ARTICLE IN PRESS

International Dairy Journal 14 (2004) 527–533

Production and characterization of casein hydrolysates with a high

amino acid Fischer’s ratio using immobilized proteases
Justo Pedroche, Mar!ıa M. Yust, Hassane Lqari, Julio Giron-Calle,
! Javier Vioque,
!
Manuel Alaiz, Francisco Millan*
Instituto de la Grasa, Consejo Superior de Investigaciones Cient!ıficas, Avda. Padre Garcia Tejero 4, Sevilla 41012, Spain
Received 15 June 2003; accepted 3 November 2003

Abstract

A procedure using immobilized enzymes has been developed to obtain protein hydrolysates with a high Fischer’s ratio (branched-
chain amino acids/aromatic amino acids (AAA)) from bovine casein. Pre-digestion with trypsin was followed by treatment with
chymotrypsin, which generated a hydrolysate enriched in peptides with AAA at the carboxyl end. Carboxypeptidase A was then
used to remove these AAA. A fraction, which represents 24% of total hydrolyzed proteins, with a Fischer’s ratio of 30.6, and
Phe+Tyr content below 1.4% of the total, was purified from this hydrolysate by gel filtration in a Biogel P2 column. This material
could be used for the treatment of patients suffering from certain hepatic encephalophaties, tyrosinemia and phenylketonuria.
r 2003 Published by Elsevier Ltd.

Keywords: Casein; Trypsin; Chymotrypsin; Carboxypeptidase A; Protein hydrolysate; Fischer’s ratio

1. Introduction absorption of hydrolysates can be more effective than

Enzymatic hydrolysis is frequently used to improve
functional and nutritional properties of food proteins.
Protein hydrolysates can be classified into three major
groups depending on the degree of hydrolysis, which
determines their applications: hydrolysates with a low
degree of hydrolysis with improved functional proper-
ties, hydrolysates with a variable degree of hydrolysis
that are used mostly as flavourings, and extensive
hydrolysates that are mostly used as nutritional supple-
ments and in special medical diets (Vioque, Clemente,
Pedroche, Yust, & Milla! n, 2001). These extensive
hydrolysates have received a great deal of attention in
recent years, and are main constituents of geriatric
products, high-energy supplements, enteral and parent-
eral solutions, and hypoallergenic foods. The use of
protein hydrolysates as a source of proteins for
hospitalized patients has been steadily increasing for
the last two decades (Morbahan & Trumbore, 1991).
Protein hydrolysates present several advantages as
constituents of medical diets. The gastrointestinal
*Corresponding author. Tel.: +34-5-4611550; fax: +34-5-4616790.
E-mail address: frmillan@cica.es (F. Mill!an).
the absorption of either intact protein (Ziegler et al.,
1990) or free amino acids (Silk et al., 1979). In addition,
the osmotic pressure of peptides is lower than that of the
corresponding free amino acids (Mahmoud, 1994);
hydrolysis can be used also to produce hypoallergenic
hydrolysates (Cordle, 1994).
Hydrolysates containing peptides with a high ratio of
branched-chain amino acids (BCAA) to aromatic amino
acids (AAA), referred to as Fischer’s ratio, are used in
specific medical diets (Fischer, 1990) for the treatment of
patients with liver diseases, including hepatic encephalo-
pathy (Kawamura-Yasui et al., 1999; Hemeth, Steindl,
.
Ferenci, Roth, & Hortnalg, 1998). Hepatic encephalo-
pathy causes liver malfunction which results in increased
protein catabolism and a deficit of branched chain
amino acids. In addition, the blood concentration of
AAA increases. These amino acids can then reach the
brain in high amounts, where they can mimic the effects
of certain neurotransmitters and/or compete with
others, resulting in several brain disorders (Hazell &
Butterworth, 1999). Protein hydrolysates with a
Fischer’s ratio higher than 20, and contents of tyrosine
plus phenylalanine not exceeding 2% of total amino
acids, are used for the treatment of these patients (Okita,

0958-6946/$ - see front matter r 2003 Published by Elsevier Ltd.

doi:10.1016/j.idairyj.2003.11.002
 ARTICLE IN PRESS
528 J. Pedroche et al. / International Dairy Journal 14 (2004) 527–533
Watanabe, & Nagashima, 1985). A diet high in BCAA
and/or low in AAA is also recommended in phenylk-
etonuric and tyrosinemic patients (Pietz et al., 1999),
and has been used with success in the treatment of
diabetes (Karabatas, Fabiano de Bruno, Pastorale,
Lombardo, & Basabe, 2000). This type of diet is also
beneficial as a nutritional support during prolonged
physical exercise (Mero, 1999).
However, the preparation of protein hydrolysates
with such a highly specific composition is not an easy
task, and requires the use of a number of proteases in
order to produce a well-defined digestion pattern. Also,
it is of interest to use proteins with a high nutritional
value as a starting material for the preparation of this
type of hydrolysates. Casein fulfills this requirement
because of its equilibrated amino acid composition.
Protein hydrolysates with a high Fischer’s ratio have
been generated using the protein zein and soluble
proteases, but the resulting hydrolysates did not have
the recommended optimal composition for clinical use
(Tanimoto, Tanabe, Watanabe, & Arai, 1991). Casein
has been used for the preparation of hydrolysates with a
high Fischer’s ratio as well. Thus, a hydrolysate with a
Fischer’s ratio of 31.97 was obtained after hydrolysis
using the enzymes thermolysin and pepsin (Adachi,
Kimura, Murakami, Matsuno, & Yokogoshi, 1991).
The production of this type of hydrolysates with good
yields requires the use of a series of protease prepara-
tions with a very specific target sequence. Considering
that these proteases are usually expensive, immobilizing
them would be a clear advantage from the economical
point of view, since immobilized enzymes can be easily
recovered and reused. Compared to soluble enzymes,
immobilized proteases present additional advantages.
Thus, immobilized enzymes allow for the process to be
performed in milder, more controlled conditions, and
the production of secondary metabolites originating
from enzyme autolysis is prevented. Also, immobilized
enzymes do not need to be inactivated by heat or
acidification, which may be damaging for the final
product. The goal of this research was to develop a
procedure for the production of protein hydrolysates
with a high Fischer’s ratio using casein and a series of
immobilized proteases.
2. Materials and methods
2.1. Materials
Bovine casein was purchased from Merck (Darmstad,
Germany); bovine trypsin (E.C. 3.4.21.4), a-chymotryp-
sin (E.C. 3.4.21.1), benzoyl-l-tyrosine p-nitroanilide
(BTNA), N-benzoyl-l-arginine p-nitroanilide (BANA),
hippuril-l-phenylalanine (HP), and glycidol from Sigma
Chemical Co. (St. Louis, MO); and carboxypeptidase A
(E.C. 3.4.17.1) from Serva (Heidelberg, Germany).
A supply of 10% beads crosslinked (10 BCL) agarose
was donated by Hispanagar SA (Madrid). Organic
solvents and all other chemical reagents were of
analytical grade.
2.2. Activation of agarose gels
The activation of agarose gels was achieved according
to the procedure described by Guisan (1988) with
following modifications: A 30 mL volume of agarose
gel (0.7 g of swollen agarose is roughly equivalent to
1 mL) was suspended in 6 mL distilled water plus 10 mL
1.7 m NaOH containing 284 mg NaBH4. This resulted in
reducing conditions that are necessary to avoid oxida-
tion of the gel. Then, 7.2 mL glycidol were added
dropwise to this suspension, which was kept on ice, in
order to reach a 2 m final concentration. The whole
suspension was gently stirred overnight at room
temperature. The activated gel was washed with
abundant water (pH 7) and 300 mL water containing
300 mmoles NaIO4 mL 1 gel to achieve multipoint
attachment. This oxidative reaction was allowed to
proceed for 2–3 h with stirring at room temperature. The
glyceryl groups obtained in the etherification reaction by
glycidol are oxidized specifically by periodate mol to
mol. The number of aldehyde groups that are produced
can be determined by measuring the NaIO4 that is not
consumed in the reaction by titration with IK.
2.3. Immobilization of the enzymes
Immobilization of the enzymes was carried out
according to Blanco and Guisa! n (1989), with some
modifications. Volumes of 10 mL activated agarose gel
containing 30 mg enzyme mL 1 gel (except for carbox-
ypeptidase A: 2 mg enzyme mL 1 gel) were dispersed in
100 mL 0.2 m sodium bicarbonate. The reaction mixture
was gently stirred at room temperature for 180 min.
Aliquots of the supernatants and whole suspensions
were withdrawn at different times and catalytic activity
was measured. Immobilized enzymes were then reduced
by addition of sodium borohydride (100 mg). After
gentle stirring for 30 min at room temperature, the
resulting immobilized enzymes were washed with
abundant distilled water to eliminate residual sodium
borohydride. These gel beads containing immobilized
enzymes were used for obtaining the protein hydro-
lysates.
2.4. Enzymatic assays
2.4.1. Trypsin
The activity of the soluble or immobilized enzyme
(20 mg mL 1) was assayed by following the increase of
absorbance at 405 nm which accompanies hydrolysis of
 ARTICLE IN PRESS
J. Pedroche et al. / International Dairy Journal 14 (2004) 527–533
the synthetic substrate BANA (75 mL soluble or
suspended enzyme were added to 2.5 mL 50 mm sodium
phosphate 40% ethanol, pH 7, containing 200 mL
BTNA 1 mm in 50 mm sodium phosphate 40% ethanol
pH 7 at room temperature).
2.4.2. a-chymotrypsin
The activity of the soluble or immobilized enzyme
(20 mg mL 1) was assayed by following the increase of
absorbance at 415 nm which accompanies hydrolysis of
the synthetic substrate BTNA (75 mL soluble or im-
mobilized enzyme were added to 2.5 mL 50 mm sodium
phosphate 40% ethanol, pH 7, containing 25 mL BTNA
40 mm in hexane:dioxane 1:1 (v/v) at room temperature).
2.4.3. Carboxypeptidase A
The activity of the soluble or immobilized enzyme
(2 mg mL 1) was assayed following the increase of
absorbance at 254 nm which accompanies hydrolysis of
the synthetic substrate HP (75 mL of sample were mixed
with 2.5 mL of 1 mm HP in 50 mm sodium phosphate pH
7 at room temperature).
2.5. Degree of hydrolysis
The degree of hydrolysis was calculated by determi-
nation of free amino groups by reaction with TNBS
(Adler-Nissen, 1979). The total number of amino groups
were determined in a sample 100% hydrolyzed by
incubation with 6 n HCl at 120 C for 24 h.
2.6. Protein determination
Amounts of protein and peptides were determined by
elemental analysis as % nitrogen content  6.25, using a
LECO CHNS-932 analyzer (St. Joseph, MI, USA).
2.7. Fast protein liquid chromatography
Lyophilized samples (1 g) were dissolved in 0.1 m
sodium borate 0.2 m sodium chloride buffer pH 8.3
(10 mL). Gel filtration was carried out in an FPLC
system equipped with a Superose 12 HR 10/30 column
from Amersham Pharmacia LKB Biotechnology (Up-
psala, Sweden). Samples at a protein concentration of
1.6 mg mL 1 were injected using a 200 mL loop. Borate
buffer at a flow rate of 0.4 mL min 1 was used for
elution, which was monitored at 280 nm. Blue dextran
(2000 kDa, Pharmacia Biotech.), catalase (240 kDa,
Serva, Heidelberg, Germany), bovine serum albumin
(67 kDa), ovalbumine (43 kDa), ribonuclease (13.7 kDa),
citochrome C (12.5 kDa, Pharmacia Biotech.) and
bacitracine (1.45 kDa, Sigma Chemical Co.) were used
as molecular weight standards.
529
2.8. Hydrolysis of casein
Casein was hydrolyzed batchwise in a fluidized bed
reactor containing gel beads with immobilized enzymes.
Immobilized trypsin (1 g) was added to a casein solution
(2% w/v in NH4HCO3 50 mm) (50 mL), which had been
previously adjusted to pH 8.0 using NH4OH 12% w/v,
and the solution was taken to 50 C. After incubation for
2 h, immobilized trypsin was removed by filtration, and
immobilized a-chymotrypsin was added to the hydro-
lysed casein. Incubation was continued for another 2 h
at the same pH and temperature. Finally, immobilized
carboxypeptidase A was added after removal of a-
chymotrypsin by filtration; incubation was continued
for two more hours at the same pH and temperature.
2.9. Isolation of fractions with a high Fischer’s ratio from
the casein hydrolysate
The casein hydrolysate was filtered trough a 5 kDa
membrane using an Amicon cell and injected into a
Biogel P2 (BIORAD, CA, USA) gel filtration column
(2  55 cm2) at a flow rate of 10 mL h 1 using 50 mm
ammonium bicarbonate as eluent. Fractions were
collected every 10 min. Cytochrome C (12,384 Da),
bacitracin (1400 Da), Val4-angiotensin (917 Da), Arg–
Lys–Glu–Val–Tyr (693 Da) and Trp–Gly (261 Da) were
used as molecular weight standards for determination of
molecular mass.
2.10. Amino acid analysis
Samples (2 mg) were hydrolyzed by addition of 6 n
HCl (4 mL) and incubation at 110 C for 24 h in tubes
sealed under nitrogen. Amino acids were determined
after derivatization with diethyl ethoxymethylenemalo-
nate by high-performance liquid chromatography
(HPLC) according to the method of Alaiz, Navarro,
Giron, and Vioque (1992).
2.11. Electrophoresis
Tricine-sodium dodecyl sulfate polyacrylamide gel
electrophoresis was performed according to the method
of Sch.agger and von Jagow (1987) with minor modifica-
tions. The separating gel consisted of a 20% T, 6% C
gel, where the composition of the acrylamide mixtures is
defined by the letters T (total percentage concentration
of acrylamide and bisacrylamide) and C (percentage
concentration of the crosslinker relative to the total
concentration T) according to Hjerten (1962). The
stacking gel consisted of a 4% T, 3% C gel. The length
of the separating and stacking gels were 6 and 2 cm,
respectively, with a gel thickness of 1 mm. Electrophor-
esis was performed at a constant voltage of 60 V for
stacking and 120 V for separation. Protein bands were
 ARTICLE IN PRESS
530 J. Pedroche et al. / International Dairy Journal 14 (2004) 527–533

fixed in 20% methanol, 8% acetic acid for 15 min, and
stained by incubation in 0.25% coomassie brilliant blue
G in 45% methanol, 10% acetic acid for 24 h.
Destaining of the gels was performed using 10% acetic
acid.
3. Results and discussion
3.1. Hydrolysis of casein
Hydrolysis of casein for elimination of AAA was first
attempted by using a two-step process in which casein
was first treated with chymotrypsin and then with
carboxypeptidase A. The following peptidic bonds are
most susceptible to hydrolysis by chymotrypsin: Tyr–
Xaa, Trp–Xaa, Phe–Xaa, and Leu–Xaa. Thus, a protein
hydrolysate generated using this enzyme should be
enriched in peptides with AAA in carboxyterminal
position. Additional treatment with carboxypeptidase A
should provide a pool of free AAA, and peptides with
fewer aromatic residues than the original ones. Never-
theless, this two-step approach using chymotrypsin and
carboxypeptidase A resulted in a low degree of
hydrolysis (results not shown). In addition, the peptide
fractions that were obtained had Fischer’s ratios below
8, not reaching the suggested optimum Fischer’s ratio
of around 20 (Okita et al., 1985). The low yield of
hydrolysis may be due to a poor accessibility of
chymotrypsin to the aromatic amino residues or to
denaturation of the enzyme. After ruling out possible
denaturation of the enzyme by analysis of chymotrypsin
activity using the synthetic substrate BTNA (results not
shown), it was concluded that a low accessibility to the
aromatic residues was the reason why hydrolysis was
not efficient (Bai, Ge, & Zhang, 1999).
In order to facilitate chymotrypsin digestion by
improving exposure of aromatic residues, a predigestion
of casein with immobilized trypsin was carried out.
Digestion by trypsin does not destroy the target sites for
chymotrypsin because it hydrolyzes preferentially Arg–
Xaa and Lys–Xaa bonds. An additional advantage of
using trypsin is that it does not generate peptides with
aromatic residues in aminoterminal position, which
cannot be hydrolyzed by carboxypeptidase A. Hydro-
lysis by trypsin produced a 7.4% DH, which compares
well with a theoretical maximum of 10.2%. These DH
values are given as % of Lys and Arg residues in casein
(Table 1), so that a 7.4% DH means that 73.0% of the
available Lys and Arg residues were hydrolyzed. FPLC
gel filtration chromatography of a casein hydrolysate
obtained by treatment with trypsin shows degradation
of the main fraction of casein (Fig. 1). These results were
confirmed by SDS-PAGE (Fig. 2). Thus, the main band
observed in SDS-PAGE of casein, which corresponds to
a-casein, was degraded by trypsin.
Because hydrolysis by trypsin was satisfactory, it was
concluded that access of the immobilized enzymes to the
substrates is not hampered by diffusion of the substrates
into the gels. Nevertheless, hydrolysis by chymotrypsin
was not very effective (Table 1) even after treatment
with trypsin. Thus, over a theoretical maximum of
36.0% DH, chymotrypsin yielded a DH of 6.2%,
representing a 17.3% efficiency. It is possible that the
hydrophilic nature of the agarose gel makes it difficult
for the AAA, Trp, Phe and Tyr, to approach
immobilized chymotrypsin. In addition to this possible
problem, it should be considered that hydrolysis by a-
chymotrypsin is inhibited when the target sequences are
close to histidine or methionine residues, and that the
hydrolysis is not performed at all when the adjacent
amino acid is proline, which is the most abundant amino
acids in casein (Blow, 1971; Keil, 1992).
Despite its low efficiency, hydrolysis by chymotrypsin
produced changes in the protein pattern as observed by
FPLC gel filtration chromatography (Fig. 1c) and SDS-
PAGE (Fig. 2). An increase in fractions of lower
molecular weight (3 and 7 kDa) and the disappearance
of high molecular weight proteins was observed by
FPLC analysis. SDS-PAGE analysis showed that al-
most all protein bands remaining after trypsin hydro-
lysis disappeared, and a smear of bands with molecular
weight between 6 and 14 kDa was observed, which is
consistent with FPLC analysis.

Table 1
Degree of hydrolysis of the casein hydrolysates obtained by sequential treatment with trypsin, chymotrypsin and carboxypeptidase A

Enzyme treatment Theoretical maximum Observed DHb(%) Observed DH/theoretical

DHa(%) DH (efficiency)

Trypsin 10.2 7.470.6 73.0

Chymotrypsinc 17.3 6.2 36.0
Trypsin +Chymotrypsin 27.5 13.670.7 50.0
Trypsin +Chymotrypsin +Carboxypeptidase A — 27.071.5 —

Data are given as percentages and represent the mean7standard deviation of three experiments.
a
Maximum number of hydrolyzable amino acid bonds deduced from the amino acid composition of casein.
b
Determined by reaction with TNBS.
c
Calculated by substraction (digestion with trypsin and chymotrypsin minus digestion with trypsin).
 ARTICLE IN PRESS
J. Pedroche et al. / International Dairy Journal 14 (2004) 527–533 531

0.2 et al., 2002) that hydrolysis with carboxypeptidase A for

0.15
30 min to 2 h releases those amino acids for which the
enzyme exhibits the highest specificity, normally tyr-
0.1 osine, phenylalanine and tryptophan. If the hydrolysis is
0.05 allowed to proceed for a longer time, other amino acids
0 are released. Thus, after 8 h, valine, isoleucine, threo-
(a) 0 5 10 15 20 25 30 35 nine, alanine, and lysine are released, and after 24 h of
hydrolysis, the amino acids serine, glycine and histidine
0.12 are released as well. Considering these results, the
0.1
treatment with carboxypeptidase for 2 h, which results
0.08
0.06 in a DH of 27.2%, is most likely causing release of
0.04 AAA, which would be exposed in the carboxyterminal
0.02 position due to the previous action of chymotrypsin.
Absorbance (280 nm)

0 The final hydrolysate obtained by hydrolysis with

(b) 0 5 10 15 20 25 30 35 trypsin, chymotrypsin and carboxypeptidase A should
0.12 be rich in di- and tripeptides. The FPLC gel filtration
0.1 profile of this hydrolysate is shown in Fig. 1d, which
0.08 also illustrates the release of aromatic residues such as
0.06 Tyr, Phe and Trp. SDS-PAGE analysis provided a
0.04 pattern similar to that obtained using trypsin and
0.02
chymotrypsin, with a smear of peptides with molecular
0
(c) 0 5 10 15 20 25 30 35 weights ranging from 6 to 14 kDa. In fact, the FPLC
chromatography profiles shown in Fig. 1c, and d were
0.5
Tyr similar with the exception of the peaks corresponding to
0.4 Tyr, Phe and Trp in Fig. 1d, which attenuates the signal
0.3 of the remaining peptides.
Trp
0.2
0.1 Phe
3.2. Purification of peptides with high Fischer’s ratio
0
0 5 10 15 20 25 30 35 For the isolation of these peptides, the final casein
(d) Volume elution (mL) hydrolysate obtained after sequential treatment with
Fig. 1. FPLC gel filtration chromatography of native casein (a), and trypsin, chymotrypsin and carboxypeptidase A was
casein hydrolysates obtained by treatment with trypsin (b), trypsin and loaded on a Biogel P2 gel filtration column (Fig. 3).
chymotrypsin (c) and trypsin, chymotrypsin, and carboxypeptidase A The eluate was divided into six fractions (FI, FII,
(d). Tyr, tyrosine; Phe, phenylalanine; Trp, tryptophan. FIII, FIV, FV and FVI). The protein contents and

kDa 1 2 3 4 5 6 kDa 0.9 F I F II F III F IV F V F VI

0.8
67.0
43.0 0.7
Absorbance (280 nm)

30.0
0.6
20.1 17.0
14.4 0.5
14.4 10.7
8.1 0.4
6.2
0.3

0.2

Fig. 2. SDS-PAGE analysis of bovine casein (2), and casein 0.1

hydrolysates obtained by treatment with trypsin (3), trypsin and
0
chymotrypsin (4), and trypsin, chymotrypsin, and carboxypeptidase A
0 10 20 30 40 50 60 70 80 90
(5). Low molecular weight marker kit (1), Peptide marker kit (6).
Elution volume (mL)

The final digestion with carboxypeptidase A released Fig. 3. Biogel P2 gel filtration chromatography of the final hydrolysate
a variety of free amino acids, depending on the duration obtained by sequential treatment of casein with trypsin, chymotrypsin
of the treatment. We previously reported (Pedroche and carboxypeptidase A.
 ARTICLE IN PRESS
532 J. Pedroche et al. / International Dairy Journal 14 (2004) 527–533

approximate molecular weights of the proteins and/or
peptides included in each fraction are shown in Table 2.
Fraction I represents 57% of total proteins eluted from
the column. It is constituted by proteins and peptides
with molecular weights ranging from 1800 to 10,000 Da,
which were not completely digested by trypsin and
chymotrypsin. Fraction II contains peptides between
1400 and 1800 Da and represents 17% of total proteins
eluted. Fraction III constitutes 24% of total proteins
and presents a profile of peptides from 500 to 1400 Da.
Fractions IV, V and VI correspond to peptides below
500 Da and free amino acids, and represent 2% of total
proteins.
The amino acid composition of each of these fractions
is shown in Table 3. The Fischer’s ratio of fractions FI
and FII is low, although higher than that of native
casein (3.3, 2.8 and 2.0, respectively). On the other hand,
fraction FIII shows low amounts of AAA, only 0.6%
Tyr and 0.3% Phe, in comparision to 3.9% and 4.4%,
Table 2
Protein recovery and molecular weight of the fractions isolated by
Biogel P-2 gel filtration chromatography
% Protein recoverya Molecular weight
range (Da)
Fraction I 5773.7 10,000–1800
Fraction II 1772.4 1800–1400
Fraction III 2474.1 1400–500
Fractions IV, V and VI 270.5 o500
Data are given as percentage and represent the mean7standard
deviation of three experiments.
a
Protein content determined by elemental analyses.
respectively, in native casein. This was expected
considering the profile observed in the Biogel P2
chromatogram for this fraction, with a low absorbance
at 280 nm. In addition, the levels of branched chain
amino acids were above those observed in the
original casein, with a total of 39.8% in FIII as
compared to 16.9% in casein. As a result, the Fischer’s
ratio in FIII is 48.2 and Phe and Tyr represent 0.9% of
the total. Fractions FIV, FV and FVI contained small
peptides and free amino acids, mainly Arg, Phe, Tyr,
Lys and Trp, respectively. These amino acids are the
specific targets of the enzymes trypsin and chymotryp-
sin, and are released after the action of carboxypepti-
dase A.
4. Conclusions
Fraction FIII, which represents 24% of total proteins
hydrolyzed, had a high Fischer’s ratio and low content
in aromatic amino acids, as required for the treatment
of hepatic encefalopathies. Also, because of its low
content in Phe and Tyr, it could be useful for the
treatment of phenylketonuria and tyrosinemia. The
sequential hydrolytic procedure that has been described
here takes advantage of immobilizing highly specific
proteases. The substrate that has been used, casein,
has a high nutritional value and is readily available.
A very specific product has been obtained that can
be applied to the treatment of different metabolic
disorders.

Table 3
Amino acid composition of native casein and fractions purified by gel filtration (Biogel P-2) chromatography

Amino acids FI FII FIII FIV FV FVI Casein

Aspartic 8.374.3 6.272.0 3.271.4 0.370.2 0.0 0.0 6.070.6

Glutamic 18.478.1 15.271.9 11.171.5 2.272.4 0.570.0 0.0 18.571.7
Serine 8.874.2 8.070.8 5.571.4 3.970.3 0.670.2 0.970.2 6.070.4
Histidine 2.370.9 3.371.7 2.271.4 4.971.7 0.270.1 0.0 2.570.2
Glicine 2.170.9 2.870.8 2.070.8 1.770.7 0.470.1 1.270.3 2.570.1
Treonine 6.270.9 4.571.0 3.571.2 1.270.4 0.570.1 0.370.2 4.570.5
Arginine 3.371.0 1.970.6 5.172.8 15.476.2 7.571.8 7.172.4 3.470.2
Alanine 3.671.1 4.270.9 4.271.1 1.170.4 0.870.0 1.470.7 2.970.1
Proline 7.670.8 8.670.7 6.370.4 3.670.4 1.870.2 0.0 18.972.4
Tyrosine 2.670.7 4.570.9 0.670.3 0.670.4 66.474.7 2.170.3 3.971.1
Valine 8.171.9 8.370.4 11.171.3 3.572.1 0.570.2 1.471.0 4.771.4
Methionine 3.571.9 2.071.5 1.770.9 0.870.3 2.370.3 1.570.1 2.470.2
Cystine 0.370.2 0.970.3 0.370.1 1.970.7 1.570.1 0.770.1 0.570.1
Isoleucine 6.671.6 6.670.9 6.471.5 1.770.4 0.370.0 0.370.1 3.970.6
Leucine 8.371.9 11.1741.8 22.373.0 8.373.1 1.570.6 3.570.2 8.370.5
Phenylalanine 4.371.2 4.771.5 0.370.1 45.272.7 7.972.4 6.771.1 4.470.5
Lisine 5.770.7 7.073.1 14.470.8 2.871.1 3.170.3 1.270.2 6.870.7
Tryptophan 0.0 0.0 0.170.0 0.370.1 4.370.6 71.773.7 0.770.2
Fischer’s ratioa 3.3 2.8 48.2 0.3 0.03 0.6 2.0

Data are given as percentages and represent the mean7standard deviation of three samples.
a
Fischer’s ratio=(Val+Ile+Leu)/(Tyr+Phe).
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J. Pedroche et al. / International Dairy Journal 14 (2004) 527–533
Acknowledgements
We are indebted to J.M. Guisa! n and R. Ferna! ndez-
Lafuente for their help with enzyme immobilization.
This work was supported by grant AGL 2001-0526
(F.M.) and AGL 2002-02836 (J.G.) from CICYT, and
! y Cajal contract (J.G.).
a Ramon
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