Animal Nutrition 4 (2018) 401e409

Contents lists available at ScienceDirect

Animal Nutrition
journal homepage: http://www.keaipublishing.com/en/journals/aninu/

Original Research Article

Development of an in vitro protein digestibility assay mimicking the

chicken digestive tract*
Dervan D.S.L. Bryan*, Dawn A. Abbott, Henry L. Classen*
Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, S7N 5A8, Saskatchewan, Canada

a r t i c l e i n f o a b s t r a c t

Article history: It is difficult to obtain in vivo digestion kinetics data of high protein ingredients using chickens. Collecting
Received 16 March 2018 kinetics data requires repeated sampling of digesta from the small intestine during the digestion process,
Received in revised form which is not easily accomplished due to the anatomical structure of chicken digestive tract. An in vitro
17 April 2018
technique is proposed for measuring the digestion kinetics of protein sources fed to chickens. The
Accepted 18 April 2018
Available online 7 May 2018
method has a 30 min gastric and 3 h intestinal phase. Five hundred milligram crude protein (CP)
equivalent of each meal sample (CP ¼ % N  6.25) was digested with pepsin (28,260 units) in 50 mL
polyethylene centrifuge tubes for 30 min in a shaking water bath (150 strokes/min; 30 mm stroke length)
Keywords:
Protein digestion rate
at 41  C. The 6.5 mL pancreatin was selected as the enzyme concentration for the intestinal phase, during
Soybean meal which time 500 mL aliquots were collected at 0, 15, 30, 45, 60, 90, 120, 150, 180 and 240 min. Samples
Corn gluten meal were diluted 1:820 with HCl and sodium acetate buffer, and then mixed with ninhydrin reagent (2:1) at
Corn distillers dried grains with solubles 100 ± 2  C for 15 min and spectrometric readings taken at 568 nm. To validate the assay, 5 replications of
Fish meal soybean meal (SBM), corn gluten meal (CGM), corn distillers dried grains with solubles (CDDGS), porcine
Porcine meal meal (PCM), fish meal (FM) and casein (CA) were digested. The digestion data were modeled with PROC
NLIN procedure, and the intra coefficient of variation (CV) assessed using PROC MEANS of SAS 9.4. The
digestion values at 180 min were SBM 95 ± 4, FM 93 ± 3, PCM 68 ± 4, CGM 82 ± 3 and CDDGS 70 ± 2.
Intra CV for SBM, CGM, CDDGS, PCM and FM were 5%, 5%, 12%, 10% and 2%, respectively. The estimated
fractional digestion rates for SBM, CGM, CDDGS, FM and PCM were 0.023, 0.013, 0.009, 0.024 and 0.013,
respectively. In conclusion, the proposed in vitro technique estimated the rate and extent of the digestion
of CP for the meals with low intra CV.
© 2018, Chinese Association of Animal Science and Veterinary Medicine. Production and hosting
by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction quality of protein in broiler diets has increased in importance, with

quality being defined by amino acid digestibility and balance
Broiler chickens have been extensively selected for rapid growth (Ravindran and Bryden, 1999). It is a general consensus among
and as a consequence the ability of the birds to deposit body protein poultry nutritional researchers that the jejunum and proximal
has increased dramatically (Zuidhof et al., 2014). Concurrently, the ileum are the major sites for amino acid absorption. However, little
information can be found pertaining to how much protein from
common ingredients gets digested in the proximal and distal por-
tions of the small intestine.
*
Presented at Annual Poultry Science Meeting, Louisiana, USA, July 12, 2016. In vivo assays are considered to be the gold standard for
* Corresponding authors.
E-mail addresses: [email protected] (D.D.S.L. Bryan), [email protected]
assessing ingredient nutritional quality in poultry (Fuller, 1991).
(H.L. Classen). In vivo estimation of protein quality of a feed ingredient is normally
Peer review under responsibility of Chinese Association of Animal Science and achieved by feeding the ingredient to the intended animal while
Veterinary Medicine. assessing the extent to which nutrients are absorbed by the ter-
minal intestine. Protein quality can also be evaluated using in vitro
chemical methods (Boisen and Eggum, 1991). In vitro assays are less
expensive, can evaluate more ingredients, and are less time
Production and Hosting by Elsevier on behalf of KeAi
consuming than in vivo assays. Historically, the focal point of

https://doi.org/10.1016/j.aninu.2018.04.007
2405-6545/© 2018, Chinese Association of Animal Science and Veterinary Medicine. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
402 D.D.S.L. Bryan et al. / Animal Nutrition 4 (2018) 401e409

assessing protein quality for chickens has been based on the extent The reagents used in this study were obtained from the
of digestion, and as a result, little data are available on the rate at following sources. Tin (II) chloride dehydrate (CAS 107-21-1),
which proteins are digested and absorbed. benzoic acid (CAS 65-85-0), glacial acetic acid (CAS 64-19-7), tri-
The rate of digestion of protein along the digestive tract has chloroacetic acid solution (Sigma T0699), hemoglobin (Sigma
been known to have significant biological effects in other species H2625), pepsin (P7125-100 g; CAS 9001-75-6), Na-benzoyl-L-
(Boirie et al., 1997; Ørskov and McDonald, 1979) and the same could arginine ethyl ester (Sigma B4500), Trizma Base (Sigma T1503), N-
be true for poultry. In vivo protein nutritional research in humans benzoyl-L-tyrosine ethyl ester (Sigma B6125), methanol (Sigma
suggested that the sequential breakdown of proteins having M1775), N-succinyl-Ala-Ala-Ala-p-nitroanilide (Sigma S4760) and
different digestion rates modulated tissue protein synthesis and ninhydrin (CAS 485-47-2) were obtained from Sigma Chemical Co.
deposition (Boirie et al., 1997). The sequential breakdown of protein (St. Louis, MO, USA). Ethylene glycol (CAS 107-21-1), sodium hy-
into intermediate peptides is considered to be the rate limiting step droxide (CAS 1310-73-2), sodium acetate trihydrate (CAS 6131-90-
for the digestion and absorption of soybean meal (SBM) protein in 4), calcium chloride (CAS 10035-04-8), guar gum (CAS 9000-30-0)
poultry diets (Sklan and Hurwitz, 1980), which might also be the and hydrochloric acid (7647-01-0) were obtained from Fisher Sci-
case for other protein sources commonly fed to poultry. Therefore, entific (Pittsburgh, PA, USA). The liquid bovine pancreatin (62,500
the degradation kinetics and bioavailability of proteins are both USP trypsin units/mL) was purchased from RENCO (10 London
important factors, which could be considered when trying to Street, Eltham 4322, New Zealand).
maximize yield in poultry production.
Extensive research is available on the extent of digestibility for 2.1. Colorimetry assay
various high protein ingredients estimated using in vivo and in vitro
procedures. However, information on the degradation character- 2.1.1. Ninhydrin reagent composition
istics of feed ingredients (in vivo or in vitro) for poultry is scarce and A 4 mol/L sodium acetate buffer was prepared by dissolving
this type of research is often limited to human research (Dangin 544 g of sodium acetate trihydrate in 100 mL of warm glacial acetic
et al., 2001; Koopman et al., 2009). Most in vivo techniques used acid and then Millipore water was added to make a total volume of
to evaluate protein degradation in humans and other animal 1,000 mL. The tin (II) chloride solution was prepared by adding 1.2 g
research require the use of expensive isotope labeling of pure tin (II) chloride to 12 mL of ethylene glycol and then vortexing to
proteins and tracers (Boirie et al., 1997). Less expensive and time dissolve all the tin (II) chloride. To prepare the ninhydrin reagent,
consuming in vitro methods have been used to obtain protein 9.75 g of ninhydrin were dissolved in 366 mL ethylene glycol, and
digestion data in ruminant species (Boila et al., 1980) and may have then 122 mL 4 mol/L sodium acetate buffer were added. This so-
value for poultry. Currently, there is no in vitro method which es- lution was mixed for 5 min with a magnetic stir bar before the
timates protein degradation kinetics for poultry. addition of 12 mL of tin (II) chloride solution and mixing for another
The purpose of this research was to develop an in vitro protein 5 min.
digestibility assay for poultry specific, which could predict the
degradation kinetics of high protein feed ingredients commonly fed 2.1.2. Validation of ninhydrin reagent
to poultry. A multi-enzymatic digestion technique using gastric and The absorbance spectrum for SBM, casein (CA), corn distiller's
intestinal digestion phases was defined and validated. The digestive dried grains with solubles (CDDGS) and corn gluten meal (CGM)
tract transit time in broiler chickens has been reported to be 2 to were determined as follows. All samples were ground to pass
3.5 h (Hughes, 2008; Svihus et al., 2002), so the optimum enzyme through a 0.5 mm screen using a Retsch Ultra Centrifugal Mill ZM
to substrate concentration that resulted in the most effective de- 200 (Haan, Germany). The CP contents of all the meals were
gree of digestion within 3 h was used as a criterion for the assay. determined as N  6.25 using the Dumas method, where N con-
The condition for the colorimetric assay used to evaluate the degree tents were determined using a Leco nitrogen analyzer (Model
of digestion was optimized. The effectiveness of the in vitro di- 601e500e100, Serial # 3211, Leco Corporation, St. Joseph, MA,
gestions technique on a variety of high protein ingredients was USA). Samples (500 mg CP equivalent) were weighed and placed in
tested. This in vitro protein digestibility assay was developed to individual 100 mL Pyrex glass bottles (No.14395) after 6 mol/L
predict the rapidly and slowly undigested protein fractions of in- hydrochloric acid was added at 4 mL per 100 mg of sample weight.
gredients, as well as the rate and extent of digestion of the proteins. The samples were gently mixed by swirling, capped and placed in
an oven at 110  C for 24 h. After 24 h hydrolysis, samples were
2. Material and methods allowed to cool to room temperature and then filtered through
Whatman Grade 601 filter paper. An aliquot of the sample was
The following methods illustrate the stages which were collected after filtering and the pH was adjusted to 7 ± 0.5 with
involved in the development of the proposed in vitro assay. The first sodium hydroxide. The filtered sample was diluted with Millipore
stage describes an appropriate colorimetry assay for identifying water to give 0.36 mg CP per mL based on the initial 500 mg CP of
changes in a protein sample due to hydrolysis of peptide bonds. The the sample that was hydrolyzed.
chemical composition of the reagent, its shelf life and wavelength Each sample (100 mL) was mixed with 1,900 mL of Millipore
sensitivity during reactions were evaluated and optimized for the water and 1,000 mL of ninhydrin reagent in disposable glass culture
in vitro assay. The second stage involved the establishment of the tubes (borosilicate glass 16  100 mm, No. 14-961-29). A blank
conditions for the in vitro assay gastric and intestinal digestion sample with 2,000 mL of Millipore water and 1,000 mL of ninhydrin
phase. The composition of the buffers which were compatible to reagent was prepared. Glass marbles were placed on top of each
the enzymes used in the gastric and intestinal phase was identified. tube and the tubes were placed in a boiling water bath for 10 min.
The optimal units of pepsin for the gastric phase were elucidated The tubes were allowed to cool for 5 min before 200 mL of sample
using a dose response study over a 30 min digestion time frame. were pipetted in triplicate into a 96 well plate (Falcon 353910 U-
Selection of enzyme dose in the pancreatin for the intestinal phase Bottom well). The samples were read from 200 to 999 nm at 1 nm
was based on a dose response study building on the gastric phase wavelength interval using a microplate reader (Epoch 2, BioTeck,
results. The final stage of the research provides validation data for USA) set at 22  C.
high protein ingredients using the colorimetry procedure and the The concentration detection limits for the ninhydrin reagent
two-stage in vitro digestion assay. with a lysine standard were identified as follows. The lysine
 D.D.S.L. Bryan et al. / Animal Nutrition 4 (2018) 401e409 403

standard was prepared and diluted over the range from 0.25 to the sample with the 10 mmol/L HCl solution. After mixing, 1.5 mL
410 mg/mL. One milliliter of each dilution was mixed with 500 mL of pepsin solution with either 14, 130, 21,195, 28,260, 42,390 or 49,455
ninhydrin reagent in disposable glass culture tubes. A blank tube units of pepsin were added to 6 replicate tubes plus 3 blank tubes
was prepared by replacing the diluted sample with Millipore water. per enzyme concentration. All tubes were vortexed and 0.5 mL
Marbles were placed on the tubes before being placed in a boiling sample was taken for electrophoresis, and another 0.5 mL was
water bath according to the process described previously. A 200 mL placed in 20 mL of sodium acetate buffers (pH 6.5) for colorimetric
volume of sample was pipetted into a 96 well plate and read at evaluation. The tubes were placed in a shaking water bath (150
maximum absorbance (OD value) identified during the previous strokes/min; 30 mm stroke length) at 41  C for 30 min. After the
spectrum scan of the samples. gastric phase digestion, 0.5 mL sample was taken for electropho-
The shelf life of the ninhydrin reagent was evaluated over resis. Another 0.5 mL of sample was placed in 20 mL of sodium
304 d. A CA standard was prepared from the hydrolyzed CA sample. acetate buffer for colorimetric evaluation.
A fresh batch of ninhydrin reagent was prepared on the morning of The samples for electrophoresis were placed in a boiling water
day 1 and placed in a dark glass bottle wrapped in aluminum foil. At bath for 15 min immediately after collection to denature the pepsin
16:00 the CA standard was reacted with the reagent as outlined in and then samples were centrifuged at 2,140  g (Beckman Allegra 6
the absorbance spectrum test above, and then the ninhydrin re- model, Beckman Coulter, Inc. California, USA) for 1 min at 21  C. An
agent was placed on a shelf for storage at room temperature aliquot was taken from the supernatant of all samples and used for
(22 ± 3  C). This test was repeated on days 10, 14, 120, and 304 after electrophoresis. All samples were analyzed in a non-reducing
the first test was conducted. condition using an Agilent 2100 Bioanalyzer system (Agilent
Technologies, Lexington, USA) and the Protein 230 Chip assay
2.2. In vitro digestion assay following the manufacture's protocol.
The samples for colorimetric analysis were vortexed before
The in vitro assay method had a 30 min gastric and 3 h intestinal centrifuging at 2,568  g for 10 min at 21  C. A 100 mL aliquot of the
phase mimicking digestion in chickens based on previous research sample was diluted with 1,900 mL of Millipore water in disposable
(Svihus et al., 2002; Hughes, 2008). The ratio of optimum enzyme glass culture tubes and then 1 mL of ninhydrin reagent was added.
to substrate was verified for the gastric phase and the intestinal A marble was placed on top of the tubes before heat treatment. The
phase using enzyme dose response assays with SBM as the model cool reaction mixture (approximately 2 mL) was read in 4.5 mL
protein. Soybean was selected as the model protein because it is the disposable plastic cuvettes (Cat. No. 14,955,129 Fisherbrand) using a
most widely used protein source in poultry diets worldwide and its Genesys 20 spectrophotometer UVeVis (Termo Fisher Scientific
volume in production accounts for more than 69% of the world's Inc., Waltham, USA).
total protein source for animal feed (USDA, 2016).
2.2.3. Pancreatin dose response assay
2.2.1. Buffer compositions The pancreatin used was a liquid bovine enzyme (65,000 trypsin
Multiple buffer compositions were evaluated in preliminary units/mL) from RENCO (New Zealand). The activity of trypsin
studies to test their interaction with the colorimetry reagent and (30,667 BAEE units/mL), chymotrypsin (2,157 BTEE units/mL) and
their impact on the stability of enzymes. Sodium acetate buffers elastase (7 units/mL) were determined using Sigma EC 3.4.21.4, EC
with pH 12.5 and 6.5 were the most suitable for maintaining 3.4.21.1 and EC 3.4.21.36 assays, respectively. Six pancreatin levels
enzyme activity of the glycerol based pancreatin and compatibility (1, 3, 5, 6.5, 7.5 and 9 mL) were evaluated in the intestinal phase for
with the ninhydrin reagent used in this study. To prepare 1 L of a the enzyme dose response assay. Six replicate tubes and 3 blank
10 mmol/L HCl solution, 833 mL of concentrated HCL were mixed tubes per enzyme level were used during the pancreatin dose
with 999.167 mL of Millipore water. A 0.1 mol/L calcium chloride response assay.
solution was prepared by dissolving 33.3 g of calcium chloride in All samples were digested for 30 min using the selected pepsin
300 mL Millipore water. A benzoic acid solution was prepared by concentration identified in the pepsin dose response assay. A
dissolving 5.8 g of benzoic acid into 2 L of Millipore water. For the 500 mL volume of 4.9 mol/L sodium hydroxide solution was added
sodium acetate buffers preparation, 27.2 g of sodium acetate tri- to each tube immediately after gastric digestion. Sodium acetate
hydrate were dissolved in 500 mL benzoic acid solution. The pH buffer (pH 12.5) was added to each tube and the pH was adjusted to
was adjusted to 12.5 or 6.5 using saturated sodium hydroxide so- 7.5. The selected volume of pancreatin solution was added to the
lution (50%; wt/wt) and then the volume of the solution was made respective tubes to bring the final volume of the tubes up to
up to 2 L with Millipore water followed by the addition of 8 mL 26.5 mL. All tubes were vortexed and 0.5 mL sample was taken for
0.1 mol/L calcium chloride solution. All buffers were stored in the electrophoresis. Another 0.5 mL was taken from the tubes for
refrigerator until use. colorimetric evaluation and placed in 10 mL 10 mmol/L HCl solu-
tion, the mixture was then vortexed followed by the addition of
2.2.2. Pepsin dose response assay 10 mL of sodium acetate buffers (pH 6.5). Three marbles were
Pepsin activity was determined using the Sigma enzymatic placed in each tube and the tubes were placed in a shaking water
assay for pepsin (3.4.23.1). One unit of pepsin was defined as a bath (150 strokes/min; 30 mm stroke length) at 41  C for 180 min.
change in DA280 of 0.001 per min at pH 2.0 and 37  C measured as During the intestinal digestion phase, a 0.5 mL aliquot was taken for
trichloroacetic acid soluble products using hemoglobin as the colorimetry assay evaluation at 15, 30, 40, 60, 90, 120, 150 and
substrate. Pepsin was dissolved in 10 mmol/L HCl solution to give 180 min. At 180 min of digestion, a 0.5 mL aliquot was taken from
9,420, 14,130, 18,840, 28,260 or 32,970 units per mL of freshly each tube for electrophoresis.
prepared solution, which was used on the day of preparation. The
pepsin dose response assay was carried out in 50 mL polyethylene 2.2.4. In vitro assay validation
screw cap centrifuge tubes (VWR 21008-178). The assay intra-variability was evaluated using high protein
A 500 mg CP (N  6.12) equivalent of SBM sample was placed in feed ingredients. The ingredients selected for the validation study
centrifuge tubes with 50 mg of guar gum and 8.5 mL of 10 mmol/L were SBM, CGM, CDDGS, PCM, and FM; CA was used as a control
HCl solution. The tubes were vortexed to evenly mix and saturate because it represents a pure protein source. All samples were
404 D.D.S.L. Bryan et al. / Animal Nutrition 4 (2018) 401e409

ground to pass through a 0.5 mm screen before proximate anal- 3. Results

ysis. The moister content of all meal samples were determined
using method 990.03 (AOAC International, 2006). Protein sources 3.1. Validation of ninhydrin reagent
were analyzed for N using a Leco nitrogen analyzer (Model
601e500e100, Serial # 3211, Leco Corporation, St. Joseph, MA, Two major peaks were identified after a full spectrum scan of
USA) according to the combustion method 990.03 (AOAC the reactions between the ninhydrin reagent and the samples as
International, 2006) using 6.25 as the conversion factor to calcu- illustrated in Fig. 1. The first peak span was from 300 to 450 nm
late CP. Soybean meal was analyzed for trypsin inhibitor activity while the second peak was from 500 to 650 nm. The reaction was
following method 22-40 (AOAC International, 2006). All meals monitored for 30 min during which time there was no change in
were analyzed for protein dispersibility index (PDI) as outlined by the OD reading of the second peak. However, the first peak OD
Johnson (1970) and protein solubility in 0.2% potassium hydroxide decreased with time and by 30 min it was no longer present.
solution (Araba and Dale, 1990). The calcium and magnesium Evaluation of the second peak data from 500 to 630 nm (Fig. 2)
contents of all meal samples were analyzed using inductively using the PROC REG function of SAS 9.4 revealed that the inflection
coupled plasma optical emission spectrometry (ICP-OES) after point for all the samples was 568.
total acid digestion with HCl. The relationship of the ninhydrin reaction with the lysine stan-
A subset of each meal sample was hydrolyzed with 6 mol/L HCL dard was used to determine the detection limits of the reaction
as outlined in the validation of ninhydrin reagent section. The (Fig. 3). Below 2 mg/mL lysine, the absorbance values did not show a
protein content of the samples was calculated as N  6.25, and then linear trend, and above 400 mg/mL, the detector of the spectropho-
500 mg CP equivalent of each ground sample were placed in 5 tometer was saturated. The R2 value of the point between the lysine
replicate tubes. The samples were digested using the optimum standard concentrations and OD values obtained at each concen-
pepsin and pancreatin concentrations identified during the 2 dose tration was 0.97 for the range from 2 to 400 mg/mL of lysine. This
response assays. The 0.5 mL aliquots for the colorimetry assay were inferred that the OD reading of the sample was a good predictor of
only collected during the intestinal digestion phase at 0, 15, 30, 40, the amount of free amino and carboxyl group present in the reaction.
60, 90, 120, 150 and 180 min. Aging the ninhydrin reagent in dark bottles shielded from light,
kept the reagent relatively stable up to 120 days (Fig. 4). It took
14 d for the reagent to stabilize and during which time there was a
2.3. Calculations and statistics 0.119 OD reduction in the absorbance reading.

The protein digestibility of the samples was calculated using the

OD of the digested sample and the OD of the totally hydrolyzed
sample as follows:

Protein digestibility ð%Þ ¼

ðOD of digested sampleÞ=ðOD of 6 mol=L HCl hydrolyzedsampleÞ
100;

where OD ¼ the absorbance at 568 nm.

The absolute percentage of CP digested per minute was calcu-
lated using the following rate formula:

Protein digested per min ¼

½Digestible CP time ðxÞ  CP time ð yÞ=½Time ðxÞ  Time ð yÞ;

where x and y represent different time points during the 180 min Fig. 1. Absorbance spectrum from 150 to 950 nm for ninhydrin reagent reaction with
digestion period. casein, soybean meal (SBM), corn gluten meal (CGM), and corn distiller's dried grain
All the protein digestibility data were fitted to the following with solubles (CDDGS) hydrolyzed with 6 mol/L HCl at 100  C for 24 h.
modified 2 tail compartmental statistical models proposed by
Ørskov and McDonald (1979) using the PROC NLIN procedure of
SAS 9.4: P ¼ A þ B ð 1  ekd  t Þ, where P ¼ CP digested at a
specific time point, A ¼ rapidly digested CP fraction, B ¼ slowly
digested CP fraction, kd ¼ the rate at which B is digested over time
(fractional rate per min). This constant was set to negative since the
data represented increasing protein digestion over time, and
t ¼ time. The undigested fraction of the proteins UD was calculated
as 100  (A þ B) and the potential digestibility (PD) of the protein
was calculated as (A þ B).
The spectrum scan data were analyzed for the maximum in-
flection point using the PROC REG procedure. Correlation analysis
was performed between calculated digestibility and the models
predicted digestibility using PROC CORR, and the means of the ki-
netic constants were compared using the PROC MIXED procedure
of SAS 9.4 with probability of P  0.05 considered significant. If Fig. 2. Maximum absorbance spectrum of the ninhydrin reagent reaction with casein,
significant differences were found between means, LSD means soybean meal (SBM), corn gluten meal (CGM), and corn distillers dried grain with
statement was used to separate treatment means. solubles (CDDGS) with 6 mol/L HCl at 100  C for 24 h.
 D.D.S.L. Bryan et al. / Animal Nutrition 4 (2018) 401e409 405

Fig. 3. Relationship between the concentrations of lysine standard and absorbance

Fig. 5. Effects of pepsin concentration (units) on the molecular weight distribution of
values when reacted with ninhydrin reagent.
peptide from soybean meal digested for 30 min at 41  C. The ladder represents protein
and peptide fragments with molecule weights measured in kilo-Daltons (kDa). Each
mean represents 6 replicates per treatment.

Fig. 6. Effects of pepsin concentration (units) on CP hydrolysis (%) from soybean meal
digested for 30 min at 41  C. Each mean represents 6 replicates per treatment.

Fig. 4. Effects of ninhydrin reagent storage time on the absorbance reading of hy-
drolyzed casein. aed Means ± standard deviation with different letters are significantly
different (P < 0.05) and n ¼ 6.

3.2. Enzyme dose response assay

Increasing the concentration of pepsin from 14,130 to 49,455

units reduced the polypeptides between 46 and 28, 63 to 46 and 95
to 63 kDa according to the ladder standards (Fig. 5). This reduction
resulted in an increase in the concentration of peptides between 12
and 7 kDa and it confirmed that hydrolysis had taken place. The
colorimetry assay data presented in Fig. 6 had a similar trend to
what was observed for the peptide concentration between 7 and
12 kDa. By dividing the units of pepsin used in the assay by the
percentage CP hydrolyzed (Fig. 6), the CP hydrolyzed per unit of
enzyme can be calculated. This resulted in 0.188%, 0.189%, 0.163%,
0.116% and 0.114% hydrolyzed CP per unit of enzyme for the 5
enzyme concentrations, respectively. Fig. 7. Effects of pancreatin concentrations (1 mL ¼ 30,667 BAEE units of trypsin; 2,157
BTEE units of chymotrypsin, and 7 units of elastase) on the digestibility of soybean
The pepsin concentration of an in vitro assay can be selected
meal CP over 180 min of the intestinal phase at 41  C after predigesting with 28,260
based on a number of criteria. In this study, taking the cost of the units of pepsin. Each time point represents 6 replicates per treatment.
pepsin into consideration, having a minimum of 4% CP hydrolysis in
the gastric phase and the ability of the selected pepsin concentration hydrolysis per unit of enzyme, which indicated that those levels of
to produce a typical digestion curve (Fig. 7) in the intestinal phase enzyme might not be economical since the concentrations were
were the basic criteria for this assay. The 28,260 units of pepsin were almost doubled. In a preliminary study, the 4 lowest pepsin con-
selected because the units of pepsin below that level did not achieve centrations were used to digest a sample of SBM, then a standard
4% CP hydrolysis during the 30 min of the assay. Concentration above 7 mL volume of pancreatin was used for the intestinal phase of the
28,260 units gave a substantial reduction in the percentage of CP digestion. The digestion of the samples was monitored over a 3-h
406 D.D.S.L. Bryan et al. / Animal Nutrition 4 (2018) 401e409

period. The 28,260 units of pepsin gave a time-dependent digestion CDDGS was similar to all samples evaluated. The CV for fraction (B)
curve, which we assumed to be the case in vivo, while the 42,390 was higher for CDDGS when compared to the other ingredients.
units curve was very steep (data not shown). Therefore, the 28,260 The SBM and FM samples had the highest (P  0.05) fractional
units of pepsin was selected as the standard pepsin concentration digestion rate (rate at which faction [B] was digested over time; kd)
because the pepsin efficiency measured as percentage CP hydro- compared to all other samples. The CDDGS had a higher (P  0.05)
lyzed per unit of enzyme was drastically reduced after 28,260 units fractional digestion rate compared to CA, but all other samples
of pepsin. The shape of the digestion curved from the preliminary were intermediate. The CV for the fractional digestion rate was the
data gave a gradual digestion with time and the extent of hydrolysis lowest for CDDGS and SBM. The trend observed for the absolute
for the SBM by 28,260 units were above 4%. digestion rate (adr), which was calculated by dividing the extent of
The criteria for the selection of the pancreatin was based on the digestion by the total digestion time, was different from that of the
extent of hydrolysis which mimic that of in vivo SBM CP digestion by fractional digestion rate. The SBM had a higher (P  0.05) absolute
poultry. The 7.5 and 9 mL pancreatin gave the highest degree of hy- digestion rate compared to all other samples. The absolute diges-
drolysis which was above 90% at the end of the intestinal incubation tion rates for PCM and CDDGS were similar, but lower (P  0.05)
time (180 min). Both the 7.5 and 9 mL pancreatin treatments also had than those of all the other ingredients. The CV for the absolute
the steepest digestion curve over time. The 1, 3 and 5 mL volumes digestion rate of the samples was similar.
were only able to hydrolyze less than 60% of the CP in the SBM samples The undigested protein fraction was calculated as the difference
after 180 min incubation. The digestion curve from the 6.5 mL volume between the total protein content of the sample and the total
of pancreatin was more gradual over time (Fig. 7). Approximately 81% protein digested. There was more (P  0.05) undigested protein in
of the CP in the SBM sample was hydrolyzed by the 6.5 mL volume of the PCM sample than all other samples except for the CDDGS which
pancreatin at the end of the 180 min intestinal digestion phase. was intermediate. Numerically, lower CV was seen for the undi-
A preliminary literature search suggested that SBM samples gested protein of FM and SBM compared to the other samples.
from 4 different countries had an average in vivo CP digestibility of The potential digestibility (PD) of samples equals the sum of
82% (Ravindran et al., 2014). The percentage of CP hydrolyzed by fraction (A and B) and values were higher (P  0.05) for Ca, FM, SBM
the 6.5 mL of pancreatin was similar to the 82%, and the digestion and CGM than those for PCM. The value for CDDGS was interme-
curve was more gradual over time, which is assumed to be the case diate and not different from any of the protein sources tested. The
for protein digestion in vivo. The shape of the curve also provided coefficients of variation for the potential digestibility of the samples
the opportunity to obtained relevant digestion kinetic data from were generally low, but PCM and CDDGS values were twice those of
the assay. Based on the criteria listed above, the 6.5 mL of the other values. The actual CP digestibility values calculated using
pancreatin (199,335.5 BAEE units of trypsin; 14,020.5 BTEE units the OD values of the samples at 180 min of intestinal digestion
chymotrypsin, and 445.5 units elastase) was selected as the opti- expressed as a percentage of the OD values after 24 h acid hydro-
mum enzyme dosage for the intestinal phase of the assay. lysis of the samples ranged from 68% to 90%. After modeling the
data, the predicted CP digestibility of the samples ranged from 60%
to 84%. The correlation R2 value between meal actual CP di-
gestibility and the model's predicted CP digestibility were above 0.9
3.3. In vitro assay validation for all the meals evaluated (Tables 3 and 4).

The composition and chemical properties of the feed in- 4. Discussion

gredients used in this assay are shown in Table 1. These data are
presented in order to give the reader a clearer overview of the 4.1. Colorimetry assay
status of the ingredients that were used. Ingredient composition
(mineral, CP and DM contents) was similar to values previously The oxidative deamination of an amino acid to form Ruhemann's
reported for samples used as poultry feed ingredients (National purple is a complex reaction with a wide absorbance spectrum
Research Council, 1994). (Bottom et al., 1978). The nitrogen from the amino acids is incor-
The rapidly digested CP fraction (A) was higher (P  0.05) for FM porated in the bluish-violet pigment after reacting with ninhydrin in
and PCM than SBM and CDDGS, while other protein fractions were the presence of tin (II) chloride dehydrate as a reducing agent
intermediate (Table 2). The coefficient of variation (CV) for fraction (Bottom et al., 1978). The full spectrum scan of this reaction reveals
(A) of the samples was numerically higher for CDDGS, CGM and that all the samples tested had maximum OD reading at 568 nm, so
SBM than that for FM, PCM and CA. The CV for fraction (B) which this OD was chosen as the OD for the colorimetry assay. Identifying
represents the proportion of the proteins digested over time was this OD provides an opportunity for the assay to increase its sensi-
higher (P  0.05) for CA, FM, SBM and CGM than that for PCM, while tivity and precision in detecting the amino and carboxyl end of
peptide bonds as they are broken during hydrolysis. It is possible
that the first peak identified during the spectrum scan was as a
Table 1
Feed ingredient composition and chemical properties as fed (%).
result of intermediate products of the reaction.
An ethylene glycol sodium acetate base was chosen for the
Item Meals
ninhydrin reagent because it provided a stable reagent and it is easy
CA FM PCM SBM CGM CDDGS to make. The reagent does not require a nitrogen atmosphere and
Dry mater 98.0 89.2 95.4 89.2 90.6 97.7 similarly it is not required for storage, unlike dimethyl sulfoxide
Crude protein 90.2 67.2 62.0 45.3 62.1 28.3 base reagents (Moore, 1968). The ninhydrin reagent is susceptible
Calcium ND 3.54 4.32 0.50 0.10 0.06 to light during storage, and in this study, it took up to 14 days
Magnesium ND 0.33 0.22 0.29 0.05 0.34
for the reagent to stabilize and provide a constant OD reading.
Trypsin inhibitor, TIU/g ND ND ND 4,335 ND ND
Protein dispersability index ND 32 25 15 15 2 If the reagent is stored in a dark sealed bottle, it can be stored up to
Protein solubility ND 45 39 78 24 28 120 d and still give good OD readings. Even though there was a
CA ¼ casein; FM ¼ fish meal; PCM ¼ porcine meal; SBM ¼ soybean meal;
reduction in the OD readings of the reagent over time, this would
CGM ¼ corn gluten meal; CDDGS ¼ corn distillers' dried grain with solubles; only be of significance if OD values from different digestion runs
ND ¼ not determined. were being compared directly. The reagent is very sensitive in
 D.D.S.L. Bryan et al. / Animal Nutrition 4 (2018) 401e409 407

Table 2
Digestion kinetic constant of meals generated with the in vitro digestion data1.

Item A2, % B2, % Kd2, h1 Adr2, %/min UD2, % PD2, %

Mean CV Mean CV Mean CV Mean CV Mean CV Mean CV

a a b b b a
CA 16.9 16 71.9 5 0.018 18 0.443 3.5 13.0 27 87.0 5
FM 13.2ab 9 70.6a 4 0.024a 20 0.463b 4.0 16.1b 9 83.9a 2
PCM 13.9ab 11 55.2b 7 0.013bc 20 0.340c 3.8 30.9a 23 69.1b 10
SBM 6.5c 30 78.8a 5 0.023a 13 0.507a 5.0 14.6b 15 85.4a 5
CGM 10.3bc 20 72.7a 7 0.013bc 20 0.433b 5.0 17.1b 27 82.9a 5
CDDGS 8.1c 24 66.8ab 12 0.009c 12 0.346c 4.1 25.1ab 31 74.9ab 12
SEM 1.2 3.6 0.001 0.008 3.5 3.5
ANOVA
P-value <0.0001 0.0023 <0.0001 <0.0001 0.0039 0.0039

CV ¼ coefficient of variation; CA ¼ casein; FM ¼ fish meal; PCM ¼ porcine meal; SBM ¼ soybean meal; CGM ¼ corn gluten meal; CDDGS ¼ corn distillers' grain with solubles.
aec
Means within a column with different superscripts are significantly different (P < 0.05).
1
Data were fitted to the model proposed by Ørskov and McDonald (1979): A þ B (1  e-kdt).
2
A ¼ rapidly digested CP fraction; B ¼ slowly digested CP fraction; kd ¼ the rate at which the B fraction is digested over time; UD ¼ undigested fraction calculate as
100  (A þ B); PD ¼ potential digestible fraction calculated as A þ B; adr ¼ absolute digestion rate (percentage of protein digested per min from 0 to 180 min); SEM ¼ standard
error of means where n ¼ 6.

Table 3
Actual and predicted digestibly coefficient of meals over 180 min.1
2
Time, min Actual coefficient Predicted coefficient

CA SBM FM CDDGS CGM PCM CA SBM FM CDDGS CGM PCM

0 12 ±3.1 5 ± 2.9 9 ± 3.1 7 ± 1.3 4 ± 1.8 7 ± 2.5 0 0 0 0 0 0

15 27 ± 6.5 31 ± 6.1 30 ± 5.1 15 ± 6.4 27 ± 2.5 28 ± 2.6 19 25 25 9 15 12
30 50 ± 2.7 52 ± 1.6 54 ± 3.0 29 ± 1.2 38 ± 3.0 34 ± 3.3 33 43 43 18 27 22
45 56 ± 4.0 59 ± 2.3 58 ± 3.1 31 ± 1.9 43 ± 5.0 38 ± 2.8 45 55 55 25 37 31
60 67 ± 2.4 67 ± 2.5 64 ± 8.6 37 ± 6.8 49 ± 6.9 45 ± 3.0 54 64 64 31 45 37
90 69 ± 7.1 67 ± 3.9 66 ± 3.7 38 ± 6.8 50 ± 3.3 45 ± 2.2 66 75 74 42 57 48
120 69 ± 2.1 71 ± 1.3 71 ± 1.8 49 ± 3.0 62 ± 1.8 48 ± 2.9 74 80 79 49 66 55
180 93 ± 3.2 95 ± 3.9 93 ± 3.2 70 ± 1.8 82 ± 3.0 68 ± 3.7 82 84 83 60 75 62

CA ¼ casein; SBM ¼ soybean meal; FM ¼ fish meal; CDDGS ¼ corn distillers' dried grain with solubles; CGM ¼ corn gluten meal; PCM ¼ porcine meal.
1
Means ± standard error of means where n ¼ 6.
2
Model ¼ A þ B (1  e-kdt) where A, B and kd are (CA ¼ 16.93, 71.9 and 0.018; SBM ¼ 6.5, 78.8 and 0.023; FM ¼ 13.2, 70.6 and 0.024; CDDGS ¼ 8.1, 66.8 and 0.009;
CGM ¼ 10.3, 72.7 and 0.013; PM ¼ 13.9, 55.2 and 0.013), respectively.

Table 4 makes it possible to track changes in the hydrolysis of the CP sam-

Pearson correlation coefficients between model predicted and actual digestibility of ples over time as more free a amino and carboxyl groups are
meals over 180 min of digestion.
exposed. In theory, the OD intensity is directly proportional to the
Model predicted digestibility Actual in vitro digestibility degree of hydrolysis, which has occurred as seen in Fig. 3. If the OD
CA SBM FM CDDGS CGM PM from the total hydrolysis of an ingredient is known, the degree of
hydrolysis can be calculated using the OD values. The very low
CA 0.97
<0.011 detection limit of the reagent means any small change in the con-
SBM 0.97 centration of amino acids or available amino acids, and carboxyl side
<0.011 group will induce a large change in OD reading. This can produce
FM 0.97
large variation in the reading of a sample if pipetting is not accurate;
<0.011
CDDGS 0.97
therefore, it is advisable to read samples in triplicate when using the
<0.011 reagent as outlined in this colorimetry assay. Proper controls and
CGM 0.97 blank samples should be run with every batch of samples that goes
<0.011 into the water bath in order to generate a correction factor for any
PM 0.95
change in temperature of the water bath during the assay.
<0.011

CA ¼ casein; SBM ¼ soybean meal; FM ¼ fish meal; CDDGS ¼ corn distillers' dried
grain with solubles; CGM ¼ corn gluten meal; PCM ¼ porcine meal.
4.2. Enzyme dose response assay
1
P-value.
One of the most important elements of an enzymatic in vitro
assay is the enzyme to substrate ratio at a known enzyme activity
detecting a amino acids, and ammonia, so proper precaution must (Boisen and Eggum, 1991). The pepsin dose response assay sug-
be taken to prevent amino acid or ammonia contamination of so- gested that the greatest change in the degree of hydrolysis over the
lutions used to make the reagent and buffers. 30 min was between 14,130 and 28,260 units of pepsin to 500 mg of
Due to the sensitivity of the reagent, the relationship between CP. When the pepsin concentration increased above 28,260 units,
the concentrations of free a amino and carboxyl group in solution the equivalent change in the degree of hydrolysis per unit of
with the OD reading is linear from 2 to 400 mg. The maximum enzyme addition was reduced. Using a pepsin concentration which
concentration from that range was at the upper limit of the detector maximizes the hydrolysis achieved per unit of enzyme can help to
in the spectrophotometer used in the study. This close relationship develop the most economical assay.
408 D.D.S.L. Bryan et al. / Animal Nutrition 4 (2018) 401e409

Electrophoresis data of the gastric phase sample presented in Porcine meal also tends to have high levels of arginine (Wang and
Fig. 5 suggests that the 2 tertiary structures normally seen in pro- Parsons, 1998), which could mean that more carboxypeptidase B is
teins extracted from soybean seed were subdivided into 5 major needed to break arginine bonds present in small peptides. Most
peptides and many smaller groups in the meal. The major shift in likely, the processing conditions during the rendering process could
these peptides of the meal due to pepsin concentration was seen at have damaged the PCM proteins, which makes them more resistant
the lower molecular weight 11S globulins 38 to 39 kDa compared to to digestion (Wang and Parsons, 1998).
the 7S fractions 62 to 90 kDa. Similar results were observed by Yang The CDDGS potential digestibility values were the second lowest
et al. (2016) after peptic digestion of SBM isolated CP. The authors of all ingredients evaluated, but they were in the range for in vivo
suggested that 11S glycinin was more susceptible to pepsin diges- values previously reported for CDDGS in broilers (Adedokun et al.,
tion because of its lower surface hydrophobicity and less b-sheet 2015). Corn products like CDDGS are known to contain zein which
secondary structures (Yang et al., 2016). Nevertheless, electropho- is a prolamine that is insoluble in water and resistant to most
resis and the colorimetry assay both show that hydrolysis had taken proteolytic enzymes except alcalase (Shukla and Cheryan, 2001).
place after pepsin digestion of the SBM. The level of zein present in the protein fraction could reduce the
The digestion kinetic data obtained is dependent to a large extent protein digestibility of CDDGS during gastric and pancreatic
on the shape of the digestion curve during the intestinal phase. The digestion. Apart from the zein content of the CDDGS, the drying
pancreatin concentration which produced a curve fitting the model process used during the postharvest of corn has been shown to
proposed by Ørskov and McDonald (1979) and gave a value reduce its protein digestible (Barrier-Guillot et al., 1993).
approximating in vivo CP digestion for SBM at the end of the
digestion period were considered to be key criteria for the pancre-
atin concentration selection. Colorimetric testing of samples from 4.4. Assay advantages and disadvantages
7.5 to 9 mL pancreatin after 180 min gave OD values which were
higher than the OD values of SBM sample totally hydrolyzed (Data The in vitro assay presented in this work for measuring CP di-
not shown). This suggested that at those higher concentrations of gestibility is not the first of its kind. Other 2 stages in vitro methods
pancreatin, there might have been auto-hydrolysis of enzyme after have been previously described for measuring CP digestibility in
180 min of digestion. Even after 240 min of intestinal digestion, the poultry (Clunies and Leeson, 1984; Ravindran and Bryden, 1999).
6.5 mL pancreatin did not produce OD values which were higher The main problems with those assays lie in the length of the 4 h
than those of the total hydrolysis sample. The 1, 3 and 5 mL enzyme gastric digestion period, which is not representative of poultry
concentrations gave final digestibility values below the average of in vivo digestion, the use of just a single enzyme and the lack of
82% in vivo digestibility for SBM (Ravindran et al., 2014). The 6.5 mL information pertaining to the activity of the major enzymes in the
pancreatin was selected as the enzyme concentration for the in- pancreatin used. All in vitro assays will suffer from various degrees of
testinal phase, based on the shape of its digestion curve, the extent uncertainty due to the complexity of simulating the mechanism
of digestion mimicking SBM in vivo digestion and the stability of the which are involved in the digestion process of proteins. However,
enzyme after 180 min during the intestinal digestion phase. in vitro assays based on enzymatic digestion can provide meaningful
characterization of feed ingredients (Ravindran and Bryden, 1999).
4.3. In vitro assay validation One of the major disadvantages of the current assay is that it
requires a minimum of 3 people to collect the samples during the
Based on fractional digestion rates (kd) values, SBM and FM can intestinal phase. The sequential timing of sample collection is
be classified as rapidly digested protein sources and CDDGS slowly affected by the length of time required for sample collection and
digested. The kd value represented the rate at which faction (B) of processing. For example, the lowest sample interval that was ach-
the proteins were digested over time assuming that the process ieved in the assay was 15 min with 4 people conducting the assay
followed the first order of kinetics. The absolute digestion rate (adr) with 30 digestion tubes. Due to the sensitivity of the ninhydrin
is a different kind of measurement which assumed that the rate of reagent, proper pipetting skills are needed, and all buffers and so-
digestion is linear. The data presented in Fig. 7 suggested that the lution used in the assay must be free of ammonia, peptide, proteins
rate at which the protein was digested followed the first order of and ammo acids. During the color development stage of the assay,
kinetics which is typical of most biological reactions and therefore the water bath should always be at boiling to obtain consistent
is a true representation of that process. sample color development.
The animal based protein ingredients tend to have higher frac- Most in vitro digestion methods suffer from some degrees of
tion (A). It is possible that this difference relates to a higher pro- imprecision. The assay presented in this study has the following
portion of peptides or free amino acids in animal than plant based advantages, many samples can be analyzed in a short time frame, it is
ingredients. Another reason for the difference between fraction (A) relatively inexpensive and easy to perform in a basic animal nutri-
of plant and animal ingredients might relate to the nature of the tion lab, and no special training is needed to use required equipment.
proteins in these meals. Plants tend to store protein in vacuoles in The assay can be easily transferred to an automated platform for
cells which are often surrounded by a fiber matrix (Staswick, 1994), running the entire assay. This would significantly reduce the number
while animal proteins do not have a fiber matrix associated with the of personnel needed to collect the kinetic data. The level of precision
protein and there are also free amino acids and peptides present in between sample collection time intervals would be increased, and
extracellular space of animal tissue. These factors could have made the timing interval could also be reduced below 15 min.
the animal based proteins more susceptible to enzymatic hydrolysis The digestibility assay was able to generate kinetic data for all
than the plant proteins. Predicting fraction (A) produced higher the ingredients tested because their digestion curves over time all
variability in the plant based ingredients compared to animal based followed the first order kinetics plot. The model developed from the
ingredients, but the reason for this is still to be determined. digestion constant was able to predict the actual in vitro digestion
The potential digestibility was quite similar for all the in- of the meals over time with a high degree of accuracy. However, it
gredients except for PCM, which was lower than all the other should be noted that the digestion constants generated for each
samples. It is possible that the PCM meal has a higher elastin and meal only represent that specific sample and may not predict the
collagen content, which would require more elastase to hydrolyze response of other samples of the same ingredient. Based on the
this meal than the 445.5 units present in pancreatin that was used. correlation coefficients in Table 2, it is safe to say that the constants
 D.D.S.L. Bryan et al. / Animal Nutrition 4 (2018) 401e409 409

generated from the model reflected the digestion characteristic of Araba M, Dale NM. Evaluation of protein solubility as an indicator of over processing
soybean meal. Poult Sci 1990;69:76e83. https://doi.org/10.3382/ps.0690076.
those samples tested.
Barrier-Guillot B, Jondreville C, Chagneau AM, Larbier M, Leuillet M. Effect of heat
drying temperature on the nutritive value of corn in chickens and pigs. Anim
4.5. Implication on future research Feed Sci Technol 1993;41:149e59.
Boila RJ, Erfle JD, Sauer FD. Evaluation of the two-stage technique for the in vitro
estimation of the dry matter digestibility of corn silage. Can J Anim Sci 1980;60:
This study provides a basic assay, which can be used to generate 367e78.
kinetic data for high protein poultry feed ingredients in a short time Boirie Y, Dangin M, Gachon P, Vasson M-P, Maubois J-L, Beaufre re B. Slow and fast
frame. It is well known that protein digestion rate modulates tissue dietary proteins differently modulate postprandial protein accretion. Proc Natl
Acad Sci 1997;94:14930e5.
protein synthesis and deposition, but this process is still unknown Boisen S, Eggum BO. Critical evaluation of in vitro methods for estimating di-
in poultry due to the lack of kinetic data for high protein in- gestibility in simple-stomach animals. Nutr Res Rev 1991;4:141e62. https://
gredients. Data from this assay can be used to develop diets for doi.org/10.1079/NRR19910012.
Bottom CB, Hanna SS, Siehr DJ. Mechanism of the ninhydrin reaction. Biochem Educ
studying the metabolic response of poultry to specific ingredient 1978;6:4e5.
digestion characteristics. More research is needed to test the assay Clunies M, Leeson S. In vitro estimation of dry matter and crude protein di-
inter-variability and to develop more precise digestion constants gestibility. Poult Sci 1984;63:89e96. https://doi.org/10.3382/ps.0630089.
Dangin M, Boirie Y, Garcia-Rodenas C, Gachon P, Fauquant J, Callier P, et al. The
for each high protein ingredient, which would be representative of digestion rate of protein is an independent regulating factor of postprandial
the ingredient and not the sample. protein retention. Am J Physiol Endocrinol Metab 2001;280:E340e8.
Fuller MF. In vitro digestion for pigs and poultry. Wallingford, England: C.A.B. In-
ternational; 1991.
5. Conclusions
Hughes RJ. Relationship between digesta transit time and apparent metabolisable
energy value of wheat in chickens. Br Poult Sci 2008;49:716e20. https://
A multi-enzymatic in vitro protein digestion technique doi.org/10.1080/00071660802449145.
mimicking the chicken digestive tract was defined and validated. Johnson D. Functional properties of oilseed proteins. J Am Oil Chem Soc 1970;47:
402e7. https://doi.org/10.1007/BF02632475.
The effectiveness of the in vitro digestion technique was tested on a Koopman R, Crombach N, Gijsen AP, Walrand S, Fauquant J, Kies AK, et al. Ingestion
variety of high protein ingredients. The in vitro protein digestibility of a protein hydrolysate is accompanied by an accelerated in vivo digestion and
assay predicted the rapidly, slowly and undigested protein fraction of absorption rate when compared with its intact protein. Am J Clin Nutr 2009;90:
106e15. https://doi.org/10.3945/ajcn.2009.27474.
ingredients, as well as the rate and extent of digestion of the pro- Moore S. Amino acid analysis: aqueous dimethyl sulfoxide as solvent for the
teins. The in vitro assay described in this study can be used to study ninhydrin reaction. J Biol Chem 1968;243:6281e3.
the digestion kinetic of high protein ingredients fed to poultry. National Research Council. Nutrient requirements of poultry. 9 rev ed. Washington,
DC: NRC, National Academy Press; 1994.
Ørskov ER, McDonald I. The estimation of protein degradability in the rumen from
Conflicts of interest incubation measurements weighted according to rate of passage. J Agric Sci
1979;92:499e503. https://doi.org/10.1017/S0021859600063048.
Ravindran V, Abdollahi MR, Bootwalla SM. Nutrient analysis, metabolizable energy,
The authors declare no conflicts of interest. and digestible amino acids of soybean meals of different origins for broilers.
Poult Sci 2014;93:2567e77.
Acknowledgements Ravindran V, Bryden WL. Amino acid availability in poultrydin vitro and in vivo
measurements. Aust J Agric Res 1999;50:889e908.
Shukla R, Cheryan M. Zein: the industrial protein from corn. Ind Crop Prod 2001;13:
The authors would like to acknowledge the National Science and 171e92.
Engineering Research Council (NSERC), Industrial Research Chair Sklan D, Hurwitz S. Protein digestion and absorption in young chicks and turkeys.
Program for financial support for this project (Grant No. IRCSA J Nutr 1980;110:139e44.
Staswick PE. Storage proteins of vegetative plant tissues. Annu Rev Plant Biol
452664-12). Funding for this program was derived from Aviagen, 1994;45:303e22.
Canadian Poultry Research Council, Chicken Farmers of Saskatch- Svihus B, Hetland H, Choct M, Sundby F. Passage rate through the anterior digestive
ewan, NSERC, Ontario Poultry Industry Council, Prairie Pride Nat- tract of broiler chickens fed on diets with ground and whole wheat. Br Poult Sci
2002;43:662e8. https://doi.org/10.1080/0007166021000025037.
ural Foods Ltd., Saskatchewan Egg Producers, Saskatchewan USDA. Major protein meals: world supply and distribution. U S Dep Agric Foreign
Hatching Egg Producers, Saskatchewan Turkey Producers, Sofina Agric Serv; 2016. https://apps.fas.usda.gov/psdonline/app/index.html#/app/
Foods Inc. and the University of Saskatchewan. downloads. [Accessed 18 September 2017].
Wang X, Parsons CM. Effect of raw material source, processing systems, and pro-
cessing temperatures on amino acid digestibility of meat and bone meals. Poult
References Sci 1998;77:834e41.
Yang Y, Wang Z, Wang R, Sui X, Qi B, Han F, et al. Secondary structure and subunit
Adedokun SA, Jaynes P, Payne RL, Applegate TJ. Standardized ileal amino acid di- composition of soy protein in vitro digested by pepsin and its relation with
gestibility of corn, corn distillers' dried grains with solubles, wheat middlings, digestibility. BioMed Res Int 2016;2016:1e11. https://doi.org/10.1155/2016/
and bakery by-products in broilers and laying hens. Poult Sci 2015;94:2480e7. 5498639.
https://doi.org/10.3382/ps/pev226. Zuidhof MJ, Schneider BL, Carney VL, Korver DR, Robinson FE. Growth, efficiency,
AOAC International. Official methods of analysis. 18th ed. Gaithersburg, MD: AOAC and yield of commercial broilers from 1957, 1978, and 20051. Poult Sci 2014;93:
Int.; 2006. 2970e82. https://doi.org/10.3382/ps.2014-04291.