FCR in low protein basal diets. Similarly addition of enzymes improved serum protein and retention of protein.


Overall enzyme supplementation has resulted in improved performance through better FCR and protein retention. Alginate process of coating needs to be further investigated for thermal stability and appropriate release of enzymes in the digestive tract.


The authors are thankful to the Department of Biotechnology, Government of India, for financially supporting the research work.


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