Apparent total tract and ileal amino acids digestibility values of vegetal protein

meals with dietary protease to broiler diet

Antonio G. Bertechini†,1, , Felipe Santos Dalolio†, Julio C.C. Carvalho†, Andressa C. Carvalho†,
Jose O.B. Sorbara‡
Animal Science Department, Federal University of Lavras, Minas Gerais, Brazil; and ‡DSM Nutritional
†

Products, São Paulo, Brazil

ABSTRACT: This experiment was carried out kg resulting in 15,000 unit of PROT/kg. The total
to study the effect of dietary exogenous mono- collection of excreta was held during 3 d, after 5
component protease on the coefficient apparent d for adaptation of broilers at the diets. The ileal
total tract (ATTD) and apparent ileal (AID) di- content was collected on d 21, after slaughter of
gestibility of amino acids of corn, soybean meal birds. The enzyme increased (P < 0.05) the ATTD
(SBM), and full fat soybean meal (FFSM) in and AID of most amino acids contained in SBM
broilers. A total of 400 males Cobb-500 (14 d of and FFSM. The digestibility of cysteine, gly-
age) were equally allocated in 80 metabolic cages cine, proline, and threonine had higher (P < 0.05)
(50 cm × 50 cm × 45 cm) in a completely random- ATTD and AID in all tested ingredients with the
ized design and a semi-controlled environment. use of protease. On average, the dietary protease
Eight treatments (basal diet with or without a increased in 5.19% and 3.86% the total and ileal
protease and three ingredients replacing the basal digestibility of amino acids, respectively. It was
diets in 40% to corn and 30% to SBM and FFSM, concluded that the dietary protease exerts major
with and without protease), with 10 replicates each effects on toasted full-fat soybean, followed by
were evaluated. The protease was added at 200 mg/ soybean meal and corn to broilers.
Key words: corn, dietary enzyme supplementation, ileal amino acids digestibility, full-fat soy-
bean meal, soybean meal

© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society
of Animal Science.
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribu-
tion, and reproduction in any medium, provided the original work is properly cited.
Transl. Anim. Sci. 2020.4:1-8
doi: 10.1093/tas/txaa219

INTRODUCTION they have digestibility of amino acids ranging

from 80% to 90% (NRC, 1994; Rostagno et al.,
Most poultry diets are formulated with 2017). Although these ingredients are considered
vegetable ingredients such as corn, soybean high in protein digestibility, there is still a portion
meal (SBM), and toasted full-fat soybean meal of nutrients, particularly amino acids, that are not
(FFSM), being these ingredients considered pri- used during bird´s metabolism and can contribute
mary in America. There is a large knowledge of for excretion of nitrogen to the environment
the contents of metabolizable energy and amino (Stefanello et al., 2016; Cowieson et al., 2018).
acids digestibility of these feedstuffs. In general, Differences on ileal digestibility between feed-
stuffs are common in the literature. Coefficients
Corresponding author: bertechini@ufla.br
1 of apparent ileal digestible (AID) for lysine con-
Received August 6, 2020. tained in SBM, for example, can vary of 0.810
Accepted November 19, 2020. to 0.901 in diets with no enzyme addition. For
1
2 Bertechini et al.

FFSM the variations are greater, from 0.798 Experimental Procedures

(Clarke and Wiseman, 2005) to 0.856 (Rostagno
et al., 2017), due to the type of processing and fat Eight treatments (basal diet with or without a
content of this feedstuff. These difference between protease and three ingredients replacing the basal
ingredients of diets can influence the efficiency of diets with and without protease), with 10 replicates
meat production of broilers. each were evaluated. The replacement of basal diet
The industrial protease production caused ex- for the tested ingredients were 40% for corn and
pectations for its use since amino acids represent 30% for SBM and FFSM (Matterson et al., 1965).
high costs in the diets for animals. In broilers, due The protease used was Ronozyme ProAct 75,000
to intensive breeding, the dietary nutritional needs PROT U/g (EC. 3.4.21, DSM Nutritional Products,
are higher, mainly of amino acids. Thus, the use of Basel, Switzerland), an alkaline serine hydrolase
exogenous enzymes such as protease may contribute derived from Nocardiopsis prasina and produced by
to the increased efficiency of use of dietary protein. Bacillus licheniformis. The protease was added to
Studies with proteases in diets with several diets a expense of the inert (kaolin) at 200 mg/kg,
ingredients for broilers have shown increase in resulting in 15,000 unit of protein U/kg. The ana-
digestibility of methionine, cysteine, histidine, lyzed basal diet and the ingredients were in Table 1.
valine, isoleucine, leucine, serine, glycine, and The total collection of excreta was held during
proline (Tejedor et al., 2001; Kocher et al., 2003; 3 d (19, 20, and 21 d of age of broilers), after 5 d for
Bertechini et al., 2009; Carvalho et al., 2009; Angel adaptation of broilers at the diets. The collections
et al., 2011; Freitas et al., 2011; Kamel et al., 2015). were made twice a day, in order to avoid possible
In order to contribute to the upgrading of chem- fermentation of excreta. The beginning and end
ical composition tables and digestibility of feed- of the collection were scored using the ferric oxide
stuffs for broilers, the objective of this study was to
evaluate the effect of dietary monocomponent pro- Table 1. Analyzed composition and mean particle
tease on coefficient of apparent total tract digest- size (MPS) of basal diet and ingredients used in the
ibility (ATTD) and AID of amino acids of corn, study (as-fed basis)a
SBM and FFSM in broilers diets. Item (g kg‒1) Basal diet Corn SBM FFSM
Dry matter, g kg‒1 899.5 880.1 892.6 894.8
MATERIAL AND METHODS Crude protein, g kg‒1 210.5 78.0 458.7 325.8
Crude fat, g kg‒1 55.3 37.7 16.5 208.3
All experimental procedures in this study were Crude fiber 35.3 16.8 57.5 56.0
approved by the Committee for Use of Animals Ash, g kg‒1 28.2 13.2 49.8 43.8
in Research of the Federal University of Lavras, Urease activity – 0.05 0.07
Minas Gerais, Brazil. Gross energy, MJ kg‒1 12.6 16.2 17.1 21.1
Amino acids, g kg‒1
Birds and Housing Alanine 9.5 5.8 18.7 15.9
Aspartic acid 17.2 3.1 30.9 26.8
A total of 400 male Cobb-500 chicks of 1 Arginine 12.5 3.5 31.0 27.8
Cysteine 3.7 1.8 5.5 5.7
d of age, obtained from commercial hatchery,
Glycine 7.9 3.8 18.7 15.2
properly vaccinated against Marek’s were used.
Glutamic acid 33.9 5.4 53.9 36.4
The birds were raised in the litter (wood shav-
Histidine 4.8 1.9 10.8 9.8
ings) from 0 to 13 d of age, feed with corn–soy- Isoleucine 8.3 2.9 20.7 16.6
bean meal diet formulated according to the Leucine 16.2 8.8 33.7 27.5
recommendations of Rostagno et al. (2017). At Lysine 12.3 2.4 28.1 23.2
14 d of age, the birds (488 ± 10 g of live weight) Methionine 4.7 1.7 6.4 4.6
were randomly placed into 80 metabolism cages Phenylalanine 9.3 3.5 23.2 18.1
(50 cm × 50 cm × 45 cm) with controlled en- Proline 11.6 7.8 22.7 19.0
vironment (average of 24.8 ± 1.3 °C of tem- Serine 8.9 2.9 24.1 18.5
perature, 65.4 ± 2.1% of relative humidity and Threonine 7.6 3.0 18.6 14.5
24L:0D light program). The cages were equipped Tyrosine 7.4 2.2 16.7 12.5
Valine 10.0 3.8 22.6 18.5
with individual feeder, nipple drinkers and metal
Total AA 185.8 73.5 418.1 288.2
tray for excreta collection. Feed and water were
MPS, µm ± sd 755±7 765±5 785±8 727±7
provided for ad libitum during all experimental
period (14–21 d). a
Amino acid analyzed by HPLC method.

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 Protease on vegetal protein meal 3

(1%) in the diet. Marked excreta were the reference

for the beginning and ending of period of collec- [(AA/Celite) d − (AA/Celite) i]
AID =
tion. The collected excreta were placed in pre-la- (AA/Celite) d
beled plastic bags and stored in a freezer at ‒4 °C
where ATTD is the apparent total tract digest-
until the end of the experimental period. Then, the
ibility of individual AA (%), AID is the apparent
samples were thawed, weighed, homogenized, and
ileal digestibility of individual AA (%), (AA/
aliquots of 300 g were predried in oven with forced
Celite)d) is the dietary ratio of amino acid to Celite
ventilation at 55 °C during 72 h. Subsequently,
and (AA/Celite)i is the ratio of amino acid to Celite
they were weighed and milled in an analytical mill
in ileal digesta.
“Willey” (Tecnal, Croton TE-625, Piracicaba, São
The differentials of the enzyme effects on each
Paulo, Brazil), together with the diets and tested in-
ingredient expressed as a g kg‒1 were calculated.
gredients for laboratorial analysis. The gross energy
(GE) was determined using a calorimeter (model
1261, Parr Instrument Company, Moline, IL, Statistical Analysis
USA) and expressed in MJ GE/kg natural matter.
Analyses of dry matter and crude protein (CP) were To compare the effect of dietary enzyme sup-
made according to the methodology described in plementation for each tested ingredient, data were
AOAC (2000) and analysis of AA made in HPLC analyzed by ANOVA using the General Linear
(SHIMADZU, Model LC-10AT, Shimadzu Corp., Models procedure SAS (2004). Differences were
Kyoto, Japan) methodology (White et al., 1986; considered significant when P < 0.05 and means
Hagen et al, 1989). were compared by the Least Significant Difference
For determination of AID of amino acid, with test.
24 h prior to collection of the ileum of broilers,
diets were mixed with 1% Celite marker (Scoot RESULTS
and Boldaji, 1997). This indigestible marker
was provided to the animals until the time of The dietary protease increased (P < 0.05) the
slaughter. At 21 d of age, four birds of each cage coefficient of ATTD and AID of amino acids in
were slaughtered by cervical dislocation to obtain all tested ingredients, however, in a different way.
the ileal content. Immediately after slaughter, the In corn, there were increases in ATTD, more spe-
ileum was exposed by abdominal incision and cifically to cysteine, glycine, isoleucine, proline,
a segment of 15 cm, starting at 1.0 cm from the serine, threonine, and valine (Table 2). In general,
ileo-cecal junction, was sectioned. With a light increases in ATTD range from 4.05% to 11.7% be-
hand pressure, the content of this segment was tween the different amino acids. For coefficient of
collected in plastic containers properly identi- AID, the dietary protease increased (P < 0.05) in-
fied, sealed, and then stored in a freezer at ‒4 °C. creases range from 2.32% to 8.28%. The greatest
Posteriorly, the ileal contents were lyophilized increases in the ATTD were observed for glycine
under vacuum and at a temperature of ‒40 °C for (11.7%) and the threonine (9.9%), while for AID,
72 h. After this, samples were macerated and for- the greatest increases occurred with glycine (8.3%)
warded for analysis of AA and acid-insoluble ash and serine (7.4%).
(Scoot and Boldaji, 1997) to determine the values​​ For SBM, protease supplementation increased
of AID of amino acid. (P < 0.05) the ATTD and AID of total amino acids,
more specifically of arginine, cysteine, glycine, his-
tidine, lysine, methionine, proline, threonine, tyro-
Digestibility Procedures of Amino Acids
sine, and valine (Table 3). In general, increases in
All digestibility coefficients of amino acids ATTD range from 1.04% to 12.09% between the
were determined on dry matter basis. ATTD and different amino acids, while for AID, increases
AID were calculated as follows: range from 2.38% to 10.02%. The greatest increases
in the ATTD were observed for glycine (12.09%),
ATTDAA = ATTDAAbasal diet
tyrosine (10.62%), and cysteine (10.23%), while for
(ATTDAAtest diet − ATTDAAbasal diet ) AID the greatest increases occurred with alanine
+
Level of inclusion, % (10.02%) and cysteine (9.91%).
For FFSM, the dietary protease increased
(AIDAAtest diet − AIDAAbasal diet ) (P < 0.05) the ATTD and AID of all amino acids,
AIDAA = AIDAAbasal diet +
Level of inclusion, % except tyrosine (ATTD and AID), arginine, aspartic
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4 Bertechini et al.

Table 2. Coefficients of ATTD and AID of amino acids of corn with or without dietary protease
Amino acids ATTD† AID†
Protease, ppm 0 200 SEM 0 200 SEMa
Alanine 0.793 0.828 0.0137 0.823 0.835 0.0071
Arginine 0.815 0.836 0.0122 0.837 0.844 0.0131
Aspartic acid 0.845 0.846 0.0065 0.902 0.911 0.0077
Cysteine 0.750b 0.792a 0.0053 0.836b 0.855a 0.0088
Glutamic acid 0.907 0.918 0.0042 0.896 0.901 0.0056
Glycine 0.659b 0.737a 0.0110 0.687b 0.744a 0.0155
Histidine 0.855 0.860 0.0031 0.884 0.907 0.0076
Isoleucine 0.822b 0.852a 0.0033 0.857b 0.877a 0.0079
Leucine 0.883 0.895 0.0029 0.874 0.887 0.0067
Lysine 0.878 0.880 0.0022 0.872 0.882 0.0086
Methionine 0.873 0.875 0.0050 0.872 0.884 0.0036
Phenylalanine 0.871 0.881 0.0026 0.900 0.910 0.0056
Proline 0.825b 0.860a 0.0053 0.843b 0.881a 0.0076
Serine 0.795b 0.833a 0.0049 0.832b 0.894a 0.0088
Threonine 0.722b 0.793a 0.0085 0.813b 0.871a 0.0105
Tyrosine 0.869 0.878 0.0033 0.919 0.934 0.0078
Valine 0.814b 0.847a 0.0071 0.799b 0.824a 0.0096
Total AA 0.838b 0.860a 0.0031 0.851b 0.874a 0.0045
Total × ileal‡ 0.839B 0.865A

†
Different lower case letters within a row indicate significantly different means by least significant difference test (P < 0.05).
‡
Different capital letters in the line indicate significantly different means by F test (P < 0.05). The data is from 10 replicates.
a
SEM: standard error of the mean.

Table 3. Coefficients of ATTD) and AID of amino acids of SBM with or without dietary protease
Amino acids ATTD† AID†
Protease, ppm 0 200 SEM 0 200 SEMa
Alanine 0.710 0.751 0.0093 0.767b 0.844a 0.0092
Arginine 0.796b 0.866a 0.0084 0.869b 0.933a 0.0135
Aspartic acid 0.879 0.901 0.0085 0.880 0.884 0.0173
Cysteine 0.592b 0.652a 0.0121 0.816b 0.896a 0.0199
Glutamic acid 0.705 0.719 0.0098 0.814 0.831 0.0138
Glycine 0.806b 0.903a 0.0091 0.868b 0.928a 0.0139
Histidine 0.779b 0.828a 0.0091 0.850 0.862 0.0108
Isoleucine 0.836b 0.873a 0.0039 0.856 b 0.894a 0.0119
Leucine 0.827 0.847 0.0060 0.825 0.833 0.0166
Lysine 0.889b 0.926a 0.0124 0.862b 0.930a 0.0129
Methionine 0.785b 0.834a 0.0057 0.851b 0.893a 0.0132
Phenylalanine 0.865 0.879 0.0039 0.832b 0.880a 0.0156
Proline 0.775b 0.820a 0.0082 0.759b 0.817a 0.0114
Serine 0.854 0.884 0.0065 0.797 0.822 0.0259
Threonine 0.857b 0.876a 0.0076 0.788b 0.852a 0.0103
Tyrosine 0.784b 0.868a 0.0098 0.824 0.843 0.0137
Valine 0.836b 0.870a 0.0070 0.817b 0.880a 0.0111
Total AA 0.793b 0.833a 0.0081 0.818b 0.868a 0.0145
Total × ileal‡ 0.813B 0.848A

†
Different lower case letters within a row indicate significantly different means by least significant difference test (P < 0.05).
‡
Different capital letters in the line indicate significantly different means by F test (P < 0.05). The data is from 10 replicates.
a
Standard error of the means.

acid, isoleucine, tyrosine, and valine (only AID) for AID, increases range from 1.73% to 7.29%. The
(Table 4). Increases in ATTD range from 3.13% to amino acids that had greater increases with the use
11.32% between the different amino acids, while of the enzyme for the ATTD were the glutamic
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 Protease on vegetal protein meal 5

Table 4. Coefficients of ATTD and AID of amino acids of FFSM with or without dietary protease
Amino acids ATTD† AID†
Protease, ppm 0 200 SEM 0 200 SEMa
Alanine 0.734b 0.812a 0.0059 0.779b 0.796a 0.0060
Arginine 0.817b 0.874a 0.0028 0.888 0.891 0.0028
Aspartic acid 0.849b 0.909a 0.0122 0.910 0.928 0.0120
Cysteine 0.796b 0.837a 0.0122 0.792b 0.849a 0.0124
Glycine 0.897b 0.942a 0.0055 0.889b 0.928a 0.0117
Glutamic acid 0.617b 0.686a 0.0039 0.722b 0.755a 0.0038
Histidine 0.812b 0.854a 0.0029 0.899b 0.923a 0.0028
Isoleucine 0.826b 0.881a 0.0058 0.863 0.864 0.0060
Leucine 0.836b 0.884a 0.0068 0.894b 0.922a 0.0071
Lysine 0.899b 0.927a 0.0071 0.902b 0.935a 0.0072
Methionine 0.809b 0.862a 0.0082 0.850b 0.874a 0.0080
Phenylalanine 0.874b 0.908a 0.0066 0.934b 0.951a 0.0067
Proline 0,790b 0.864a 0.0061 0.838b 0.881a 0.0059
Serine 0.846b 0.889a 0.0037 0.900b 0.932a 0.0035
Threonine 0.805b 0.867a 0.0082 0.881b 0.919a 0.0080
Tyrosine 0.886 0.899 0.0051 0.946 0.957 0.0049
Valine 0.798b 0.862a 0.0071 0.758 0.762 0.0069
Total AA 0.817b 0.868a 0.0064 0.862b 0.886a 0.0068
Total × ileal 0.843B 0.0065 0.874A 0.0048

†
Different lower case letters within a row indicate significantly different means by least significant difference test (P < 0.05).
‡
Different capital letters in the line indicate significantly different means by F test (P < 0.05) The data is from 10 replicates.
a
Standard error of the means.

acid (11.3%), alanine (9.92%), proline (9.33%), and with 24.1% in the SBM). In these cases, the effects
valine (8.05%), and for AID the cysteine (7.29%), of dietary protease were more effective. Greater
proline (5.12%), glutamic acid (4.6%), and glycine protease effect was also observed by Dessimoni
(4.35%). et al. (2019) when it reduced dietary protein levels.
The addition of the enzyme increased the total
digestibility of both fecal and ileal amino acids
DISCUSSION
in all the feedstuffs studied. Other studies have
The dietary protease supplementation was ef- also indicated the effectiveness of this enzyme in
ficient in to increase the use of amino acids for increasing the total digestibility of the amino acids
broilers. To the corn, in average, an increase of of corn (Bertechini et al., 2009) and of diets based
5.59% and 4.63% for ATTD and AID, respectively. on corn-soybean meal (Angel et al., 2011). In the
For SBM, these increases were 6.58% and 6.82% previous paper, the authors reduced the dietary CP
and for FFSM the increase was 6.49% and 3.67%, levels by 10% in relation to the control diet and
respectively. The chemical composition of the feed- observed the same performance of the birds when
stuffs used in the present study are similar to those compared to the control group.
shown in the feed composition tables (NRC, 1994; In general, the effects of dietary protease were
Rostagno et al., 2017), except to the CP content of different on each amino acid and on each feed-
the FFSM, that was lower than that observed in the stuff. The most significant effects of the enzyme
literature. Besides good source of AA, the FFSM were observed in FFSM and followed by SBM
has a high fat content which may influence the ef- and corn. At FFSM, 70% of amino acids had
fects of exogenous protease (Duke and Evanson, increase in the ileal digestibility values, 65% of
1972; Li and Sauer, 1994). amino acids in the SBM and only 41% of amino
The protease used in the present study is a acids contained in the corn. Only cysteine, glycine,
serine hydrolase. The increase of digestibility of this proline, and threonine had increases in digest-
amino acid was observed only in the corn and in ibility in all feedstuffs, regardless of the method
the FFSM. This result may be related to the lower of determination of digestibility (ATTD or AID).
content of this amino acid contained in these feeds Although glycine, threonine, and proline are pre-
(2.9% in the corn, 14.0% in the FFSM compared sent in large amounts in the intestinal fluids, there
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6 Bertechini et al.

was an increase in the fecal and ileal digestibility Higher values of ileal digestibility of amino acids
of these nutrients. Glycine corresponds to about were observed with FFSM (87.4% vs. 85.2% for corn
90% of the total amino acids secreted in the bile and 84.3% for SBM). This result may be related to
(Souffrant, 1991), whereas 95% of the intestinal the higher oil content contained in FFSM. Hulan
mucus is composed by glycoprotein rich in threo- and Bird (1972) reported that the increase of the
nine, serine and proline (Neutra and Forstner, dietary fat influences the activities of protease, lipase,
1987). This result shows that the relationship be- and amylase in pancreatic juice. Duke and Evanson
tween digestible amino acids can be altered with (1972) reported that fats are powerful inhibitors of
the use of exogenous enzymes in the diet. gastric emptying. According to Li and Sauer (1994),
In this sense, several studies have shown that the delay of gastric emptying also delay the passage
dietary amino acids can modify the metabolic of the digestion through the small intestine, increas-
changes in the organisms. Grimble et al. (1992) ing the digestibility of the nutrients. However, in the
indicated that cysteine, a sulfur amino acid, is present study there were no differences between fecal
of prime importance in facilitating increases in and ileal digestibilities when considering total amino
liver glutathione (GSH), Zn and protein con- acids. Similar results were found by Honda et al.
tent in rats. In broilers, Tsiagbe et al. (1987) in- (2009; 2010) working with roosters fed on diets con-
dicated that cysteine is an important nutrient for taining fats ranging from 3% to 10%.
the immune response, modulating lymphocyte, The use of nutritional matrices with average in-
and macrophage functions (Droge et al., 1991). crements for all amino acids is an inconsistency, since
Furthermore, feeding l-cysteine increases tissue enzymes are specific and increase the digestibility of
GSH level, which may be beneficial for growth amino acids differently. The ideal is to use a nutri-
of chicks reared in conditions of oxidative stress tional matrix that contemplates the differences in the
(Tsiagbe et al., 1987). By other hand, excess levels action of the enzyme in each amino acid.
of cysteine and homocysteine can induce tibial The ileal digestibility over total is adopted to
dyschondroplasia in broiler chicks. Thus, stud- minimize the effects of the microbiota present in
ies with exogenous enzyme supplementation in the large intestine (Ten Doeschate et al., 1993).
broilers are need. However, the use of the ileal digestibility technique
When the ATTD technique was applied, the is costly and, in most cases, involves the sacrifice of
major effects of the protease were for FFSM, large numbers of birds. Thus, studies of ileal and
followed by SBM and corn (Figure 1). With re- fecal digestibility of each amino acid in birds are
spect to the AID technique, it was verified that important so that future studies can establish the
the FFSM presented a smaller increment in rela- relationships between the different values and be
tion to the SBM. This result may be related to the able to predict the values of ileal digestibility from
higher fiber content contained in the SBM (6.5% fecal digestibility.
vs. 3.4% contained in the FFSM) or the oil con-
tained in the FFSM (18.4% vs. 3.8% for corn and
1.2% for SBM). CONCLUSION
The use of dietary protease influences in a dif-
9
ferent way the total and ileal digestibility of amino
8 acid in feedstuffs for broilers. Most evident effects
7 are observed with higher crude protein feedstuffs.
The digestibility of cysteine, glycine, proline, and
6
threonine are more influenced by the addition of
Improved, %

5 this exogenous protease in the diet. The mean of

4 protease effects for all amino acids and in all in-
3 gredients studied were 5.19% and 3.86% for ATTD
and AID, respectively. The total collection method
2
was lower by 3.7% of amino acids digestibility than
1 the ileal method for vegetal ingredients.
0
Corn SBM FFSM Average
ACKNOWLEDGMENTS
Protein meals

Figure 1. Protease effect (%) on ATTD and AID of the feed The authors thank DSM Nutritional Products,
ingredients. São Paulo, Brazil, for the financial support and the
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 Protease on vegetal protein meal 7

enzyme provided to conduct this work. Likewise, of amino-acids in food. J. Assoc. Off. Anal. Chem.
they are grateful for CNPq that granted support 72:912–916.
Honda, K., H. Kamisoyama, Y. Isshiki, and S. Hasegawa.
for students. All authors provided intellectual input 2009. Effects of dietary fat levels on nutrient digestibility
and reviewed this manuscript. at different sites of chicken intestines. J. Poult. Sci. 46:
Conflict of interest statement. The authors de- 291–295. doi.org/10.2141/jpsa.46.291
clare no conflict of interest. Honda, K., H. Kamisoyama, S. Kubo, T. Motoori, and
S. Hasegawa. 2010. Effects of dietary fat levels on
amino acid digestibility at different sites of chicken in-
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AOAC. 2000. Official methods of analysis. 17th ed. 102.4.459
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