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Original Research
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
Introduction
There is increasing attention on the supplementation of animal as efficiently as they could be, there is a cost to
animal feed with specific exogenous enzymes to improve both the producer and the environment, due to higher
animal performance and efficiency of meat and egg pro- levels of excreted intact nutrients which are lost to the
duction whilst decreasing pollution from intensive farms animal (Chiou et al., 2007; Kalmendal et al., 2012). By
(Bedford, 2000; Mehri et al., 2011; Doherty et al., 2014). improving digestion and the absorption of nutrients,
Enzyme supplementation allows the feed producer there is a reduction in the volume of manure produced
greater flexibility in the type of raw materials that can and a lowering of phosphorus and nitrogen excretion
confidently be used in feed formulation (Bedford, (Fidelis Fru-Nji, 2011).
2000; Vieira et al., 2011). In many animal production sys- Proteases are protein-digesting enzymes that are used
tems the biggest single cost is feed, and on-farm profit- in animal nutrition to break down storage proteins in
ability can depend on the relative cost and nutritive various plant materials and proteinaceous anti-nutrients
value of the feed ingredients available (Barletta, 2010; in vegetable proteins (Hajati, 2010). This process facili-
Zakaria et al., 2010). If feeds are not digested by the tates improved nitrogen utilisation which can cause a
decrease in diet protein content and in turn reduce the 1 M Acetic acid
content of nitrogen in manure (Isaksen et al., 2010; This solution was prepared by diluting glacial acetic acid
Vieira et al., 2011). Proteases have also been found to with distilled water in a volumetric flask (Reagent 2).
assist in the breakdown of non-starch polysaccharides
(NSP) and storage proteins that bind to starch, releasing
0.1 M Acetate buffer pH 4.7
bound starch that can then be digested by the animal
(Hayashi et al., 2004; Ljubojević et al., 2011). Reagent 1 (25 ml) and Reagent 2 (50 ml) were added to a
The quantification of feed additive proteases following 1 l beaker containing a stir bar and approximately 800 ml
supplementation to feed has proved technically challenging of distilled water. The pH was adjusted to 4.7 ± 0.02 with
(Südekum et al., 2010; Sheehan 2010). Conventional meth- 0.1M HCl and the solution was diluted to volume with
odologies involve colorimetric or fluorimetric testing distilled water.
which can be time consuming, expensive and laborious
(McCleary, 2001; Sheehan 2010). There remains a need 0.5 M Sodium acetate
for a suitable method for the detection of protease activity.
This solution was prepared by diluting Reagent 1 in dis-
The criteria for such a method would be that it is robust
tilled water in volumetric flask.
enough to allow the detection of protease in feed additives
Journal of Applied Animal Nutrition
procedure was repeated to prepare a solution with 50 and of four tyrosine concentrations was determined. The
25 µg tyrosine/ml by using 50 ml and 25 ml, respectively, concentrations (x axis) were plotted against absorbance
of the 100 µg tyrosine/ml solution. (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
Enzyme samples
terms of absorbance per µg tyrosine.
A representative commercial enzyme (Vegpro®, Alltech One HUT (haemoglobin unit on a tyrosine basis) of
Inc, USA) sample was prepared by collecting ten 1 kg proteolytic (protease) activity was defined as that amount
samples throughout a production run. The samples of enzyme that produces, in 1 min under the specified
were then mixed to make one representative sample. conditions, a hydrolysate with an absorbance at 275 nm
Commercial Vegpro® test solutions were prepared by that is the same as that of a solution containing
dissolving a 1 g sample of the relevant test substance in 1.10 µg/ml tyrosine in 0.006 N HCl.
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 Calculation of the tyrosine curve factor
the linear range. Subsequently, 5 ml portions of haemo- Tyrosine curve factor = Total volume (ml)
Journal of Applied Animal Nutrition
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 tests taken in each laboratory. (Figures 1 and 2).
4°C + 2°C. 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
Assay 2: The determination of the activity of protease the shortest feasible interval and will exceed the repeat-
directly in animal feedstuffs ability limit r in not more than 5% of the cases.
The assay used a 10 g sample of pelleted starter chicken Intra-laboratory reproducibility was considered as the
feed, with an extraction volume of 40 ml. The extraction absolute difference between two single test results on
procedure involved grinding the sample in a motor and identical test material reported by two laboratories that
pestle and then solubilising it in acetate buffer solution will exceed the reproducibility limit R in not more
and shaking for 15 minutes. The solution was then cen- than 5% of the cases. To study repeatability, fifteen deter-
trifuged at 2,000 G for five minutes at 4°C for an incu- minations of Vegpro® were undertaken. The results of
bation time of 6 hrs with the enzyme feed supplement: this experiment are given in Table 2.
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 – Figure 1. Calibration of tyrosine between 0 and 150 µg/ml as measured by
including the Limit of Quantification (LOQ) and Limit of spectrophotometer (absorbance 275 nm) in Laboratory 2
Detection (LOD); c) Uncertainty and d) Accuracy
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®
* 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 substrate that had been denatured with TCA. The LOD
involved preparing a standard calibration curve with tyro- was calculated using the formula; LOD = 3 SD of the
sine three times over three consecutive months to deter- blank test (enzyme + substrate). The LOD was demon-
mine the absorbance and linearity over the range. The strated to be 1.35 HUT/ml. The LOQ was calculated
results of the absorbance observed are presented in Table 3. using the formula; LOQ = 10 SD of the blank test (enzyme
The calibration curve over the range of 0–150 µg/ml of + substrate). The limit of quantification was found to be 4.5
tyrosine yielded good linearity with an R2 of 0.9998 HUT/ml. The LOD and LOQ were also calculated separ-
(Figure 3). The experiment undertaken to determine intra- ately by Laboratory 2 under different conditions. Three rep-
laboratory repeatability was similar to take undertaken for licate determinations were conducted on three separate
repeatability only the repeat testing was carried out on 15 days over the course of a week. The results, tabulated in
consecutive days (Table 4). The RSD was 4.08% which Table 5, were calculated as 3 HUT/ml and 8 HUT/ml
was within the established standard limit of 5%. for the LOD and LOQ respectively.
Table 3. Matrix 1: Study of intra-laboratory reproducibility - calibration with tyrosine (absorbance 275 nm)
Concentration Cm (µg/ml)
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.
Table 4. Matrix 1: Protease analyses performed on 15 different days on the feed additive preparation Vegpro®:
* 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
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
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 vided in Table 10. An LOQ of 1,000 HUT/kg was cho-
calibration range of 0 to 22,000 HUT/kg feed, necessitat- sen for the method. However, the adequacy test gives an
ing re-evaluation of the upper range concentration. The estimated calculated uncertainty associated with this
range 0 to 17,500 HUT/kg feed was considered accept- point that is greater than 46.6%. It was decided that a
able, with the quadratic model giving a R2 value of higher LOQ would be more suitable. The SD and uncer-
0.9993. 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
Uncertainty
range. In a secondary independent test, the LOQ calcu-
The uncertainty of the experimental data was established lated using the conventional equation LOQ = 10 SD.
as a determinant from various calibration ranges The LOQ calculated by this method was found to be
made under conditions that satisfy reproducibility. The 2,000 HUT/kg and therefore, this value was established
standard deviation (SD) was calculated for each of the as the LOQ for this method. The LOD was calculated as
range points. Table 9 shows the uncertainty determined 700 HUT/kg.
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 Matrix effect
various range endpoints, there was uncertainty ranging The objective of this study was to determine whether the
from 10 to 20%. Over the supplementation rate ranging method was effective in determining protease activity in a
from 2,000 to 17,500 HUT/kg, the method provides
an expanded estimated calculated uncertainty equal to Table 10. Matrix 2 (protease in feedstuffs): Validation of the limit of
quantification (LOQ) and determination of the Uncertainty % at
+20% in relative value. 1,000 HUT/kg
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
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
variety of poultry feedstuffs. The feedstuffs were repre- uptake. Hitherto, it had proved difficult to analyse the
sentative commercial soy-base diets for different phases activity of protease in animal feed additives and directly
of poultry growth. The method was tested using a in animal feedstuffs. This paper validates an analytical
range of enzyme concentrations in chicken starter feed; method based on the hydrolysis of haemoglobin and
that same range of enzyme concentrations was used to involved measuring the proteolytic activity which is
test a series of feed matrices. Enzyme activity levels expressed as haemoglobin units of the tyrosine basis
were then calculated. Activity levels determined empiric- (HUT).
ally were plotted against the theoretical levels. A linear It has been demonstrated that the principle of the
regression was plotted corresponding to the activity levels method can be applied on two separate matrices; namely,
recovered versus the theoretical levels added. The data for the determination of the activity of protease in feed
for this study is shown in Table 11 and Figures 5. To additives; and for the determination of the activity of
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.
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 G.P. Dillon et al.
protease in animal feedstuffs. Both of the matrices were Glenney P. and Filer K. (2005) Development of an analytical method
for the analysis of acid proteases in feed samples. Poster Presentation
validated separately and independently and cover the at ADSA/ASAS Joint Annual Meeting.
parameters such as linearity, precision, sensitivity, accur- Hajati H. (2010) Effects of enzyme supplementation on performance,
acy and matrix effects. The tests show that the method- carcass characteristics, carcass composition and some blood para-
meters of broiler chicken. American Journal of Animal and Veterinary
ology is fit for purpose and therefore suitable to Sciences 5 (3), 221–227.
determine the activity of protease in both assays. The Hayashi K., Saleh F., Ohtsuka A. and Tanaka T. (2004)
study therefore meets the objective in providing a Carbohydrates are Digested by Proteases present in enzyme prepara-
tions during in vitro digestion. Journal of Poultry Science 41, 229–235.
method that can be applied to protease for routine testing Isaksen M.F., Cowieson A.J. and Kragh K.M. (2010) Starch- and
and in quality control conditions. Protein-degrading Enzymes: Biochemistry, Enzymology and
Characteristics Relevant to Animal Feed Use, In: Partridge G.G.;
Bedford M.R. (Eds.), Farm Animal Nutrition, 2nd edition.; Publisher:
CABI North American Office, Cambridge, MA 02139, USA, 85–95.
Acknowledgements Johnson F.N. (1996) General tests and Assays, in: Food Chemicals
Codex. Publisher: National Academy Press, Washington, USA,
The authors wish to acknowledge the work of O. 812–813.
Locqueneux, M.D. L’Hotellier and K. Michel on perform- Kalmendal R. and Tauson R. (2012) Effects of a xylanase and protease,
ing the validation work at the Institute of Environmental individually or in combination, and an ionophore coccidiostat on per-
formance, nutrient utilisation, and intestinal morphology in broiler
Engineering and Biotechnology, Bordeaux, France. The
Journal of Applied Animal Nutrition