production and characterization of casein hydrolysates with a high

8/6/2019 Production and Characterization of Casein Hydrolysates With a High

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International Dairy Journal 14 (2004) 527533

Production and characterization of casein hydrolysates with a high

amino acid Fischers ratio using immobilized proteases

Justo Pedroche, Mar!a M. Yust, Hassane Lqari, Julio Gir !on-Calle, Javier Vioque,Manuel Alaiz, Francisco Mill !an*

Instituto de la Grasa, Consejo Superior de Investigaciones Cient!ficas, 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 Fischers 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 Fischers 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; Fischers ratio

1. Introduction

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, & Mill!an, 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

absorption of hydrolysates can be more effective thanthe 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 Fischers 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, & H .ortnalg, 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

Fischers 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,

ARTICLE IN PRESS

*Corresponding author. Tel.: +34-5-4611550; fax: +34-5-4616790.

E-mail address: [email protected] (F. Mill!an).

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

doi:10.1016/j.idairyj.2003.11.002


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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 prolongedphysical 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 Fischers 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 Fischers ratio as well. Thus, a hydrolysate with a

Fischers 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 easilyrecovered 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 Fischers 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

1.7m 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 2m 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

300mmoles NaIO4 mL1 gel to achieve multipoint

attachment. This oxidative reaction was allowed to

proceed for 23 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 Guis!an (1989), with some

modifications. Volumes of 10 mL activated agarose gel

containing 30 mg enzyme mL1 gel (except for carbox-

ypeptidase A: 2 mg enzyme mL1 gel) were dispersed in

100 mL 0.2m 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

(20mgmL1) was assayed by following the increase of

absorbance at 405 nm which accompanies hydrolysis of

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J. Pedroche et al. / International Dairy Journal 14 (2004) 527533528


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the synthetic substrate BANA (75mL soluble or

suspended enzyme were added to 2.5 mL 50 mm sodium

phosphate 40% ethanol, pH 7, containing 200 mL

BTNA 1mm in 50 mm sodium phosphate 40% ethanol

pH 7 at room temperature).

2.4.2. a-chymotrypsinThe activity of the soluble or immobilized enzyme

(20mgmL1) was assayed by following the increase of

absorbance at 415 nm which accompanies hydrolysis of

the synthetic substrate BTNA (75mL soluble or im-

mobilized enzyme were added to 2.5 mL 50 mm sodium

phosphate 40% ethanol, pH 7, containing 25mL 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

(2mgmL1) was assayed following the increase of

absorbance at 254 nm which accompanies hydrolysis ofthe 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 120C for 24h.

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 0.1m

sodium borate 0.2m 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

1.6mgmL1 were injected using a 200 mL loop. Borate

buffer at a flow rate of 0.4mL min1 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 (1.45kDa, Sigma Chemical Co.) were used

as molecular weight standards.

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 50C. 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 Fischers 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

(255cm2) at a flow rate of 10mLh1 using 50mm

ammonium bicarbonate as eluent. Fractions were

collected every 10 min. Cytochrome C (12,384 Da),

bacitracin (1400 Da), Val4-angiotensin (917 Da), Arg

LysGluValTyr (693 Da) and TrpGly (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 6n

HCl (4 mL) and incubation at 110C 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

ofSch.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

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J. Pedroche et al. / International Dairy Journal 14 (2004) 527533 529


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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, TrpXaa, PheXaa, and LeuXaa. 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 Fischers ratios below

8, not reaching the suggested optimum Fischers 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 chymotrypsinactivity 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 LysXaa 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 theavailable 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 chymotrypsinproduced 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.

ARTICLE IN PRESS

Table 1

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

Enzyme treatment Theoretical maximum

DHa(%)

Observed DHb(%) Observed DH/theoretical

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.aMaximum number of hydrolyzable amino acid bonds deduced from the amino acid composition of casein.bDetermined by reaction with TNBS.cCalculated by substraction (digestion with trypsin and chymotrypsin minus digestion with trypsin).



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The final digestion with carboxypeptidase A released

a variety of free amino acids, depending on the duration

of the treatment. We previously reported (Pedroche

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

30 min to 2 h releases those amino acids for which the

enzyme exhibits the highest specificity, normally tyr-

osine, phenylalanine and tryptophan. If the hydrolysis is

allowed to proceed for a longer time, other amino acids

are released. Thus, after 8 h, valine, isoleucine, threo-

nine, alanine, and lysine are released, and after 24 h ofhydrolysis, the amino acids serine, glycine and histidine

are released as well. Considering these results, the

treatment with carboxypeptidase for 2 h, which results

in a DH of 27.2%, is most likely causing release of

AAA, which would be exposed in the carboxyterminal

position due to the previous action of chymotrypsin.

The final hydrolysate obtained by hydrolysis with

trypsin, chymotrypsin and carboxypeptidase A should

be rich in di- and tripeptides. The FPLC gel filtration

profile of this hydrolysate is shown in Fig. 1d, which

also illustrates the release of aromatic residues such as

Tyr, Phe and Trp. SDS-PAGE analysis provided a

pattern similar to that obtained using trypsin and

chymotrypsin, with a smear of peptides with molecular

weights ranging from 6 to 14 kDa. In fact, the FPLC

chromatography profiles shown in Fig. 1c, and d were

similar with the exception of the peaks corresponding to

Tyr, Phe and Trp in Fig. 1d, which attenuates the signal

of the remaining peptides.

3.2. Purification of peptides with high Fischers ratio

For the isolation of these peptides, the final casein

hydrolysate obtained after sequential treatment with

trypsin, chymotrypsin and carboxypeptidase A wasloaded on a Biogel P2 gel filtration column (Fig. 3).

The eluate was divided into six fractions (FI, FII,

FIII, FIV, FV and FVI). The protein contents and

ARTICLE IN PRESS

67.0

43.0

30.0

20.1

14.4

kDa 1 2 3 4

6.2

17.0

14.4

8.110.7

5 6 kDa

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

hydrolysates obtained by treatment with trypsin (3), trypsin and

chymotrypsin (4), and trypsin, chymotrypsin, and carboxypeptidase A

(5). Low molecular weight marker kit (1), Peptide marker kit (6).

0

0.020.04

0.06

0.08

0.1

0.12

0 5 10 15 20 25 30 35

0

0.02

0.04

0.06

0.08

0.1

0.12

0 5 10 15 20 25 30 35

0

0.05

0.1

0.15

0.2

0 5 10 15 20 25 30 35

0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20 25 30 35

Absorbance(280nm)

Volume elution (mL)

Tyr

Phe

Trp

(a)

(b)

(c)

(d)

Fig. 1. FPLC gel filtration chromatography of native casein (a), and

casein hydrolysates obtained by treatment with trypsin (b), trypsin and

chymotrypsin (c) and trypsin, chymotrypsin, and carboxypeptidase A

(d). Tyr, tyrosine; Phe, phenylalanine; Trp, tryptophan.

F V F VIF IVF IIIF IIF I

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Elution volume (mL)

Absorbance(280nm)

0 10 20 30 40 50 60 70 80 90

Fig. 3. Biogel P2 gel filtration chromatography of the final hydrolysate

obtained by sequential treatment of casein with trypsin, chymotrypsin

and carboxypeptidase A.



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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 between1400 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 Fischers 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%,

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 Fischersratio 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 Fischers 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.

ARTICLE IN PRESS

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 10,0001800

Fraction II 1772.4 18001400

Fraction III 2474.1 1400500

Fractions IV, V and VI 270.5 o500

Data are given as percentage and represent the mean7standard

deviation of three experiments.aProtein content determined by elemental analyses.

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 2.970.1Proline 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

Fischers 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.aFischers ratio=(Val+Ile+Leu)/(Tyr+Phe).



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Acknowledgements

We are indebted to J.M. Guis !an and R. Fern !andez-

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

a Ram!

on y Cajal contract (J.G.).

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Alaiz, M., Navarro, J., Giron, J., & Vioque, E. (1992). Amino acid

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