Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol87.Issue3-4.Oct2000:
Animal Feed Science and Technology
87 (2000) 173±186
The in¯uence of hydrochloric acid concentration
and measurement method on the determination
of amino acid levels in soya bean products
David M. Albin*, Jennifer E. Wubben, Vince M. Gabert
Department of Animal Sciences, University of Illinois at Urbana-Champaign,
1207 W. Gregory Drive, Urbana, IL 61801, USA
Received 14 September 1999; received in revised form 10 March 2000; accepted 16 August 2000
Abstract
Accurate determination of amino acid levels in soya products facilitates optimum diet
formulation and amino acid supplementation. A study was carried out to investigate the effect of
hydrochloric acid (HCl) concentration and method of amino acid measurement on the measurement
of amino acid levels. Six different soya bean products were evaluated. Hydrolysis was carried out
with ®ve different acid concentrations (1, 3, 6, 9 and 12 M HCl). Amino acids were analysed by
both ion-exchange chromatography with ninhydrin detection and pre-column derivatisation with
phenyl isothiocyanate. Both acid concentration and measurement method affected (P < 0:05)
measurements of most amino acids. Standard hydrolysis conditions (hydrolysis in 6 M HCl at
1108C for 24 h) rarely provided the maximal amino acid values, and thus, correction factors were
calculated to standardise amino acid levels by dividing the maximum value by the value obtained
with 6 M HCl. Therefore, when determining amino acid concentrations, the patterns of release and
degradation of amino acids, as well as correction factors, should be considered to obtain the most
accurate values. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Amino acids; Soya bean products; Measurement methods; Acid concentration
1. Introduction
The amino acid levels in feedstuffs used in animal diets must be determined to
understand the nutritional value of these ingredients and to optimise diet formulations.
Commonly, an acid hydrolysis of the samples is performed. Then, amino acids are
*
Corresponding author. Tel.: 1-217-333-9749; fax: 1-217-333-7088.
E-mail address: [email protected] (D.M. Albin).
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 2 0 4 - 2
174
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
measured with high pressure liquid chromatography using pre-column derivatisation with
phenyl isothiocyanate (PITC), or ion-exchange chromatography (IEC) with post-column
ninhydrin reaction (Bidlingmeyer et al., 1984; Elkin and Wasynczuk, 1987; Fountoulakis
and Lahm, 1998).
The release of amino acids from proteins with acid hydrolysis is the most important
step in the determination of amino acid concentrations. Standard hydrolysis procedures
involve 24 h acid hydrolysis in 6 M HCl at 1108C (Fountoulakis and Lahm, 1998; Gehrke
et al., 1985; Heinrikson and Meredith, 1984; Rowan et al., 1992). Hydrolysis time (16±
72 h) has been shown to affect the release and degradation of amino acids in a diet for
growing pigs, and standard hydrolysis conditions rarely provided the maximum amino
acid concentration (Rowan et al., 1992). Other acids, such as methanesulfonic acid and ptoluenesulfonic acid, have been used to hydrolyse proteins, and the effects of time and
temperature on hydrolysis have been investigated (Fountoulakis and Lahm, 1998).
Soya bean products are used widely in animal nutrition (Emmert and Baker, 1995).
However, the effect of acid concentration on amino acid measurements in commonly
used soya protein products has not been investigated. The purposes of the present study
were threefold. The ®rst purpose was to determine the effect of acid concentration
on amino acid measurements in soya bean products. Secondly, the effect of method
(PITC versus IEC) on amino acid measurements was examined. Finally, for amino acids
levels that were not maximised with hydrolysis in 6 M HCl, correction factors were
calculated.
2. Materials and methods
2.1. Procedure
Representative samples of soya bean meal from two cropping years (SBM96, SBM97),
soya protein concentrate (SPC; Arcon F 65±301, Archer-Daniels-Midland, Decatur, IL),
soya protein isolate (SPI; Ardex 66±960, Archer-Daniels-Midland, Decatur, IL), whole
soya beans (WholeSB; Williams 82 variety) and soya bean hulls (Soya hulls) were
obtained. Homogenous samples (approximately 10 g) of each were ®nely ground in a
coffee bean grinder (Mr. Coffee, Model # IDS-50, Bedford Heights, OH) with
approximately 10 ml liquid nitrogen for about 20 s, mixed and stored frozen at ÿ108C.
2.2. Chemical analysis
Triplicate samples of each soya bean product were used to determine dry matter and
crude protein (N 6:25) according to procedures outlined by AOAC (1990). The
following chemicals were used for amino acid analyses: water (HPLC grade, cat. # W54); methanol (HPLC grade, cat. # A452-4); acetonitrile (HPLC grade, cat. # A998-4);
disodium hydrogen phosphate (anhydrous, certi®ed A.C.S., cat. # S374±500); ophosphoric acid (HPLC grade, cat. # A260±500); sodium acetate trihydrate (HPLC grade,
cat. # S220±1); concentrated hydrochloric acid (37%, cat. # A144±212); glacial acetic
acid (reagent A.C.S., A38c±212), all purchased from Fisher Scienti®c, St. Louis, MO. All
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
175
amino acids; phenyl isothiocyanate (protein sequencing grade, cat. # P-1034); and
triethylamine (cat. # 13,206±3), were obtained from Sigma±Aldrich, St. Louis, MO.
Triplicate samples of SBM96, SBM97, WholeSB, and Soya hulls (at least 200 mg),
and triplicate samples of SPC and SPI (at least 100 mg) were accurately weighed into
screw-capped test tubes (15 ml, Pyrex, cat. # 9826±16x, Corning, NY) with te¯on-lined
caps. HCl (12 ml) were added to the tubes. The acid concentrations used in this study
were the following: 1, 3, 6, 9 and 12 M. The tubes were purged with N2 for 10 s, mixed
and hydrolysed in an oven (Fisher Scienti®c, Model # 500 Series, Pittsburgh, PA) at
1108C for 24 h. After removal from the oven, the samples were allowed to cool. Once the
samples had cooled, 0.5 ml of a-amino butyric acid (AABA, 50 mM) and 0.5 ml of
norleucine (Nor, 50 mM) were accurately weighed on a balance and added to each tube.
The tubes were inverted 200 times and centrifuged at 1100 g for 10 min to pellet
debris. Then, the hydrolysates were diluted in a 1:2.5 ratio by adding 300 ml of distilled,
deionised water to a 200 ml aliquot of hydrolysate in a 1.5 ml microfuge tube (Fisher
Scienti®c, cat. # 05±408±10, Pittsburgh, PA). Amino acid standards (2.5 mM) were
prepared by weighing individual amino acids into a 250 ml volumetric ¯ask and
dissolving them in 0.1 M HCl. Three standards were used. Standard 1 consisted of Asp,
Ser, Gln, Citrulline, Arg, AABA, Val, Ile, Nor and Trp. Standard 2 consisted of Glu, Asn,
Taurine, Thr, Pro, AABA, Met, Leu, Nor and Ornithine. Standard 3 consisted of
Hydroxyproline, Gly, His, Ala, AABA, Tyr, Cys, Nor, Phe, Lys and Homoarginine.
Standards were prepared to accommodate analyses of hydrolysates and physiological
samples. Samples and standards were prepared for PITC and IEC analysis.
The procedures for amino acid determination with PITC were the following: 20 ml of
diluted hydrolysates or 10 ml of either standard 1, 2 or 3 were pipetted into polypropylene
tubes (Fisher Scienti®c, cat. # 1495910AA, St. Louis, MO). They were allowed to dry
under vacuum overnight in a freeze-drier (Labconco, Model # 77500, Kansas City, MO).
The samples were re-dried by adding 20 ml of 1:1:1 (v/v/v) methanol:water:triethylamine
to each sample. The tubes were held at a 458 angle and spun several times to resolubilise
amino acids. The tubes were then allowed to vacuum-dry for 4 to 6 h. Finally, the samples
were derivatised by adding 20 ml of 7:1:1:1 (v/v/v/v) methanol:water:triethylamine:phenyl isothiocyanate, and mixed by holding the tubes at a 458 angle and spinning them
several times. The tubes were capped and derivatisation was allowed to occur for 35 min
at room temperature (228C). The samples were vacuum-dried for 4±6 h following
derivatisation. The samples were then reconstituted in 200 ml of sample diluent, which
was composed of a mixture of 95:5 (v/v) phosphate buffer (5 mM sodium phosphate
dibasic, pH 7.4, adjusted with o-phosphoric acid):acetonitrile. The samples were vortexed
(Barnstead/Thermolyne, Type 16700 Mixer, Dubuque, IA), allowed to stand for
approximately 15 min, and then vortexed again. A pipette was used to transfer most of
the liquid to a polypropylene HPLC vial (Bio-Rad Laboratories, cat. # 223±9471,
Hercules, CA), while leaving any debris at the bottom of the microfuge tube. The
injection volume used ranged from 30 to 60 ml depending on the protein content of the
sample.
The Waters HPLC system consisted of either a 712 WISP or a 700 Satellite WISP
autosampler, two 510 pumps, a column heater (468C) and a 484 tunable absorbance
detector set at 254 nm. Peaks were identi®ed and integrated with Waters Maxima 820
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D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
software. The HPLC column was a Waters Pico-Tag1 3:9 mm 30 cm reverse-phase
column (Waters, cat. # WAT010950, Milford, MA). The packing consisted of 4 mm Silica/
C18 beads. A 4:6 mm 5 cm Supelcosil reverse-phase C18 guard column with 40 mm
Pellicular packing (Sigma±Aldrich, Supelco, cat. # 5±8232, Bellefonte, PA) was used.
Two eluents were used. Eluent A consisted of 2.5:97.5 (v/v) 70 mM sodium acetate, pH
6.55:acetonitrile. Eluent B consisted of 50:35:15 (v/v/v) acetonitrile:water:methanol.
Both eluents were vacuum ®ltered through a 0.45 mm nylon ®lter before use. The ¯ow
rate began at 1.0 ml/min. At 75 min, the ¯ow rate was increased to 1.3 ml/min. The ¯ow
rate returned to 1.0 ml/min at 76 min. The gradient which was run for the separation
consisted of 100% eluent A until 13.5 min, at which point the level of eluent A decreased
to 97%, eluent B increased to 3% (vertical change, Waters No. 11). The level of eluent A
continually decreased while eluent B increased, and this pattern is indicated in the
following: 24 min, concave curve, Waters No. 9/(A, 95%: B, 5%); 30 min, convex curve,
Waters No. 5/(A, 91%; B, 9%); 50 min, linear change, Waters No. 6/(A, 66%; B, 34%);
65 min, linear change, Waters No. 6/(A, 0%; B, 100%). The column was reequilibrated
with 100% eluent A (linear change, Waters No. 6) at 76 min for 89 min.
The procedures for amino acid determination with IEC were the following: duplicate
samples of SBM97 and SPC were utilised. Samples of hydrolysate were taken from the
same tubes that were used for the PITC procedure. Following hydrolysis, addition of
internal standards and centrifugation at 1100 g for 10 min, as described above, 200 ml
of each sample was diluted 1:5 (v/v) hydrolysate: distilled, deionised water. The diluted
samples were then pH adjusted with a buffer solution of NaOH in 10 ml sodium citrate
(2%, Beckman Instruments, Inc., Palo Alto, CA) in a ratio of 1:5 diluted sample:buffer
solution. For samples that were hydrolysed with 6 M HCl, 0.1 g of NaOH was used. For
samples that were hydrolysed with 1 and 3 M HCl, 0.01 g of NaOH was used. For
samples that were hydrolysed with 9 and 12 M HCl, 0.15 g of NaOH was used. The
pH of the samples was measured with litmus paper and found to be approximately 2.
The samples were then analysed using post-column detection with ninhydrin using
procedures that have been described previously (Spitz, 1973). It should be noted that
the internal standards were not used for calculations of amino acid concentrations with
IEC, but were used for PITC calculations only. External standards were used for the IEC
procedure.
2.3. Data analysis
One-way analysis of variance was performed following procedures described by Ott
(1993) to determine if acid concentration affected amino acid yield determined by PITC.
The means of each amino acid level at the various points in time were compared
according to the Student±Newman±Keuls multiple range test (Ott, 1993). One-way
analysis of variance was used to compare amino acid concentrations in SBM97 and SPC,
obtained with 6 M HCl, and determined with PITC and IEC (Ott, 1993). One of the
concentrations of histidine in Soya hulls determined with 6 M HCl was an outlier and was
removed from the data set. The mean of the two remaining values for histidine in Soya
hulls determined with 6 M HCl was used to complete the data set. The General Linear
Models Procedure of the Statistical Analysis Systems Institute Inc. (SAS Institute Inc.,
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
177
1988) was used for all calculations. An a level of 0.05 was used to determine statistically
signi®cant differences.
3. Results
The dry matter and crude protein (g kgÿ1, dry matter basis) contents, respectively, of
the soya bean samples were as follows: SBM96 (888, 536 g kgÿ1); SBM97 (885,
512 g kgÿ1); SPC (927, 682 g kgÿ1); SPI (907, 827 g kgÿ1); WholeSB (931, 410 g kgÿ1);
and Soya hulls (881, 116 g kgÿ1).
3.1. Acid concentration and amino acid composition
Mean amino acid values for the soya bean samples obtained using the various
acid concentrations during hydrolysis are shown in Figs. 1±4. Most amino acid
measurements were affected by acid concentration (P < 0:05) regardless of soya bean
sample. Acid concentration did not affect (P > 0:05) the release of the following
amino acids: lysine in SBM97; aspartic acid in SPC, SPI, WholeSB and Soya hulls;
threonine in Soya hulls; and phenylalanine in Soya hulls. Most amino acids exhibited
more release with acid hydrolysis in 3 M than in 1 M (P < 0:05), regardless of soya
bean sample. Aspartic acid was maximised with 1 M in SBM96 and SBM97, but it was
only signi®cant (P < 0:05) for SBM96. Maximum valine measurements were obtained
with acid concentrations greater than 6 M, but the differences were only signi®cant
(P < 0:05) for SBM96, SPC and SPI. Isoleucine was also maximised at concentrations
greater than 6 M, except in SBM97 and SPC, and the differences were only signi®cant
(P < 0:05) for SBM96 and SPI. There was a trend for serine concentrations to be
maximised (P > 0:05) with 3 M for SBM96 and Soya hulls, while threonine
concentrations were maximised with 9 M for SBM96, SBM97 and SPI. Threonine
release was greater (P < 0:05) with 9 M acid for SBM96 only. There was a trend
for tyrosine to be maximised (P > 0:05) with 3 M in SBM96, SBM97, SPC and
WholeSB. Acid hydrolysis with 9 and 12 M degraded (P < 0:05) tyrosine in all soya
bean samples. Glycine, histidine, alanine, arginine, proline, leucine, phenylalanine and
lysine tended (P > 0:05) to increase with increasing acid concentration for most soya
bean samples until 6 M HCl. There was a tendency for concentrations of isoleucine,
leucine, phenylalanine and lysine in SBM97 to decrease (P > 0:05) with hydrolysis
in acid concentrations greater than 6 M, while the concentrations of the same amino
acids increased (P > 0:05; P < 0:05 for isoleucine) in SBM96 with hydrolysis in
acid concentrations greater than 6 M. Glutamic acid increased (P < 0:05) from 1 to 3 M
in all soya bean samples, and then tended to increase or remain constant (P > 0:05) from
3 to 12 M.
3.2. Measurement method and amino acid composition
The in¯uence of measurement method on amino acid measurements of the soya bean
samples is shown in Table 1. With the exception of aspartic acid, most amino acid
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D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
Fig. 1. Effect of acid concentration on the mean yield of amino acids (Y axis, g kgÿ1 dry matter basis) from soya
bean meal 1996 (full line, shaded diamond), soya bean meal 1997 (long-dashed line, open square) and soya bean
hulls (short-dashed line, shaded circle). Error bars indicate sample standard error (n 3; for histidine in soya
bean hulls determined with 6 M HCl, one data point was an outlier. It was removed and the mean of the two
remaining values was used to complete the data set).
concentrations in SBM97 were higher (P > 0:05) when determined with PITC than when
determined with IEC. With the exception of aspartic acid and histidine, most amino acid
concentrations in SPC were numerically higher (P < 0:05) when determined with PITC
than when determined with IEC.
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
179
Fig. 2. Effect of acid concentration on the mean yield of amino acids (Y axis, g kgÿ1 dry matter basis) from soya
bean meal 1996 (full line, shaded diamond), soya bean meal 1997 (long-dashed line, open square) and soya bean
hulls (short-dashed line, shaded circle). Error bars indicate sample standard error (n 3).
3.3. Correction factors
Correction factors calculated for the soya bean samples are shown in Table 2. The
correction factors were determined by dividing the maximum amino acid measurement
by the value obtained with hydrolysis in 6 M HCl. Correction factors were only
calculated for measurements conducted with PITC. Most of the correction factors ranged
from 1.00 to 1.32. However, the correction factors for isoleucine in SBM96 and lysine in
Soya hulls were greater than 1.32.
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D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
Fig. 3. Effect of acid concentration on the mean yield of amino acids (Y axis, g kgÿ1 dry matter basis) from soya
protein isolate (full line, shaded triangle), soya protein concentrate (long-dashed line, cross) and whole soya
beans (short-dashed line, open circle). Error bars indicate sample standard error (n 3).
4. Discussion
4.1. Acid concentration and amino acid composition
It is well known that valine and isoleucine are released slowly, while serine and
threonine are continually degraded, over time during acid hydrolysis (Gehrke et al., 1985;
Rees, 1946; Rowan et al., 1992). Increasing acid concentration with ®xed hydrolysis time
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
181
Fig. 4. Effect of acid concentration on the mean yield of amino acids (Y axis, g kgÿ1 dry matter basis) from soya
protein isolate (full line, shaded triangle), soya protein concentrate (long-dashed line, cross) and whole soya
beans (short-dashed line, open circle). Error bars indicate sample standard error (n 3).
also resulted in the slow release of valine and isoleucine, as measurements of the two
amino acids increased with increasing acid concentration in the present study (Figs. 1±4).
Serine was progressively destroyed from hydrolysis with 3 M to greater acid
concentrations in only SBM96 and Soya hulls. However, threonine was maximised with
9 M HCl for SBM96, SBM97 and SPI. There was no indication of threonine degradation
with hydrolysis using 9 M, but threonine was degraded with 12 M. This study indicates
that the differences in the release and degradation of valine, isoleucine, serine and
182
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
Table 1
Amino acid levels (g kgÿ1, dry matter basis) in soya bean meal (SBM97) and soya protein concentrate (SPC)
determined using 6 M HCl for acid hydrolysis and using pre-column derivitisation with phenyl isothiocyanate
(PITC) or post-column detection with ninhydrin (IEC)
Amino acid
SBM97
SPC
PITC
Aspartic acid
Glutamic acid
Serine
Glycine
Histidine
Threonine
Alanine
Arginine
Proline
Tyrosine
Valine
Isoleucine
Leucine
Phenylalanine
Lysine
48.8
101.5
30.5
27.9
14.2
25.8
27.1
41.7
31.5
20.2
25.8
25.8
43.1
29.8
36.0
IEC
b
2.30
2.20
1.00
1.00
0.80
0.10
1.00
2.20
1.00
1.60
1.40
1.20
1.10
0.10
4.20
51.5
84.6
23.8
19.6
12.3
18.5
20.2
33.3
24.7
16.2
21.5
20.7
35.6
23.9
29.4
0.20
0.20
0.10
0.10
0.10
0.10
0.10
0.20
0.40
0.10
0.20
0.20
0.20
0.10
0.20
Sig.a
PITC
NS
71.0
123.3
35.0
31.6
15.5
28.1
31.5
50.6
37.8
24.7
33.9
36.4
57.6
41.6
60.7
**
*
**
NS
**
**
*
*
NS
*
*
*
**
NS
IEC
3.10
0.20
1.90
4.80
0.60
4.30
1.4
0.20
5.90
0.70
5.80
7.10
2.00
5.40
5.30
71.1
116.8
32.5
26.8
16.5
25.2
27.4
49.0
34.8
21.7
29.7
28.4
48.7
31.1
39.7
Sig.
0.20
0.10
0.30
0.10
0.10
0.10
0.10
2.50
0.30
0.10
0.10
0.10
0.20
0.20
0.50
NS
**
NS
NS
NS
NS
NS
NS
NS
*
NS
NS
*
NS
*
a
NS: non-signi®cant (P > 0:05).
Standard deviation (n 3).
*
P < 0:05.
**
P < 0:01.
b
Table 2
Correction factorsa for amino acids in soya bean meal (SBM96, SBM97), soya protein concentrate (SPC), soya
protein isolate (SPI), whole soya beans (WholeSB) and soya bean hulls (Soya hulls)
Amino acid
SBM96
SBM97
SPC
SPI
WholeSB
Soya hulls
Aspartic acid
Glutamic acid
Serine
Glycine
Histidine
Threonine
Alanine
Arginine
Proline
Tyrosine
Valine
Isoleucine
Leucine
Phenylalanine
Lysine
1.19
1.08
1.05
1.10
1.10
1.05
1.11
1.08
1.07
1.03
1.17
1.45
1.06
1.11
1.21
1.08
1.00
1.00
1.06
1.02
1.00
1.02
1.02
1.08
1.04
1.05
1.00
1.00
1.00
1.00
1.02
1.01
1.00
1.00
1.07
1.00
1.00
1.01
1.00
1.00
1.19
1.00
1.01
1.00
1.00
1.03
1.00
1.02
1.00
1.07
1.02
1.03
1.00
1.02
1.00
1.11
1.11
1.03
1.02
1.04
1.02
1.00
1.00
1.17
1.00
1.00
1.03
1.00
1.12
1.02
1.04
1.09
1.00
1.00
1.02
1.14
1.17
1.02
1.08
1.16
1.00
1.29
1.16
1.00
1.00
1.32
1.15
1.01
1.12
1.65
a
Correction factors determined by expressing the maximum amino acid yield as a proportion of the yield
using 6 M HCl.
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
183
threonine are, at least in part, dependent on protein source. Other researchers (Glazer
et al., 1976; Rowan et al., 1992) have attributed differences in degradation and release of
amino acids to protein source. Tyrosine, reported to be susceptible to oxidation (Finley,
1985; Gehrke et al., 1985), was degraded with the use of 9 and 12 M HCl. However,
similar concentrations of tyrosine were obtained with 6 M HCl relative to those obtained
with 3 M HCl, although some of the values were slightly lower at 6 M compared to 3 M
HCl. Rowan et al. (1992) reported similar observations concerning tyrosine, and
attributed the stability to the addition of phenol. In the present study, however, phenol was
not added prior to hydrolysis, and tyrosine was essentially not degraded with acid
concentrations as strong as 6 M HCl. Aspartic acid tended to decrease as acid
concentration increased from 1 M for samples of SBM96, SBM97 and Soya hulls,
indicating mild sensitivity of aspartic acid to acid degradation in these samples. Similar
patterns were not observed for SPC, SPI and WholeSB.
The relative stability or gradual increase in measurements of aspartic acid, glycine,
histidine, alanine, arginine, proline, leucine, phenylalanine and lysine with increasing
acid concentration used during this study re¯ected the lack of sensitivity of these amino
acids to increasing acid concentration, even using 12 M HCl.
4.2. Measurement method and amino acid composition
Different investigators have evaluated the two amino acid determination methods used
in this study, and they have reported that the two procedures provide very similar results
(Bidlingmeyer et al., 1984; Elkin and Wasynczuk, 1987; Heinrikson and Meredith, 1984).
However, in the current study, method affected amino acid measurements. For many
amino acids, in both SBM97 and SPC using hydrolysis in 6 M HCl, the PITC procedure
provided signi®cantly higher measurements. Also, it should be noted that the hydrolysis
curves for SBM97 and SPC determined with IEC followed the same patterns of release as
those shown in Figs. 1±4 (data not shown). Comparisons with published amino acid
values have indicated that the measurements determined with PITC and IEC were
comparable for amino acid levels in soya bean samples. For example, using PITC and
IEC in this study, the threonine content of SBM97 was 25.8 and 18.5 g kgÿ1, respectively
(dry matter basis). Other publications (Cavins et al., 1972; Emmert and Baker, 1995;
NRC, 1998; Rudolph et al., 1983) have reported threonine concentrations in soya bean
meal of 21.5, 20.6 and 18.6 g kgÿ1, respectively (dry matter basis). The threonine content
of SPC using PITC and IEC was found to be 28.1 and 25.2 g kgÿ1, respectively (dry
matter basis). Emmert and Baker (1995) and NRC (1998) reported threonine
concentrations in SPC of 27.3 and 31.1 g kgÿ1, respectively (dry matter basis). There
are several possible explanations for the differences in amino acid measurements
determined with the two methods in the current study. Many steps of the IEC procedure
demanded accurate volumetric measurements. Before amino acids were determined with
the IEC procedure, the hydrolysate needed to be diluted and buffered. This was done in
two different steps. As part of this procedure, external standards were used and, therefore,
dilution of standards and injection volumes needed to be accurate. The PITC procedure
did not require accurate volumetric measurements because internal standards and tube
weighing were used. Therefore, more opportunities for error existed with the IEC
184
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
procedure than when the PITC procedure was used. Despite this, it is interesting to note
that less variation due to method existed for amino acids in SPC compared to SBM97.
During soya bean processing, SPC is the result of further processing than soya bean meal.
Thus, SPC is more puri®ed and re®ned than soya bean meal, and, therefore, may contain
fewer factors than soya bean meal that could alter the determination of amino acids. Even
though both procedures provided values for most amino acids that are within the range
found in the literature, further research is necessary to increase the accuracy and decrease
the variation from procedure-to-procedure.
4.3. Correction factors
Garnett (1985) suggested the use of generalised correction factors as a simple approach
to correcting amino acid concentrations. Correction factors have been calculated by
others (Kohler and Palter, 1967; Rowan et al., 1992; Slump, 1980; Tkachuk and Irvine,
1969) to correct amino acid measurements determined with 24 h hydrolysis to the
maximum values obtained with different hydrolysis times. Recently, research projects
have begun to use correction factors to obtain amino acid measurements that are more
accurate (Lenis et al., 1990; Mroz et al., 1994). However, correction factors have not been
determined to correct the values obtained with hydrolysis in 6 M HCl to the maximum
values obtained with other acid concentrations. For hydrolysis time, correction factors for
serine, isoleucine and threonine in foods have been reported (Kohler and Palter, 1967;
Rowan et al., 1992; Slump, 1980; Tkachuk and Irvine, 1969) and ranged from 1.04 to
1.14, 1.02 to 1.21 and 1.02 to 1.08, respectively. In the present study, correction factors
for serine, isoleucine and threonine ranged from 1.02 to 1.05, 1.09 to 1.15 and 1.02 to
1.05, respectively. Rowan et al. (1992) and Slump (1980) have reported correction factors
for 24 h hydrolysis for valine of 1.20 and 1.08, respectively. In the present study, valine
correction factors ranged from 1.04 to 1.32. Most of the correction factors in this study
were similar to those determined with different hydrolysis times. For isoleucine in
SBM96 and lysine in Soya hulls, the correction factors were 1.45 and 1.65, respectively.
These should be used with caution, as comparison correction factors would suggest that
these factors are abnormally high (Rowan et al., 1992). However, there is nothing to
indicate that analytical error produced abnormal measurements of these amino acids.
In conclusion, determination of amino acid concentrations in protein sources is
affected by acid hydrolysis of the samples and the use of different amino acid
determination methods. Ion-exchange chromatography with ninhydrin detection has been
shown to provide similar results when compared to pre-column derivatization with phenyl
isothiocyanate, and the ®ndings of the current study indicate that the two methods
provided somewhat similar measurements. However, method did affect several amino
acid concentrations, indicating that variation in amino acid analysis can provide different
results. In many cases standard hydrolysis procedures (hydrolysis in 6 M HCl for 24 h at
1108C) do not provide the maximum amino acid measurements in samples. Correction
factors are necessary to determine amino acid concentrations more accurately (i.e. for
serine, threonine, isoleucine, valine) in protein sources. Further research is necessary with
more feedstuffs, as well as a range of diets, to determine ideal hydrolysis conditions and
correction factors.
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
185
Acknowledgements
The authors would like to thank Zoran Magas of LC Tech Services, Burlington, Ont.,
Canada for technical advice and maintenance of the high pressure liquid chromatography
equipment used in this study.
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87 (2000) 173±186
The in¯uence of hydrochloric acid concentration
and measurement method on the determination
of amino acid levels in soya bean products
David M. Albin*, Jennifer E. Wubben, Vince M. Gabert
Department of Animal Sciences, University of Illinois at Urbana-Champaign,
1207 W. Gregory Drive, Urbana, IL 61801, USA
Received 14 September 1999; received in revised form 10 March 2000; accepted 16 August 2000
Abstract
Accurate determination of amino acid levels in soya products facilitates optimum diet
formulation and amino acid supplementation. A study was carried out to investigate the effect of
hydrochloric acid (HCl) concentration and method of amino acid measurement on the measurement
of amino acid levels. Six different soya bean products were evaluated. Hydrolysis was carried out
with ®ve different acid concentrations (1, 3, 6, 9 and 12 M HCl). Amino acids were analysed by
both ion-exchange chromatography with ninhydrin detection and pre-column derivatisation with
phenyl isothiocyanate. Both acid concentration and measurement method affected (P < 0:05)
measurements of most amino acids. Standard hydrolysis conditions (hydrolysis in 6 M HCl at
1108C for 24 h) rarely provided the maximal amino acid values, and thus, correction factors were
calculated to standardise amino acid levels by dividing the maximum value by the value obtained
with 6 M HCl. Therefore, when determining amino acid concentrations, the patterns of release and
degradation of amino acids, as well as correction factors, should be considered to obtain the most
accurate values. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Amino acids; Soya bean products; Measurement methods; Acid concentration
1. Introduction
The amino acid levels in feedstuffs used in animal diets must be determined to
understand the nutritional value of these ingredients and to optimise diet formulations.
Commonly, an acid hydrolysis of the samples is performed. Then, amino acids are
*
Corresponding author. Tel.: 1-217-333-9749; fax: 1-217-333-7088.
E-mail address: [email protected] (D.M. Albin).
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 2 0 4 - 2
174
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
measured with high pressure liquid chromatography using pre-column derivatisation with
phenyl isothiocyanate (PITC), or ion-exchange chromatography (IEC) with post-column
ninhydrin reaction (Bidlingmeyer et al., 1984; Elkin and Wasynczuk, 1987; Fountoulakis
and Lahm, 1998).
The release of amino acids from proteins with acid hydrolysis is the most important
step in the determination of amino acid concentrations. Standard hydrolysis procedures
involve 24 h acid hydrolysis in 6 M HCl at 1108C (Fountoulakis and Lahm, 1998; Gehrke
et al., 1985; Heinrikson and Meredith, 1984; Rowan et al., 1992). Hydrolysis time (16±
72 h) has been shown to affect the release and degradation of amino acids in a diet for
growing pigs, and standard hydrolysis conditions rarely provided the maximum amino
acid concentration (Rowan et al., 1992). Other acids, such as methanesulfonic acid and ptoluenesulfonic acid, have been used to hydrolyse proteins, and the effects of time and
temperature on hydrolysis have been investigated (Fountoulakis and Lahm, 1998).
Soya bean products are used widely in animal nutrition (Emmert and Baker, 1995).
However, the effect of acid concentration on amino acid measurements in commonly
used soya protein products has not been investigated. The purposes of the present study
were threefold. The ®rst purpose was to determine the effect of acid concentration
on amino acid measurements in soya bean products. Secondly, the effect of method
(PITC versus IEC) on amino acid measurements was examined. Finally, for amino acids
levels that were not maximised with hydrolysis in 6 M HCl, correction factors were
calculated.
2. Materials and methods
2.1. Procedure
Representative samples of soya bean meal from two cropping years (SBM96, SBM97),
soya protein concentrate (SPC; Arcon F 65±301, Archer-Daniels-Midland, Decatur, IL),
soya protein isolate (SPI; Ardex 66±960, Archer-Daniels-Midland, Decatur, IL), whole
soya beans (WholeSB; Williams 82 variety) and soya bean hulls (Soya hulls) were
obtained. Homogenous samples (approximately 10 g) of each were ®nely ground in a
coffee bean grinder (Mr. Coffee, Model # IDS-50, Bedford Heights, OH) with
approximately 10 ml liquid nitrogen for about 20 s, mixed and stored frozen at ÿ108C.
2.2. Chemical analysis
Triplicate samples of each soya bean product were used to determine dry matter and
crude protein (N 6:25) according to procedures outlined by AOAC (1990). The
following chemicals were used for amino acid analyses: water (HPLC grade, cat. # W54); methanol (HPLC grade, cat. # A452-4); acetonitrile (HPLC grade, cat. # A998-4);
disodium hydrogen phosphate (anhydrous, certi®ed A.C.S., cat. # S374±500); ophosphoric acid (HPLC grade, cat. # A260±500); sodium acetate trihydrate (HPLC grade,
cat. # S220±1); concentrated hydrochloric acid (37%, cat. # A144±212); glacial acetic
acid (reagent A.C.S., A38c±212), all purchased from Fisher Scienti®c, St. Louis, MO. All
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
175
amino acids; phenyl isothiocyanate (protein sequencing grade, cat. # P-1034); and
triethylamine (cat. # 13,206±3), were obtained from Sigma±Aldrich, St. Louis, MO.
Triplicate samples of SBM96, SBM97, WholeSB, and Soya hulls (at least 200 mg),
and triplicate samples of SPC and SPI (at least 100 mg) were accurately weighed into
screw-capped test tubes (15 ml, Pyrex, cat. # 9826±16x, Corning, NY) with te¯on-lined
caps. HCl (12 ml) were added to the tubes. The acid concentrations used in this study
were the following: 1, 3, 6, 9 and 12 M. The tubes were purged with N2 for 10 s, mixed
and hydrolysed in an oven (Fisher Scienti®c, Model # 500 Series, Pittsburgh, PA) at
1108C for 24 h. After removal from the oven, the samples were allowed to cool. Once the
samples had cooled, 0.5 ml of a-amino butyric acid (AABA, 50 mM) and 0.5 ml of
norleucine (Nor, 50 mM) were accurately weighed on a balance and added to each tube.
The tubes were inverted 200 times and centrifuged at 1100 g for 10 min to pellet
debris. Then, the hydrolysates were diluted in a 1:2.5 ratio by adding 300 ml of distilled,
deionised water to a 200 ml aliquot of hydrolysate in a 1.5 ml microfuge tube (Fisher
Scienti®c, cat. # 05±408±10, Pittsburgh, PA). Amino acid standards (2.5 mM) were
prepared by weighing individual amino acids into a 250 ml volumetric ¯ask and
dissolving them in 0.1 M HCl. Three standards were used. Standard 1 consisted of Asp,
Ser, Gln, Citrulline, Arg, AABA, Val, Ile, Nor and Trp. Standard 2 consisted of Glu, Asn,
Taurine, Thr, Pro, AABA, Met, Leu, Nor and Ornithine. Standard 3 consisted of
Hydroxyproline, Gly, His, Ala, AABA, Tyr, Cys, Nor, Phe, Lys and Homoarginine.
Standards were prepared to accommodate analyses of hydrolysates and physiological
samples. Samples and standards were prepared for PITC and IEC analysis.
The procedures for amino acid determination with PITC were the following: 20 ml of
diluted hydrolysates or 10 ml of either standard 1, 2 or 3 were pipetted into polypropylene
tubes (Fisher Scienti®c, cat. # 1495910AA, St. Louis, MO). They were allowed to dry
under vacuum overnight in a freeze-drier (Labconco, Model # 77500, Kansas City, MO).
The samples were re-dried by adding 20 ml of 1:1:1 (v/v/v) methanol:water:triethylamine
to each sample. The tubes were held at a 458 angle and spun several times to resolubilise
amino acids. The tubes were then allowed to vacuum-dry for 4 to 6 h. Finally, the samples
were derivatised by adding 20 ml of 7:1:1:1 (v/v/v/v) methanol:water:triethylamine:phenyl isothiocyanate, and mixed by holding the tubes at a 458 angle and spinning them
several times. The tubes were capped and derivatisation was allowed to occur for 35 min
at room temperature (228C). The samples were vacuum-dried for 4±6 h following
derivatisation. The samples were then reconstituted in 200 ml of sample diluent, which
was composed of a mixture of 95:5 (v/v) phosphate buffer (5 mM sodium phosphate
dibasic, pH 7.4, adjusted with o-phosphoric acid):acetonitrile. The samples were vortexed
(Barnstead/Thermolyne, Type 16700 Mixer, Dubuque, IA), allowed to stand for
approximately 15 min, and then vortexed again. A pipette was used to transfer most of
the liquid to a polypropylene HPLC vial (Bio-Rad Laboratories, cat. # 223±9471,
Hercules, CA), while leaving any debris at the bottom of the microfuge tube. The
injection volume used ranged from 30 to 60 ml depending on the protein content of the
sample.
The Waters HPLC system consisted of either a 712 WISP or a 700 Satellite WISP
autosampler, two 510 pumps, a column heater (468C) and a 484 tunable absorbance
detector set at 254 nm. Peaks were identi®ed and integrated with Waters Maxima 820
176
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
software. The HPLC column was a Waters Pico-Tag1 3:9 mm 30 cm reverse-phase
column (Waters, cat. # WAT010950, Milford, MA). The packing consisted of 4 mm Silica/
C18 beads. A 4:6 mm 5 cm Supelcosil reverse-phase C18 guard column with 40 mm
Pellicular packing (Sigma±Aldrich, Supelco, cat. # 5±8232, Bellefonte, PA) was used.
Two eluents were used. Eluent A consisted of 2.5:97.5 (v/v) 70 mM sodium acetate, pH
6.55:acetonitrile. Eluent B consisted of 50:35:15 (v/v/v) acetonitrile:water:methanol.
Both eluents were vacuum ®ltered through a 0.45 mm nylon ®lter before use. The ¯ow
rate began at 1.0 ml/min. At 75 min, the ¯ow rate was increased to 1.3 ml/min. The ¯ow
rate returned to 1.0 ml/min at 76 min. The gradient which was run for the separation
consisted of 100% eluent A until 13.5 min, at which point the level of eluent A decreased
to 97%, eluent B increased to 3% (vertical change, Waters No. 11). The level of eluent A
continually decreased while eluent B increased, and this pattern is indicated in the
following: 24 min, concave curve, Waters No. 9/(A, 95%: B, 5%); 30 min, convex curve,
Waters No. 5/(A, 91%; B, 9%); 50 min, linear change, Waters No. 6/(A, 66%; B, 34%);
65 min, linear change, Waters No. 6/(A, 0%; B, 100%). The column was reequilibrated
with 100% eluent A (linear change, Waters No. 6) at 76 min for 89 min.
The procedures for amino acid determination with IEC were the following: duplicate
samples of SBM97 and SPC were utilised. Samples of hydrolysate were taken from the
same tubes that were used for the PITC procedure. Following hydrolysis, addition of
internal standards and centrifugation at 1100 g for 10 min, as described above, 200 ml
of each sample was diluted 1:5 (v/v) hydrolysate: distilled, deionised water. The diluted
samples were then pH adjusted with a buffer solution of NaOH in 10 ml sodium citrate
(2%, Beckman Instruments, Inc., Palo Alto, CA) in a ratio of 1:5 diluted sample:buffer
solution. For samples that were hydrolysed with 6 M HCl, 0.1 g of NaOH was used. For
samples that were hydrolysed with 1 and 3 M HCl, 0.01 g of NaOH was used. For
samples that were hydrolysed with 9 and 12 M HCl, 0.15 g of NaOH was used. The
pH of the samples was measured with litmus paper and found to be approximately 2.
The samples were then analysed using post-column detection with ninhydrin using
procedures that have been described previously (Spitz, 1973). It should be noted that
the internal standards were not used for calculations of amino acid concentrations with
IEC, but were used for PITC calculations only. External standards were used for the IEC
procedure.
2.3. Data analysis
One-way analysis of variance was performed following procedures described by Ott
(1993) to determine if acid concentration affected amino acid yield determined by PITC.
The means of each amino acid level at the various points in time were compared
according to the Student±Newman±Keuls multiple range test (Ott, 1993). One-way
analysis of variance was used to compare amino acid concentrations in SBM97 and SPC,
obtained with 6 M HCl, and determined with PITC and IEC (Ott, 1993). One of the
concentrations of histidine in Soya hulls determined with 6 M HCl was an outlier and was
removed from the data set. The mean of the two remaining values for histidine in Soya
hulls determined with 6 M HCl was used to complete the data set. The General Linear
Models Procedure of the Statistical Analysis Systems Institute Inc. (SAS Institute Inc.,
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
177
1988) was used for all calculations. An a level of 0.05 was used to determine statistically
signi®cant differences.
3. Results
The dry matter and crude protein (g kgÿ1, dry matter basis) contents, respectively, of
the soya bean samples were as follows: SBM96 (888, 536 g kgÿ1); SBM97 (885,
512 g kgÿ1); SPC (927, 682 g kgÿ1); SPI (907, 827 g kgÿ1); WholeSB (931, 410 g kgÿ1);
and Soya hulls (881, 116 g kgÿ1).
3.1. Acid concentration and amino acid composition
Mean amino acid values for the soya bean samples obtained using the various
acid concentrations during hydrolysis are shown in Figs. 1±4. Most amino acid
measurements were affected by acid concentration (P < 0:05) regardless of soya bean
sample. Acid concentration did not affect (P > 0:05) the release of the following
amino acids: lysine in SBM97; aspartic acid in SPC, SPI, WholeSB and Soya hulls;
threonine in Soya hulls; and phenylalanine in Soya hulls. Most amino acids exhibited
more release with acid hydrolysis in 3 M than in 1 M (P < 0:05), regardless of soya
bean sample. Aspartic acid was maximised with 1 M in SBM96 and SBM97, but it was
only signi®cant (P < 0:05) for SBM96. Maximum valine measurements were obtained
with acid concentrations greater than 6 M, but the differences were only signi®cant
(P < 0:05) for SBM96, SPC and SPI. Isoleucine was also maximised at concentrations
greater than 6 M, except in SBM97 and SPC, and the differences were only signi®cant
(P < 0:05) for SBM96 and SPI. There was a trend for serine concentrations to be
maximised (P > 0:05) with 3 M for SBM96 and Soya hulls, while threonine
concentrations were maximised with 9 M for SBM96, SBM97 and SPI. Threonine
release was greater (P < 0:05) with 9 M acid for SBM96 only. There was a trend
for tyrosine to be maximised (P > 0:05) with 3 M in SBM96, SBM97, SPC and
WholeSB. Acid hydrolysis with 9 and 12 M degraded (P < 0:05) tyrosine in all soya
bean samples. Glycine, histidine, alanine, arginine, proline, leucine, phenylalanine and
lysine tended (P > 0:05) to increase with increasing acid concentration for most soya
bean samples until 6 M HCl. There was a tendency for concentrations of isoleucine,
leucine, phenylalanine and lysine in SBM97 to decrease (P > 0:05) with hydrolysis
in acid concentrations greater than 6 M, while the concentrations of the same amino
acids increased (P > 0:05; P < 0:05 for isoleucine) in SBM96 with hydrolysis in
acid concentrations greater than 6 M. Glutamic acid increased (P < 0:05) from 1 to 3 M
in all soya bean samples, and then tended to increase or remain constant (P > 0:05) from
3 to 12 M.
3.2. Measurement method and amino acid composition
The in¯uence of measurement method on amino acid measurements of the soya bean
samples is shown in Table 1. With the exception of aspartic acid, most amino acid
178
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
Fig. 1. Effect of acid concentration on the mean yield of amino acids (Y axis, g kgÿ1 dry matter basis) from soya
bean meal 1996 (full line, shaded diamond), soya bean meal 1997 (long-dashed line, open square) and soya bean
hulls (short-dashed line, shaded circle). Error bars indicate sample standard error (n 3; for histidine in soya
bean hulls determined with 6 M HCl, one data point was an outlier. It was removed and the mean of the two
remaining values was used to complete the data set).
concentrations in SBM97 were higher (P > 0:05) when determined with PITC than when
determined with IEC. With the exception of aspartic acid and histidine, most amino acid
concentrations in SPC were numerically higher (P < 0:05) when determined with PITC
than when determined with IEC.
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
179
Fig. 2. Effect of acid concentration on the mean yield of amino acids (Y axis, g kgÿ1 dry matter basis) from soya
bean meal 1996 (full line, shaded diamond), soya bean meal 1997 (long-dashed line, open square) and soya bean
hulls (short-dashed line, shaded circle). Error bars indicate sample standard error (n 3).
3.3. Correction factors
Correction factors calculated for the soya bean samples are shown in Table 2. The
correction factors were determined by dividing the maximum amino acid measurement
by the value obtained with hydrolysis in 6 M HCl. Correction factors were only
calculated for measurements conducted with PITC. Most of the correction factors ranged
from 1.00 to 1.32. However, the correction factors for isoleucine in SBM96 and lysine in
Soya hulls were greater than 1.32.
180
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
Fig. 3. Effect of acid concentration on the mean yield of amino acids (Y axis, g kgÿ1 dry matter basis) from soya
protein isolate (full line, shaded triangle), soya protein concentrate (long-dashed line, cross) and whole soya
beans (short-dashed line, open circle). Error bars indicate sample standard error (n 3).
4. Discussion
4.1. Acid concentration and amino acid composition
It is well known that valine and isoleucine are released slowly, while serine and
threonine are continually degraded, over time during acid hydrolysis (Gehrke et al., 1985;
Rees, 1946; Rowan et al., 1992). Increasing acid concentration with ®xed hydrolysis time
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
181
Fig. 4. Effect of acid concentration on the mean yield of amino acids (Y axis, g kgÿ1 dry matter basis) from soya
protein isolate (full line, shaded triangle), soya protein concentrate (long-dashed line, cross) and whole soya
beans (short-dashed line, open circle). Error bars indicate sample standard error (n 3).
also resulted in the slow release of valine and isoleucine, as measurements of the two
amino acids increased with increasing acid concentration in the present study (Figs. 1±4).
Serine was progressively destroyed from hydrolysis with 3 M to greater acid
concentrations in only SBM96 and Soya hulls. However, threonine was maximised with
9 M HCl for SBM96, SBM97 and SPI. There was no indication of threonine degradation
with hydrolysis using 9 M, but threonine was degraded with 12 M. This study indicates
that the differences in the release and degradation of valine, isoleucine, serine and
182
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
Table 1
Amino acid levels (g kgÿ1, dry matter basis) in soya bean meal (SBM97) and soya protein concentrate (SPC)
determined using 6 M HCl for acid hydrolysis and using pre-column derivitisation with phenyl isothiocyanate
(PITC) or post-column detection with ninhydrin (IEC)
Amino acid
SBM97
SPC
PITC
Aspartic acid
Glutamic acid
Serine
Glycine
Histidine
Threonine
Alanine
Arginine
Proline
Tyrosine
Valine
Isoleucine
Leucine
Phenylalanine
Lysine
48.8
101.5
30.5
27.9
14.2
25.8
27.1
41.7
31.5
20.2
25.8
25.8
43.1
29.8
36.0
IEC
b
2.30
2.20
1.00
1.00
0.80
0.10
1.00
2.20
1.00
1.60
1.40
1.20
1.10
0.10
4.20
51.5
84.6
23.8
19.6
12.3
18.5
20.2
33.3
24.7
16.2
21.5
20.7
35.6
23.9
29.4
0.20
0.20
0.10
0.10
0.10
0.10
0.10
0.20
0.40
0.10
0.20
0.20
0.20
0.10
0.20
Sig.a
PITC
NS
71.0
123.3
35.0
31.6
15.5
28.1
31.5
50.6
37.8
24.7
33.9
36.4
57.6
41.6
60.7
**
*
**
NS
**
**
*
*
NS
*
*
*
**
NS
IEC
3.10
0.20
1.90
4.80
0.60
4.30
1.4
0.20
5.90
0.70
5.80
7.10
2.00
5.40
5.30
71.1
116.8
32.5
26.8
16.5
25.2
27.4
49.0
34.8
21.7
29.7
28.4
48.7
31.1
39.7
Sig.
0.20
0.10
0.30
0.10
0.10
0.10
0.10
2.50
0.30
0.10
0.10
0.10
0.20
0.20
0.50
NS
**
NS
NS
NS
NS
NS
NS
NS
*
NS
NS
*
NS
*
a
NS: non-signi®cant (P > 0:05).
Standard deviation (n 3).
*
P < 0:05.
**
P < 0:01.
b
Table 2
Correction factorsa for amino acids in soya bean meal (SBM96, SBM97), soya protein concentrate (SPC), soya
protein isolate (SPI), whole soya beans (WholeSB) and soya bean hulls (Soya hulls)
Amino acid
SBM96
SBM97
SPC
SPI
WholeSB
Soya hulls
Aspartic acid
Glutamic acid
Serine
Glycine
Histidine
Threonine
Alanine
Arginine
Proline
Tyrosine
Valine
Isoleucine
Leucine
Phenylalanine
Lysine
1.19
1.08
1.05
1.10
1.10
1.05
1.11
1.08
1.07
1.03
1.17
1.45
1.06
1.11
1.21
1.08
1.00
1.00
1.06
1.02
1.00
1.02
1.02
1.08
1.04
1.05
1.00
1.00
1.00
1.00
1.02
1.01
1.00
1.00
1.07
1.00
1.00
1.01
1.00
1.00
1.19
1.00
1.01
1.00
1.00
1.03
1.00
1.02
1.00
1.07
1.02
1.03
1.00
1.02
1.00
1.11
1.11
1.03
1.02
1.04
1.02
1.00
1.00
1.17
1.00
1.00
1.03
1.00
1.12
1.02
1.04
1.09
1.00
1.00
1.02
1.14
1.17
1.02
1.08
1.16
1.00
1.29
1.16
1.00
1.00
1.32
1.15
1.01
1.12
1.65
a
Correction factors determined by expressing the maximum amino acid yield as a proportion of the yield
using 6 M HCl.
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
183
threonine are, at least in part, dependent on protein source. Other researchers (Glazer
et al., 1976; Rowan et al., 1992) have attributed differences in degradation and release of
amino acids to protein source. Tyrosine, reported to be susceptible to oxidation (Finley,
1985; Gehrke et al., 1985), was degraded with the use of 9 and 12 M HCl. However,
similar concentrations of tyrosine were obtained with 6 M HCl relative to those obtained
with 3 M HCl, although some of the values were slightly lower at 6 M compared to 3 M
HCl. Rowan et al. (1992) reported similar observations concerning tyrosine, and
attributed the stability to the addition of phenol. In the present study, however, phenol was
not added prior to hydrolysis, and tyrosine was essentially not degraded with acid
concentrations as strong as 6 M HCl. Aspartic acid tended to decrease as acid
concentration increased from 1 M for samples of SBM96, SBM97 and Soya hulls,
indicating mild sensitivity of aspartic acid to acid degradation in these samples. Similar
patterns were not observed for SPC, SPI and WholeSB.
The relative stability or gradual increase in measurements of aspartic acid, glycine,
histidine, alanine, arginine, proline, leucine, phenylalanine and lysine with increasing
acid concentration used during this study re¯ected the lack of sensitivity of these amino
acids to increasing acid concentration, even using 12 M HCl.
4.2. Measurement method and amino acid composition
Different investigators have evaluated the two amino acid determination methods used
in this study, and they have reported that the two procedures provide very similar results
(Bidlingmeyer et al., 1984; Elkin and Wasynczuk, 1987; Heinrikson and Meredith, 1984).
However, in the current study, method affected amino acid measurements. For many
amino acids, in both SBM97 and SPC using hydrolysis in 6 M HCl, the PITC procedure
provided signi®cantly higher measurements. Also, it should be noted that the hydrolysis
curves for SBM97 and SPC determined with IEC followed the same patterns of release as
those shown in Figs. 1±4 (data not shown). Comparisons with published amino acid
values have indicated that the measurements determined with PITC and IEC were
comparable for amino acid levels in soya bean samples. For example, using PITC and
IEC in this study, the threonine content of SBM97 was 25.8 and 18.5 g kgÿ1, respectively
(dry matter basis). Other publications (Cavins et al., 1972; Emmert and Baker, 1995;
NRC, 1998; Rudolph et al., 1983) have reported threonine concentrations in soya bean
meal of 21.5, 20.6 and 18.6 g kgÿ1, respectively (dry matter basis). The threonine content
of SPC using PITC and IEC was found to be 28.1 and 25.2 g kgÿ1, respectively (dry
matter basis). Emmert and Baker (1995) and NRC (1998) reported threonine
concentrations in SPC of 27.3 and 31.1 g kgÿ1, respectively (dry matter basis). There
are several possible explanations for the differences in amino acid measurements
determined with the two methods in the current study. Many steps of the IEC procedure
demanded accurate volumetric measurements. Before amino acids were determined with
the IEC procedure, the hydrolysate needed to be diluted and buffered. This was done in
two different steps. As part of this procedure, external standards were used and, therefore,
dilution of standards and injection volumes needed to be accurate. The PITC procedure
did not require accurate volumetric measurements because internal standards and tube
weighing were used. Therefore, more opportunities for error existed with the IEC
184
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
procedure than when the PITC procedure was used. Despite this, it is interesting to note
that less variation due to method existed for amino acids in SPC compared to SBM97.
During soya bean processing, SPC is the result of further processing than soya bean meal.
Thus, SPC is more puri®ed and re®ned than soya bean meal, and, therefore, may contain
fewer factors than soya bean meal that could alter the determination of amino acids. Even
though both procedures provided values for most amino acids that are within the range
found in the literature, further research is necessary to increase the accuracy and decrease
the variation from procedure-to-procedure.
4.3. Correction factors
Garnett (1985) suggested the use of generalised correction factors as a simple approach
to correcting amino acid concentrations. Correction factors have been calculated by
others (Kohler and Palter, 1967; Rowan et al., 1992; Slump, 1980; Tkachuk and Irvine,
1969) to correct amino acid measurements determined with 24 h hydrolysis to the
maximum values obtained with different hydrolysis times. Recently, research projects
have begun to use correction factors to obtain amino acid measurements that are more
accurate (Lenis et al., 1990; Mroz et al., 1994). However, correction factors have not been
determined to correct the values obtained with hydrolysis in 6 M HCl to the maximum
values obtained with other acid concentrations. For hydrolysis time, correction factors for
serine, isoleucine and threonine in foods have been reported (Kohler and Palter, 1967;
Rowan et al., 1992; Slump, 1980; Tkachuk and Irvine, 1969) and ranged from 1.04 to
1.14, 1.02 to 1.21 and 1.02 to 1.08, respectively. In the present study, correction factors
for serine, isoleucine and threonine ranged from 1.02 to 1.05, 1.09 to 1.15 and 1.02 to
1.05, respectively. Rowan et al. (1992) and Slump (1980) have reported correction factors
for 24 h hydrolysis for valine of 1.20 and 1.08, respectively. In the present study, valine
correction factors ranged from 1.04 to 1.32. Most of the correction factors in this study
were similar to those determined with different hydrolysis times. For isoleucine in
SBM96 and lysine in Soya hulls, the correction factors were 1.45 and 1.65, respectively.
These should be used with caution, as comparison correction factors would suggest that
these factors are abnormally high (Rowan et al., 1992). However, there is nothing to
indicate that analytical error produced abnormal measurements of these amino acids.
In conclusion, determination of amino acid concentrations in protein sources is
affected by acid hydrolysis of the samples and the use of different amino acid
determination methods. Ion-exchange chromatography with ninhydrin detection has been
shown to provide similar results when compared to pre-column derivatization with phenyl
isothiocyanate, and the ®ndings of the current study indicate that the two methods
provided somewhat similar measurements. However, method did affect several amino
acid concentrations, indicating that variation in amino acid analysis can provide different
results. In many cases standard hydrolysis procedures (hydrolysis in 6 M HCl for 24 h at
1108C) do not provide the maximum amino acid measurements in samples. Correction
factors are necessary to determine amino acid concentrations more accurately (i.e. for
serine, threonine, isoleucine, valine) in protein sources. Further research is necessary with
more feedstuffs, as well as a range of diets, to determine ideal hydrolysis conditions and
correction factors.
D.M. Albin et al. / Animal Feed Science and Technology 87 (2000) 173±186
185
Acknowledgements
The authors would like to thank Zoran Magas of LC Tech Services, Burlington, Ont.,
Canada for technical advice and maintenance of the high pressure liquid chromatography
equipment used in this study.
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