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Validation of an Analytical Method for the Determination of the Activity of

Protease in Animal Feed Additives and in Animal Feedingstuffs

Article  in  Journal of Applied Animal Research · January 2015

DOI: 10.1017/jan.2014.10

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 Journal of Applied Animal Nutrition, Vol. 3; e1; page 1 of 10 doi:10.1017/jan.2014.10
© Cambridge University Press and Journal of Applied Animal Nutrition Ltd. 2015

Original Research

Validation of an Analytical Method for the Determination of the Activity of

Protease in Animal Feed Additives and in Animal Feedingstuffs

G.P. Dillon1*, K. Filer2 and C.A. Moran3

1
Alltech Ireland, Sarney, Summerhill Road, Dunboyne, Co Meath, Ireland
2
Alltech Inc, 3031 Catnip Hill Pike, Nicholasville, 40356 KY, USA
3
Alltech France SARL, ZI Belle Etoile, 25 allée des Sapins, 44483 Carquefou, France
Journal of Applied Animal Nutrition

Summary
The nutrient availability in animal feeds can be improved by including exogenous enzymes to the feed, either by helping break-
down anti-nutritional factors or by increasing digestibility of complex ingredients thereby releasing more nutrients for utili-
sation. This process can improve the efficiency of meat and egg production, increase animal health, decrease feeding costs
and reduce nutrients in animal waste. Proteases are protein-digesting enzymes that are used in animal nutrition to break
down storage proteins in various plant materials and proteinaceous anti-nutrients in vegetable proteins. The analysis of exogen-
ous proteases in feed additives and after they have been added to feed has proven technically challenging. Accordingly, the
purpose of this work was to validate a method for the determination of the activity of protease in animal feed additives
and supplemented animal feed. The approach used for the assay was to adapt an assay based on the hydrolysis of haemoglo-
bin. The method validations examined a range of parameters including; linearity & range; uncertainty, sensitivity, accuracy and
studies designed to highlight any possible matrix effects on various types of supplemented feed. The assay method described
herein is convenient and inexpensive and could be applied to the analysis of proteases in animal feeds during quality control
and in investigating fraudulent adulteration of feed to ensure the authenticity and traceability of the product.
Keywords: Protease method validation: enzyme: animal feed

(Received 4 July 2014 – Accepted 10 November 2014)

Introduction
There is increasing attention on the supplementation of
animal feed with specific exogenous enzymes to improve
animal performance and efficiency of meat and egg pro-
duction whilst decreasing pollution from intensive farms
(Bedford, 2000; Mehri et al., 2011; Doherty et al., 2014).
Enzyme supplementation allows the feed producer
greater flexibility in the type of raw materials that can
confidently be used in feed formulation (Bedford,
2000; Vieira et al., 2011). In many animal production sys-
tems the biggest single cost is feed, and on-farm profit-
ability can depend on the relative cost and nutritive
value of the feed ingredients available (Barletta, 2010;
Zakaria et al., 2010). If feeds are not digested by the
animal as efficiently as they could be, there is a cost to
both the producer and the environment, due to higher
levels of excreted intact nutrients which are lost to the
animal (Chiou et al., 2007; Kalmendal et al., 2012). By
improving digestion and the absorption of nutrients,
there is a reduction in the volume of manure produced
and a lowering of phosphorus and nitrogen excretion
(Fidelis Fru-Nji, 2011).
Proteases are protein-digesting enzymes that are used
in animal nutrition to break down storage proteins in
various plant materials and proteinaceous anti-nutrients
in vegetable proteins (Hajati, 2010). This process facili-
tates improved nitrogen utilisation which can cause a

*Corresponding author: gdillon@alltech.com

 2 G.P. Dillon et al.
decrease in diet protein content and in turn reduce the
content of nitrogen in manure (Isaksen et al., 2010;
Vieira et al., 2011). Proteases have also been found to
assist in the breakdown of non-starch polysaccharides
(NSP) and storage proteins that bind to starch, releasing
bound starch that can then be digested by the animal
(Hayashi et al., 2004; Ljubojević et al., 2011).
The quantification of feed additive proteases following
supplementation to feed has proved technically challenging
(Südekum et al., 2010; Sheehan 2010). Conventional meth-
odologies involve colorimetric or fluorimetric testing
which can be time consuming, expensive and laborious
(McCleary, 2001; Sheehan 2010). There remains a need
for a suitable method for the detection of protease activity.
The criteria for such a method would be that it is robust
enough to allow the detection of protease in feed additives
Journal of Applied Animal Nutrition
and in complete (compound) feed. The method would be
inexpensive and convenient to use and could be applied to
routine quality control testing and could be further utilised
by authoritive bodies and inspectorates interested in rou-
tine monitoring of food and feed quality and when inves-
tigating fraudulent adulteration of feed and ensuring the
authenticity and traceability of feed products.
The objective of the experiments described herein is to
provide a method for the detection of proteases that
meets the criteria described above and to validate the
method accordingly. The approach used for the assay
was to adapt a procedure used to determine the proteo-
lytic activity of haemoglobin, which is expressed as
haemoglobin units on the tyrosine basis (HUT)
(Johnson, 1996). The activity of protease is measured
as a function of the catabolic degradation of haemoglobin
in terms of changes to its optical rotator dispersion. The
progress of the catabolic reaction is monitored discon-
tinuously by spectrophotometric assay at predetermined
time intervals and samples are inactivated to stop the
reaction.
Materials and methods
Only reagents of recognised analytical grade were used.
Deionised or demineralised water or water of equivalent
purity (18.2 MΩ/cm at 25°C) was used.
2 M Sodium acetate
This solution was prepared by dissolving Sodium acetate
trihydrate in distilled water and was then diluted to vol-
ume in a volumetric flask (Reagent 1).
1 M Acetic acid
This solution was prepared by diluting glacial acetic acid
with distilled water in a volumetric flask (Reagent 2).
0.1 M Acetate buffer pH 4.7
Reagent 1 (25 ml) and Reagent 2 (50 ml) were added to a
1 l beaker containing a stir bar and approximately 800 ml
of distilled water. The pH was adjusted to 4.7 ± 0.02 with
0.1M HCl and the solution was diluted to volume with
distilled water.
0.5 M Sodium acetate
This solution was prepared by diluting Reagent 1 in dis-
tilled water in volumetric flask.
0.3 N HCl
This solution was prepared by diluting concentrated HCl
(I N) distilled water in a volumetric flask.
Trichloroacetic acid (TCA) solution 14% (w/v)
TCA (14.0 g) was dissolved in approximately 50 ml of
distilled water and diluted to volume with distilled
water in a 100 ml volumetric flask.
Haemoglobin solution
Haemoglobin (Sigma #H2625) (4.0g) was weighed into
250 ml beaker which contained a stir bar. 100 ml of dis-
tilled water was added and the solution was stirred for
10 minutes. A pH electrode was immersed in the solution
and the solution was adjusted to pH 1.7 with 0.3 N HCl
with continuous stirring. After 10 minutes, the pH was
adjusted to 4.7 with 0.5 M sodium acetate. A volume
of 200 ml was transferred to a volumetric flask carefully
to minimise foaming and diluted to volume with distilled
water and vortexed. Fresh haemoglobin solutions were
prepared daily.
L-tyrosine standards
L-tyrosine (100 mg) was dissolved in 60 ml 0.1 N HCl in a
150 ml beaker. This solution was transferred analytically to
a 1 l volumetric flask. After tyrosine was completely dis-
solved, the solution was diluted to volume with distilled
water. This solution had a concentration of 100 µg/ml.
A dilution containing 75 µg tyrosine/ml was prepared by
adding 75 ml of the above solution to a 100 ml volumetric
flask and bringing to volume with deionised water. This
 Method validation protease animal feed
procedure was repeated to prepare a solution with 50 and
25 µg tyrosine/ml by using 50 ml and 25 ml, respectively,
of the 100 µg tyrosine/ml solution.
Enzyme samples
A representative commercial enzyme (Vegpro®, Alltech
Inc, USA) sample was prepared by collecting ten 1 kg
samples throughout a production run. The samples
were then mixed to make one representative sample.
Commercial Vegpro® test solutions were prepared by
dissolving a 1 g sample of the relevant test substance in
acetate buffer pH 4.7 to produce a solution containing
between 9 and 22 HUT in each ml. The enzyme was
to produce an absorbance reading (ΔA below) within
the linear range. Subsequently, 5 ml portions of haemo-
Journal of Applied Animal Nutrition
globin solution was transferred by pipette into test
tubes - two for each enzyme sample, one for the enzyme
sample blank and a single substrate blank. The test tubes
were incubated for five minutes in a 40°C water bath.
Beginning at time zero, 1.0 ml of enzyme solution was
transferred by pipette to the sample reaction tubes
which contained 5 ml of the equilibrated haemoglobin
solution. The mixture was gently vortexed for 30 seconds
and then placed into the 40°C water bath. A 1.0 ml ali-
quot of enzyme solution was continually transferred by
pipette to the sample reaction tubes until all samples
have been combined. After exactly 30 min, 5 ml of
TCA solution was quickly added to each tube (sample
reaction tubes, substrate blank tubes, and enzyme blank
tubes) to stop the reaction.
Preparation of the blank control was made whereby
1 ml of the acetate buffer solution was added to each
blank tube. Each tube was vortexed for 40s and the mix-
ture was kept at room temperature for 1 hour, while vor-
texing the tubes frequently throughout the incubation
time. After 1 h, tubes were vortexed and filtered through
a funnel with Whatman filter paper (Sigma #Z241113).
The mixture was re-filtered through the same filter.
Assay calibration and determination of protease
activity
Quartz cuvettes were used to read the absorbencies of
the filtrate at 275 nm using the filtrate of the substrate
blank to zero the instrument. Where an absorbency read-
ing between 0.2 and 0.5 was not obtained, the test was
repeated using more or less enzyme preparation as neces-
sary. A calibration curve was constructed based on the
tyrosine standard. The absorbance at 275 nm for each
3
of four tyrosine concentrations was determined. The
concentrations (x axis) were plotted against absorbance
(y axis) and a linear regression was used to determine
the absorbance for a solution containing 1.10 µg tyro-
sine/ml. The slope of the curve was determined in
terms of absorbance per µg tyrosine.
One HUT (haemoglobin unit on a tyrosine basis) of
proteolytic (protease) activity was defined as that amount
of enzyme that produces, in 1 min under the specified
conditions, a hydrolysate with an absorbance at 275 nm
that is the same as that of a solution containing
1.10 µg/ml tyrosine in 0.006 N HCl.
Calculation of the tyrosine curve factor
Tyrosine curve factor = Total volume (ml)
A275 of 1.10 mg tyr/ml · Time(min)
Calculation of enzyme activity
Enzyme activity (HUT/g)
= (DA/0.0084)· 11 ml/(30 min∗ W(g )∗ F)
Where:
ΔA is the difference in absorbance (μg tyrosine per ml)
between the sample and blank.
0.0084 is the accepted absorbance of 1.10 µg ty/ml at
275 nm (i.e. tyrosine curve factor).
11 ml is the final volume of the test solution.
30 min is the reaction time.
W is the weight (g) of the original sample.
F is the dilution factor.
Protease assays
The experimentation followed the ICH procedures for a
single-laboratory validation in an accredited laboratory
with a concomitant verification of the performance
characteristics in a second, independent expert laboratory
(Thompson et al., 2002; Simone and Robouch, 2014).
The results for two assays are provided for both the
determination of the activity of protease in Vegpro®
and the determination of the activity of protease in ani-
mal feedstuffs.
Assay 1: The determination of the activity of protease
in Vegpro®
The validation of the published method (Glenney et al.,
2005) for determining protease activity in the commercial,
 4 G.P. Dillon et al.

protease containing enzyme Vegpro® was undertaken at an Table 1. Matrix 1 (protease in Vegpro®): Comparison of the calibration of
tyrosine between 0 and 150 µg/ml as measured by spectrophotometer at
independent expert laboratory (Laboratory 1). The tests absorbance 275nm at two laboratories
involved were carried out in accordance with the principles
Concentration Cm (µg/ml) Laboratory 1 Laboratory 2
established in the French Standard, Norme NF XPT 90-210
Association Française de Normalisation (AFNOR), 1999. 0 0 0
25 0.189 0.184
In addition, a separate verification study was completed in 50 0.372 0.376
a secondary laboratory (Laboratory 2) to corroborate the 75 0.55 0.562
100 0.735 0.744
results. The results of both sets of analyses are described 150 1.12 1.119
and discussed here. Slope 0.00743 0.0075
R2 0.9998 1
Coefficient (1.1 slope) 0.0082 0.00835
Conditions that applied to Laboratory 1: Enzyme
feed supplement: Vegpro® in liquid form; batch
#247639; stored at 5°C + 3°C. The calibration curves were used to determine the pre-
cision of protease activity in Vegpro®. The calibration
Conditions that applied to Laboratory 2: Enzyme over the range of 0–150 µg/ml of tyrosine yielded linear-
feed supplement: Vegpro® in liquid form; batches ity with an R2 value of 0.9998 and 1.0 respectively for the
Journal of Applied Animal Nutrition

#297922 (known) and #297923 (unknown); stored at
4°C + 2°C.
Assay 2: The determination of the activity of protease
directly in animal feedstuffs
The assay used a 10 g sample of pelleted starter chicken
feed, with an extraction volume of 40 ml. The extraction
procedure involved grinding the sample in a motor and
pestle and then solubilising it in acetate buffer solution
and shaking for 15 minutes. The solution was then cen-
trifuged at 2,000 G for five minutes at 4°C for an incu-
bation time of 6 hrs with the enzyme feed supplement:
Vegpro® in liquid form (batch #249568, where 1 ml
has a mean activity measured: 43,500 HUT/kg feed).
The blank solution was prepared by adding TCA solution
to a feed sample prior to the addition of the enzyme feed
supplement and therefore no substrate enzyme inter-
action occurs.
Specifically, the studies wished to investigate the follow-
ing validation parameters; a) Precision – including repeat-
ability and intra-laboratory reproducibility; b) Sensitivity –
including the Limit of Quantification (LOQ) and Limit of
Detection (LOD); c) Uncertainty and d) Accuracy
tests taken in each laboratory. (Figures 1 and 2).
Reproducibility was defined as the absolute difference
between two single test results found on identical test
material by one operator using the same apparatus within
the shortest feasible interval and will exceed the repeat-
ability limit r in not more than 5% of the cases.
Intra-laboratory reproducibility was considered as the
absolute difference between two single test results on
identical test material reported by two laboratories that
will exceed the reproducibility limit R in not more
than 5% of the cases. To study repeatability, fifteen deter-
minations of Vegpro® were undertaken. The results of
this experiment are given in Table 2.
Figure 1. Calibration of tyrosine between 0 and 150 µg/ml as measured by
spectrophotometer (absorbance 275 nm) in Laboratory 2

Assay 1: The determination of the activity of protease

in Vegpro®

Precision
Both laboratories generated a standard calibration curve
with tyrosine to determine a set of absorbances over a
pre-determined range. The results of the readings Figure 2. Calibration of tyrosine between 0 and 150 µg/ml as measured by
observed are presented in Table 1. spectrophotometer (absorbance 275 nm) in Laboratory 2
 Method validation protease animal feed 5

Table 2. Matrix 1: Repeatability of protease activity in the feed additive preparation, Vegpro®

Trials Blank test

Sample no. ΔA* Dilution factor Activity (HUT/ml)
A1 A2 A Substrate A Enzyme + substrate

1 0.838 0.838 0.439 0.440

2 0.838 0.841 0.432 0.433 0.407 2 500 44 360
3 0.842 0.845 0.438 0.439 0.405 2 500 44 142
4 0.851 0.848 0.432 0.439 0.411 2 500 44 797
5 0.865 0.863 0.455 0.460 0.404 2 500 44 087
6 0.877 0.866 0.440 0.444 0.428 2 500 46 652
7 0.877 0.883 0.457 0.471 0.409 2 500 44 633
8 0.866 0.887 0.448 0.453 0.424 2 500 46 215
9 0.872 0.880 0.453 0.458 0.418 2 500 45 615
10 0.879 0.867 0.458 0.459 0.414 2 500 45 179
11 0.871 0.878 0.458 0.458 0.417 2 500 45 451
12 0.863 0.871 0.449 0.451 0.416 2 500 45 397
13 0.870 0.879 0.449 0.457 0.418 2 500 45 561
14 0.852 0.864 0.450 0.455 0.403 2 500 43 978
15 0.877 0.876 0.452 0.455 0.422 2 500 45 997
Mean 0.447 0.451 0.413 45 033
Standard deviation (absolute value) 0.009 0.010 0.009 923
Journal of Applied Animal Nutrition

Standard deviation (relative value). CVr. % 2.01 2.29 2.05 2.05

Limits. ΔA Detection 3 · Sr (A enzyme + substrate) = 0.031
Quantification 10 · Sr (A enzyme + substrate) = 0.103
Limits. HUT/ml Detection 1.35
Quantification 4.51

* ΔA corresponds to the difference in absorbance (A) between the mean absorbance values A1 and A2 of the two trial and the absorbance value for an enzyme + substrate blank
test read at a wavelength of 275 nm. ΔA = (A1 + A2)/2 – ( A enzyme+substrate blank test)

The study to investigate intra-laboratory reproducibility
involved preparing a standard calibration curve with tyro-
sine three times over three consecutive months to deter-
mine the absorbance and linearity over the range. The
results of the absorbance observed are presented in Table 3.
The calibration curve over the range of 0–150 µg/ml of
tyrosine yielded good linearity with an R2 of 0.9998
(Figure 3). The experiment undertaken to determine intra-
laboratory repeatability was similar to take undertaken for
repeatability only the repeat testing was carried out on 15
consecutive days (Table 4). The RSD was 4.08% which
was within the established standard limit of 5%.
substrate that had been denatured with TCA. The LOD
was calculated using the formula; LOD = 3 SD of the
blank test (enzyme + substrate). The LOD was demon-
strated to be 1.35 HUT/ml. The LOQ was calculated
using the formula; LOQ = 10 SD of the blank test (enzyme
+ substrate). The limit of quantification was found to be 4.5
HUT/ml. The LOD and LOQ were also calculated separ-
ately by Laboratory 2 under different conditions. Three rep-
licate determinations were conducted on three separate
days over the course of a week. The results, tabulated in
Table 5, were calculated as 3 HUT/ml and 8 HUT/ml
for the LOD and LOQ respectively.

Sensitivity – LOD and LOQ Uncertainty

The LOD and LOQ were based on the determinations of Uncertainty was determined from the calibration ranges
the blank sample which contained the enzyme and the under reproducible conditions and based on the standard

Table 3. Matrix 1: Study of intra-laboratory reproducibility - calibration with tyrosine (absorbance 275 nm)

Concentration Cm (µg/ml)

Date 0 25 50 75 100 150 Coefficient (1.1 Slope)

October 17 0 0.190 0.371 0.558 0.747 1.110 0.00814
October 24 0 0.190 0.370 0.546 0.730 1.121 0.00816
November 2 0 0.200 0.384 0.564 0.751 1.122 0.00818
Slope 0.00743
R2 0.9998
Coefficient (1.1 · slope) 0.0082
 6 G.P. Dillon et al.

formula: Percentage Recovery = [(average recorded value

– expected value)/average recorded value] 100 + 100.
The result was found to be 108% (Table 6). A sample of
an unknown sample was also tested. The SD and %RSD
were found to be 134 and 1.2% respectively (Table 7).

Assay 2: The determination of the activity of protease

directly in animal feedstuffs
Validation tests were performed to study the proposed
Figure 3. Linear model of observed range from feed spiked with known con-
centrations of protease (HUT/kg feed)
method for determining protease activity in animal feed-
stuffs which had been supplemented with Vegpro®.
Specifically, the validation examined:
deviations associated with each of the range endpoints.
Uncertainty was defined as twice the ratio of the standard 1) A study of the range of concentrations covered by
deviation of the average absorbance and expressed as a the method
percentage of the reported value. The uncertainty asso- 2) An estimate of uncertainty
Journal of Applied Animal Nutrition

ciated to the results is 20% of the reported value. An 3) Verification of the choice of the quantification limit
extended uncertainty value of +8% should be applied with the calculation of the detection limit
to protease activity values. The uncertainty of the experi- 4) A study designed to highlight any possible matrix
ment was observed to be 8.16%. effects on various types of poultry feed.

Accuracy Concentration range

The accuracy of the assay was examined by undertaking six
replicate determinations of a known sample with
a theoretical activity of 20,000 HUT/ml on two consecu-
tive days. The accuracy is a measure of the closeness of
agreement between the theoretic true value and the experi-
mental value. The accuracy was calculated with the
A test feed sample was supplemented with increasing
concentrations of enzyme in the range 0 to 22,000
HUT/kg feed. The principle extraction of the feed in
an acetate buffer solution was performed. Note that
this step was not required when directly analysing the
feed additive. The extract was brought into contact

Table 4. Matrix 1: Protease analyses performed on 15 different days on the feed additive preparation Vegpro®:

Trials Blank test

No. Date of analysis ΔA* Dilution factor Activity (HUT/ml)
A1 A2 A Substrate A Enzyme + substrate

1 October 17 0.902 0.911 0.481 0.488 0.419 2 500 45 670

2 October 18 0.881 0.908 0.455 0.460 0.435 2 500 47 416
3 October 19 0.921 0.941 0.470 0.477 0.454 2 500 49 544
4 October 20 0.894 0.893 0.489 0.487 0.407 2 500 44 360
5 October 21 0.881 0.893 0.474 0.483 0.404 2 500 44 087
6 October 24 0.902 0.906 0.462 0.463 0.441 2 500 48 125
7 October 25 0.875 0.871 0.451 0.452 0.421 2 500 45 942
8 October 26 0.854 0.854 0.446 0.450 0.404 2 500 44 087
9 October 27 0.867 0.873 0.447 0.450 0.420 2 500 45 833
10 October 31 0.875 0.870 0.420 0.436 0.437 2 500 47 634
11 November 2 0.914 0.913 0.495 0.496 0.418 2 500 45 561
12 November 3 0.884 0.886 0.474 0.474 0.411 2 500 44 851
13 November 4 0.896 0.897 0.455 0.457 0.440 2 500 47 961
14 November 7 0.925 0.925 0.466 0.470 0.455 2 500 49 653
15 November 9 0.885 0.875 0.473 0.479 0.401 2 500 43 760
Mean 46 366
Standard deviation SR (absolute value) 1 892
Standard deviation SR (relative value): CVR. % 4.08
Extended uncertainty (2· CVR) 8.16

* ΔA corresponds to the difference in absorbance (A) between the mean absorbance values A1 and A2 of the two trial and the absorbance value for an enzyme + substrate blank
test read at a wavelength of 275 nm. ΔA = (A1 + A2)/2 – (A enzyme+substrate blank test)
 Method validation protease animal feed 7

Table 5. Matrix 1 (Protease in Vegpro®): LOD and LOQ analysis Table 8. Matrix 2 (protease in feedstuffs): Determination of the range of
calculation over three independent days protease concentration (HUT/kg) in feed

Sample Date Absorbance (275 nm) Information value

Size (HUT/kg feed) Surface values
1 Day 1 0.466 component Linear Quadratic
2 Day 1 0.461
3 Day 1 0.463 0 0.049 −1468.130 −67.098
4 Day 2 0.462 0.07 −1433.645 415.201
5 Day 2 0.451 0.102 −729.913 −55.204
6 Day 2 0.465 0.083 −1093.293 31.829
7 Day 3 0.455 0.077 −1087.793 −1.472
8 Day 3 0.468 2175 0.268 2565.3 2143.34
9 Day 3 0.475 0.271 2346.81 1628.68
0.287 2337.95 2256.67
Mean 0.463 0.253 2054.05 1888.6
Standard deviation 0.007 0.271 2217.22 2030.38
Slope 0.0084 4351 0.441 5751.53 4637.75
LOD 3 HUT/ml 0.445 5619.45 3860.61
LOQ 8 HUT/ml 0.438 4841.98 4378.59
0.452 5738.3 4778.9
0.465 5522.23 4647.31
Table 6. Matrix 1 (protease in Vegpro®): Accuracy of reproducible 8701 0.657 9729.7 8679.26
0.728 10942.2 9832.43
Journal of Applied Animal Nutrition

analyses determined from 12 samples on two independent days

0.704 9253.07 8630.14
0.672 9811.33 8874.02
Sample ID Results (HUT/ml)
0.701 9542.76 8619.58
13532 0.848 13247.4 13110.7
Day 1 297922-1 21136
0.856 13349.7 13486
297922-2 22340
0.969 13647.6 13517.3
297922-3 23021
0.85 13106.8 12878.9
297922-4 22288
0.931 13461.1 13324
297922-5 22550
17402 1.015 16323.2 17645
297922-6 23048
0.961 15324.5 16926.1
Day 2 297922-7 20612
1.159 16798.3 17421.5
297922-8 21319
1.035 16531.8 17696.8
297922-9 21162
1.109 16493.5 17529.2
297922-10 21921
297922-11 21738
297922-12 20167
mean 21775
SD 920 enzymatic preparation. A linear and quadratic determin-
RSD % 4.2 ation of the range of concentration was assessed. The
Accuracy % 108
data acquired is given in Table 8 and depicted in
RSD % = SD x 100/x; SD = standard deviation; X = mean Figures 3 and 4 for the linear and quadratic analysis
% Recovery = [(average recorded value – expected value)/average recorded value]
100 + 100 respectively.
Initially, a calibration graph over seven enzyme concen-
with a haemoglobin solution. After 6 h of incubation at trations were chosen within the presumed range of the
40°C, the enzymatic reaction was stopped and the method (in the range 0 to 22,000 HUT/kg feed); supple-
absorbance of the solution measured at a wavelength mentation rates and absorbencies were collected over a
of 275 nm. Under the same operating conditions, a sam- five month period. This data was used to develop linear
pling range was determined from a non-supplemented
feed to which was incorporated known doses of an

Table 7. Matrix 1 (protease in Vegpro®): Determination of protease

concentration (HUT/ml) in three unknown samples of Vegpro®

Sample ID Results (HUT/ml)

297923-1 10948
297923-2 11210
297923-3 11131
mean 11096
SD 134
%RSD 1.2 Figure 4. Quadratic model of observed range from feed spiked with known
concentrations of protease (HUT/kg feed)
 8 G.P. Dillon et al.

Table 9. Matrix 2 (protease in feedstuffs): Calibration values (absorbance 275 nm) from the range of application and matrix effects

HUT/kg
0 2,175 4,351 8,701 13,520 17,402

0.049 0.268 0.441 0.657 0.848 1.015

0.070 0.271 0.445 0.728 0.856 0.961
0.102 0.287 0.438 0.704 0.969 1.159
0.083 0.253 0.452 0.672 0.850 1.035
0.077 0.271 0.465 0.701 0.931 1.109
0.087 0.301 0.477 0.679 0.866 1.051
0.066 0.310 0.474 0.709 0.834 0.929
0.082 0.331 0.472 0.754 0.979 1.246
0.093 0.321 0.457 0.771 0.998 1.145
0.070 0.310 0.423 0.725 0.934 1.084
Average 0.078 0.292 0.454 0.710 0.906 1.073
SD 0.015 0.026 0.018 0.036 0.062 0.096
Expanded uncertainty% 38.6 17.8 7.8 10.1 13.8 17.8

and second degree (quadratic) polynomial models. A were determined. The results of this experiment are pro-
Journal of Applied Animal Nutrition

significant model error was observed over the chosen
calibration range of 0 to 22,000 HUT/kg feed, necessitat-
ing re-evaluation of the upper range concentration. The
range 0 to 17,500 HUT/kg feed was considered accept-
able, with the quadratic model giving a R2 value of
0.9993.
Uncertainty
The uncertainty of the experimental data was established
as a determinant from various calibration ranges
made under conditions that satisfy reproducibility. The
standard deviation (SD) was calculated for each of the
range points. Table 9 shows the uncertainty determined
for each concentration point. All the values of the cali-
brations made during the study of the domain of applica-
tion and the matrix effects were compiled. For the
various range endpoints, there was uncertainty ranging
from 10 to 20%. Over the supplementation rate ranging
from 2,000 to 17,500 HUT/kg, the method provides
an expanded estimated calculated uncertainty equal to
+20% in relative value.
vided in Table 10. An LOQ of 1,000 HUT/kg was cho-
sen for the method. However, the adequacy test gives an
estimated calculated uncertainty associated with this
point that is greater than 46.6%. It was decided that a
higher LOQ would be more suitable. The SD and uncer-
tainty of the concentration point of 2,000 HUT/kg was
assessed and resulting associated uncertainty was found
to be 19.6%, which was within the 20% uncertainty
range. In a secondary independent test, the LOQ calcu-
lated using the conventional equation LOQ = 10 SD.
The LOQ calculated by this method was found to be
2,000 HUT/kg and therefore, this value was established
as the LOQ for this method. The LOD was calculated as
700 HUT/kg.
Matrix effect
The objective of this study was to determine whether the
method was effective in determining protease activity in a
Table 10. Matrix 2 (protease in feedstuffs): Validation of the limit of
quantification (LOQ) and determination of the Uncertainty % at
1,000 HUT/kg

Replicate # Concentration obtained

Sensitivity (LOD and LOQ)
The LOQ was examined in accordance with the French
standard Norme NF XPT 90-210 (Association Française
do Normalisation, 1996). Two conditions are stipulated
in this standard in choosing an LOQ: 1) the average
value does not differ from the quantification limit cho-
sen, and 2) the quantification limit is not equal to zero.
A LOQ of 1,000 HUT/kg which met the demands of
the standard was selected and the SD and uncertainty
of ten replicate analyses of this concentration point
1 747
2 1114
3 1202
4 1235
5 1026
6 850
7 787
8 739
9 736
10 676
Mean 911.2
SD 212.268
Uncertainty 46.6%
 Method validation protease animal feed 9

Table 11. Matrix 2 (protease in feedstuffs): Matrix effect of 10 selected poultry feeds on protease recovery (HUT/kg feed). All diets were standard corn soy
diets with protein and energy levels as listed in the table

Size Size after Relative

Added Y addition w deviation %

Chicken grower PT ‘control’ 20% protein; 3,200 kcal/kg metabolizable energy ME 8700 9123 4.9
Chicken grower PT ‘control’ 20% protein; 3,200 kcal/kg ME 17400 19384 11.4
Chicken grower PT ‘control’ 20% protein; 3,200 kcal/kg ME 4350 4464 2.6
Chicken grower PE ‘energy’ 20% protein; 3,100 kcal/kg ME 2175 2296 5.1
Chicken grower PE ‘energy’ 20% protein; 3,100 kcal/kg ME 4350 4399 1.1
Chicken grower PE ‘energy’ 20% protein; 3,100 kcal/kg ME 13050 16148 23.7
Chicken grower PT ‘protein’ 18% protein; 3,200 kcal/kg ME 1088 1211 11.3
Chicken grower PT ‘protein’ 18% protein; 3,200 kcal/kg ME 4350 5810 29.0
Chicken grower PT ‘protein’ 18% protein; 3,200 kcal/kg ME 13050 14065 7.8
Poultry finisher DF 20% protein; 3,200 kcal/kg ME 2175 1431 −34.2
Poultry finisher DF 20% protein; 3,200 kcal/kg ME 8700 8220 −5.5
Poultry finisher DF 20% protein; 3,200 kcal/kg ME 17400 15607 −10.3
Finisher no 2 20% protein; 3,200 kcal/kg ME 4350 4127 −5.1
Finisher no 2 20% protein; 3,200 kcal/kg ME 13050 14424 10.5
Finisher no 2 20% protein; 3,200 kcal/kg ME 17400 18519 6,4
Pellet base test 20% protein; 3,200 kcal/kg ME 1088 840 −22.8
Pellet base test 20% protein; 3,200 kcal/kg ME 2175 1569 −27.9
−24.2
Journal of Applied Animal Nutrition

Pellet base test 20% protein; 3,200 kcal/kg ME 8700 6593

Layer no 5 20% protein; 2,800 kcal/kg ME 4350 3628 −16.6
Layer no 5 20% protein; 2,800 kcal/kg ME 13050 12257 −6.1
Layer no 5 20% protein; 2,800 kcal/kg ME 17400 15430 −11.3
Layer 6 – 47 20% protein; 2,800 kcal/kg ME 1088 1101 1.2
Layer 6 – 47 20% protein; 2,800 kcal/kg ME 8700 9731 11.9
Layer 6 – 47 20% protein; 2,800 kcal/kg ME 17400 17908 2.9
(Turkey) starter diet TD1 20% protein; 2,900 kcal/kg ME 1088 1595 46.6
(Turkey) starter diet TD1 20% protein; 2,900 kcal/kg ME 4350 4832 11.1
(Turkey) starter diet TD1 20% protein; 2,900 kcal/kg ME 13050 12622 −3.3
(Turkey) finisher diet TD1 17% protein; 3,200 kcal/kg ME 2175 2287 5.1
(Turkey) finisher diet TD1 17% protein; 3,200 kcal/kg ME 8700 8892 2.2
(Turkey) finisher diet TD1 17% protein; 3,200 kcal/kg ME 17400 21036 20.9

variety of poultry feedstuffs. The feedstuffs were repre-
sentative commercial soy-base diets for different phases
of poultry growth. The method was tested using a
range of enzyme concentrations in chicken starter feed;
that same range of enzyme concentrations was used to
test a series of feed matrices. Enzyme activity levels
were then calculated. Activity levels determined empiric-
ally were plotted against the theoretical levels. A linear
regression was plotted corresponding to the activity levels
recovered versus the theoretical levels added. The data
for this study is shown in Table 11 and Figures 5. To
evaluate specificity, we verified that the curve of the
line did not differ by 1 and that the original y axis
did not differ from 0. The study showed that no signifi-
cant matrix effect was present in the feed matrices
investigated.
uptake. Hitherto, it had proved difficult to analyse the
activity of protease in animal feed additives and directly
in animal feedstuffs. This paper validates an analytical
method based on the hydrolysis of haemoglobin and
involved measuring the proteolytic activity which is
expressed as haemoglobin units of the tyrosine basis
(HUT).
It has been demonstrated that the principle of the
method can be applied on two separate matrices; namely,
for the determination of the activity of protease in feed
additives; and for the determination of the activity of

Conclusions
Proteases are enzymes which can be added to animal
feed and provide benefits such as improving animal
health, digestion and facilitate the optimal utilisation of Figure 5. Matrix effect of 10 selected commercial poultry feeds on protease
feedstuffs in terms of maximising nutrient use and recovery (HUT/kg feed)
 10
protease in animal feedstuffs. Both of the matrices were
validated separately and independently and cover the
parameters such as linearity, precision, sensitivity, accur-
acy and matrix effects. The tests show that the method-
ology is fit for purpose and therefore suitable to
determine the activity of protease in both assays. The
study therefore meets the objective in providing a
method that can be applied to protease for routine testing
and in quality control conditions.
Acknowledgements
The authors wish to acknowledge the work of O.
Locqueneux, M.D. L’Hotellier and K. Michel on perform-
ing the validation work at the Institute of Environmental
Engineering and Biotechnology, Bordeaux, France. The
Journal of Applied Animal Nutrition
authors would like to thank Alltech Inc (Nicholasville,
Kentucky, USA) for their financial support of this project.
Declaration of Interest
The authors G.D. Dillon, K. Filer and C.A. Moran are
employees of Alltech Inc. which fully funded this
research.
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