Bioresource Technology 90 (2003) 249–254

Protein size distribution and inhibitory effect of wheat

hydrolysates on Neutraseâ
R. Kammoun *, S. Bejar, R. Ellouz
Laboratoire de Microbiologie Industrielle, Centre de Biotechnologie de Sfax, BP ‘‘K’’, 3038 Sfax, Tunisia
Received 15 April 2002; received in revised form 6 May 2003; accepted 8 May 2003

Abstract
Neutraseâ , used for hydrolysis of wheat proteins, was inhibited by end-product in a competitive uncompetitive way. The in-
hibition ratio depends on the progress of protein hydrolysis (degree of hydrolysis) and it remains constant beyond a degree of
hydrolysis of 7.5%. The inhibitor was separated, on Sephadex G-25 column, in three fractions (>2.4, 2.4–0.5 and <0.5 kDa) gen-
erating an inhibition ratio of 29%, 46% and 67% respectively. The peptides size distribution (<1, 1–2, 2–3 and >3 kDa) of fractions
was determined using size exclusion-high performance liquid chromatography. The analysis of obtained data, using a simple
mathematical regression, showed a correlation factor of 0.98 between the inhibition ratio and the peptides less than 1 kDa and 0.99
when considering the peptides lower than 1 kDa and higher than 3 kDa.
Ó 2003 Elsevier Ltd. All rights reserved.

Keywords: Inhibition; Neutraseâ ; Peptides; Protein hydrolysis; Peptide size

1. Introduction nursing infants or sick adults and as stimulants for

The increasing demand for wheat to produce flour,
glucose and fructose syrups, involves the simultaneous
creation of large quantities of non-starch by-products
rich in protein. Nowadays, the major market for these
protein rich by-products is animal feed. However, in-
creasing the value of these materials would be a pro-
mising way to satisfy the increasing needs of new
functional molecules essential in many applications
(Piot et al., 1988) especially in infant feeding (Mannheim
and Cheryan, 1990).
Usually protein hydrolysis studies concern the im-
provement of functional properties of the proteins, in
particular, their solubility (Adler-Nissen, 1982). Some
studies, dealing with the production of low molecular
peptides and in particular the fraction rich in di and tri-
peptides, have been reported. These peptides have the
advantage of being absorbed in the intestine without any
digestion in the stomach (Gonzalez-Tello et al., 1994b)
and have low allergenic effects. This explains their
preferential use in many formulas such as diets for
*
Corresponding author. Tel.: +216-74-27-4110; fax: +216-74-27-
5970.
E-mail address: radhouan.kammoun@cbs.rnrt.tn (R. Kammoun).
persons liable to develop allergy. According to various
authors (Gonzalez-Tello et al., 1994b; Loosen et al.,
1990; Samuelsson and Poulsen, 1987) peptide fractions
allowing a better absorption should contain a limited
range of molecular weights and a high di- and tri-pep-
tide content. In addition, to avoid allergenic effects, the
molecular weight of peptides should be below 2000 Da.
Another important factor to be taken into consider-
ation is the bitterness of peptides. As a matter of fact,
it was reported that peptides larger than 1000 Da,
derived from whey proteins, are bitter. Therefore, the
size of the much sought-after peptides for the above-
mentioned application would be preferably less than
1000 Da.
Enzymes used for protein hydrolysis can be inhibited
during reaction. Hence, several studies concerning,
mainly, inhibition of Alcalaseâ , trypsin, papain and
Neutraseâ , have been reported. Perea and Uglade (1996)
have indicated, in their study of the continuous hydro-
lysis of the whey in a membrane reactor, that Alcalaseâ
(Subtilisine A: a serine protease) is inhibited by small
peptides. However for the same substrate, the kinetic
study of the enzymatic hydrolysis reported by Gonzalez-
Tello et al. (1994a) shows the presence of an irreversible
serine-protease inhibitor. From bovine milk (from

0960-8524/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-8524(03)00130-5
250 R. Kammoun et al. / Bioresource Technology 90 (2003) 249–254
which the whey derives) Weber and Nielsen (1991) have
isolated a native serine-type protease inhibitor having a
molecular weight of 56–65 kDa. In the case of soybean
proteins, the presence of more than five trypsin inhibi-
tors was reported (Hsu et al., 1977). Product inhibition
of Alcalaseâ was also reported in the kinetic study of
enzymatic hydrolysis of corn gluten proteins (Hardwick
and Glatz, 1989). Ozbek and Lovitt (1999) have noticed
an inhibition of PROMOD 144 (a papain preparation)
by the hydrolysis product of wheat protein.
As far as we know, no study has been reported about
the nature of the inhibitor of Neutraseâ found in the
reaction mixture despite the extensive use of this enzyme
in protein hydrolysis.
In our laboratory, we developed a process for pro-
ducing glucose syrup from gruel (by-product of wheat
milling) via an amylolytic hydrolysis that leads to a by-
product rich in proteins (Bejar et al., 1992). In previous
work, we examined various enzymes in by-product
protein hydrolysis (Kammoun, 2000). This study en-
abled us to identify the effective enzymatic systems for
hydrolyzing protein matter and for building a kinetic
model for the control of hydrolysis (Kammoun et al.,
2001). More specifically, we demonstrated that the
Neutraseâ : EC 3.4.24.28, a commercial bacterial prote-
ase, is suitable to produce small peptides, although it is
inhibited by the hydrolysis products (Kammoun et al.,
1999). In the present study, the aims were to define the
effects and the nature of Neutraseâ inhibitory products
and constitutes a contribution to the understanding of
the inhibition process that limits the wheat protein hy-
drolysis.
2. Methods
2.1. Reagents and substrate
Reagents used in this study were analytic grade and
purchased from Sigma-Aldrich (Germany). The by-
product of amylolytic hydrolysis of gruel (a by-product
of wheat milling) was obtained from the ‘‘Centre de
Biotechnologie de Sfax’’ (CBS). The average content of
this product is 58% (based on dry matter) obtained by
using KjeldahlÕs method as described by AOAC (1975).
Its water content, as calculated by dehydration at 60 °C
for 24 h, is 13%.
2.2. Enzyme
The enzyme used was Neutraseâ 0.5 l (a liquid food
grade preparation from Novozymes A/S Denmark): an
endo-proteolytic enzyme of Bacillus subtilis. This en-
zyme is a metallo-protease preparation. Its density is
1.25 g/ml. It contains 0.5 Anson Unit/g (AU/g). 1 Anson
unit releases 0.001 A750 nm as TCA soluble hydrolysis
product per minute at 25 °C using denatured hemoglo-
bin as the substrate and Folin reagent. This enzyme was
used at its optimal pH and temperature (6.5 and 40 °C)
(Kammoun, 2000).
2.3. Hydrolysis
The substrate was hydrated in distilled water, heated
at 100 ° C for 10 min and subsequently cooled at room
temperature. The pH was adjusted to 6.5 with NaOH
(0.1 N) solution for subsequent hydrolysis. The experi-
ments were conducted in a 50 ml stirred batch reactor
(700 rpm) fitted with both temperature and pH controls.
The reaction was started by adding the enzyme. Protein
enzymatic hydrolysis was achieved by means of a pH-
stat technique (Adler-Nissen, 1982). The pH of the re-
action medium was maintained constant by the 718
STAT Titrino unit (Metrohm-Switzerland). Solutions of
0.5, 0.1 and 0.01 N of NaOH were used to adjust and
regulate the reaction medium. At the end of the hy-
drolysis, the amount of free amino-groups in the hy-
drolyzate was determined by the ninhydrin reagent
(Masson et al., 1986). Then, the hydrolysate was cen-
trifuged at 4500g for 15 min, and the supernatant was
separated by filtration through a 0.45 lm pore-size filter.
Finally, it was freeze-dried and the protein content was
determined by KjeldahlÕs method.
2.4. Determination of hydrolysis degree
The degree of hydrolysis (DH), defined as being the
ratio between the numbers of cleaved and total peptide
bonds, was routinely measured by the pH-stat method
(DH is proportional to the volume of base added to
maintain a pH constant) (Adler-Nissen, 1982). When we
hydrolyzed proteins at a pH near the pKa of a-amino-
groups, the DH determined by the above-mentioned
method was not accurate. By measuring the DH using a
relationship based on the amount of free amino-groups
(ninhydrin reagent) and the volume of base consump-
tion (pH-stat method) reported to protein mass in per-
cent (B
N =mp
100) for Neutraseâ 0.5 l at a pH 6.5
and 40 °C (Fig. 1), we established the following corre-
lation:
B
N
DHð%Þ ¼ 0:179

100 ð1Þ
mp
where B (liter) represents the volume of base needed to
keep the pH constant; mp (kg) is the protein mass, N
(mol/l) is the normality of the base and 0.179 (kg/mol) is
the coefficient of the correlation.
2.5. Gel filtration chromatography
The molecular weight distribution of samples was
investigated by gel filtration on Sephadex G-25 (Sigma,
 R. Kammoun et al. / Bioresource Technology 90 (2003) 249–254
Fig. 1. Relationship between the DH (%) using Neutraseâ 0.5 l (40 °C,
pH 6.5), determined by ninhydrin method and the base consumption
per the protein quantity expressed as B
N =mp
100 and measured
by pH-stat.
France) using a 75
2.0 cm column and insulin (5808
Da), vitamin B12 (1350 Da), Thiamine (377 Da) and
Gly-Tyr (238 Da) as standards. The injection quantity
was 100 mg of dried product and the elution buffer was
0.02 M sodium phosphate (pH 7.2) (Amiot et al., 1981).
This elution was carried out at a rate of 7 ml/h using a
UV detector set at 280 nm. The recovery volume was 2
ml/tube. The chromatogram obtained for each protein
hydrolysate was divided in three parts of known mole-
cular weight intervals. The corresponding fractions of
these parts were pooled and the area under each frac-
tion, proportional to the amounts of products released
by the protein hydrolysis, was determined. The pooled
fractions were concentrated by evaporation under vac-
uum followed by freeze-drying.
2.6. SE-HPLC
The range of molecular weights in samples was de-
termined by size exclusion-high performance liquid
chromatography (SE-HPLC) using the KW 802.5 Sho-
dex column (30 cm
8 mm internal diameter) purchased
from Waters (Saint-Quentin, France). The solvent was a
phosphate buffer (0.05 M) containing 0.02 M of Na2 SO4
(pH 7.5). Elution was carried out at flux of 0.5 ml/min
using a UV detector at 280 nm. The calibration of the
Shodex KW 802.5 column was achieved by injection of
10 ll volume containing 360 lg of the standards: Thy-
roglobulin 670 kDa, Gamma globulin 158 kDa, Oval-
bumin 44 kDa, Myoglobin 17 kDa, Insulin 5 808 Da,
Vitamin B12 1 350 Da, Lys-Try-Lys 437.5 Da, Penicillin
G 356 Da, Gly-Try 238 Da, L-carnosine 226 Da and
Cresol 108 Da.
2.7. Identification of the enzyme inhibition type
Lineweaver–Burk representation was used to identify
the type of inhibition (Palmer, 1984). This representa-
251
tion consisted of plotting the inverse of the initial speed
(1=V0 ) versus the inverse of the initial substrate con-
centration (1=S0 ) in the absence or in the presence of the
inhibitor (using the same quantity of the inhibitor). For
the establishment of these curves, we followed the ki-
netic of base volume added to maintain a pH constant.
This kinetic curve allows the determination of the tan-
gent at the origin. At a given normality of the base used
to control the pH, the slope value of the tangent is
proportional to the initial speed of hydrolysis. For these
experiments, we have used an end-hydrolysis product
issued from a hydrolysis having 9.3% as hydrolysis de-
gree (the hydrolysis time is about 10 h).
2.8. Determination of inhibition ratio
The inhibition ratio of different products has been
evaluated by measuring the hydrolysis degree after 10
min by means of the following equation:
DH10  DHI10
Inhibition ratio10 min ð%Þ ¼ ð2Þ
DH10
Inhibition ratio10 min : percent of inhibition after 10 min
of hydrolysis, DH10 , DHI10 : degree of hydrolysis mea-
sured after 10 min in absence and presence of the in-
hibitor respectively.
2.9. Statistical analysis
All experiments were replicated three times. Inhibi-
tion ratio10 min was regressed on percentage of fraction
using a linear regression model. This was achieved by
performing a hypothesis test of Person correlation co-
1=2 1=2
efficient (r) based on the fact that rðn  2Þ =ð1  r2 Þ
has a Student distribution with n  2 degrees of freedom
(n: number of points). This yields a critical value for r at
the 5% significance level of rC ¼ 0:95. All analysis were
performed using EXCEL Software (Office 2000 edition)
for WINDOWS.
3. Results and discussion
3.1. Effect of hydrolysis degree on inhibition
Several end-products having different hydrolysis de-
grees were prepared. Each end-product was used to test
its inhibitory effect on Neutraseâ 0.5 l. The inhibition (%
of inhibition after 10 min and noticed as inhibition
ratio10 min ) versus degree of hydrolysis showed two kinetic
zones: a DH-dependent zone, where a large increase in
inhibition ratio10 min was observed at DH lower than
7.5%, and a DH-independent zone, where DH level had
little or no effect on inhibition ratio10 min , was observed
with a DH greater than 7.5% (Fig. 2).
252 R. Kammoun et al. / Bioresource Technology 90 (2003) 249–254
Fig. 2. Hydrolysis degree effect (DH) on Neutraseâ 0.5 l inhibition.
The inhibition was calculated after 10 min in presence of the same
quantity of inhibitor. Reaction conditions: 40 °C; pH 6.5; enzyme 0.07
g/g wheat protein, inhibitor 0.33 g/g wheat protein; protein concen-
tration in the reactor 19.6 g/l and time range between 2 and 14 h.
Consequently, the inhibition ratio depended on the
degree of hydrolysis that reached a maximum value and
remained constant beyond 7.5% independent of the
hydrolysis degree. This behavior was probably linked to
the size of peptides contained in each product.
3.2. Identification of inhibition type
For this study, a high DH end-product (DH > 7.5%)
was used as inhibitor. The representation of the inverse
of the initial speed of hydrolysis (1=V0 ) versus the inverse
of the initial substrate concentration (1=S0 ) in the pres-
ence and the absence of the inhibitor, according to Li-
neweaver–Burk, has been applied to identify the type of
Neutraseâ inhibition (Fig. 3). We have tested two dif-
ferent inhibitor concentrations (4 and 5.8 g/l, which
correspond to 0.2 and 0.29 g of inhibitor, respectively).
Results showed that the Lineweaver–Burk plots, in
the presence and in the absence of inhibitor, were linear.
12000
10000
1/Vo (min.ml/mmol)
8000
6000 -0.7 -0.5 -0.3
4000
2000
0 product and its different fractions was determined while
0 0.1 0.2 0.3
1/So (ml/g)
Fig. 3. Lineweaver–Burk plot of Neutraseâ 0.5 l inhibition type. Data
are presented as inverse of the initial hydrolysis speed (1=V0 ) in absence
(O) and presence of 0.2 g (M), 0.29 g (
) of inhibitor versus the inverse
of the substrate concentration (1=S0 ). Reaction conditions: 40 °C; pH
6.5; enzyme 0.88 AU/l; protein concentration in the reactor ranges
between 5 and 65 g/l; time 10 min and volume mixture 0.03 l.
Table 1
Inhibition ratio of the Neutraseâ 0.5 l caused by the high DH end-
product and their derivative inhibitory fractions
Size (kDa) Inhibition ratio10 min (%)a
Entire inhibitor 45.5 ± 1.1
Fraction I >2.4 29.2 ± 0.7
Fraction II 2.4–0.5 46.2 ± 1.2
Fraction III <0.5 67.7 ± 1.7
The highest inhibition ratio is in bold.
a
Means values and standard deviations for three experiments.
However, the slopes of the three curves were very dif-
ferent indicating a substantial inhibition of Neutraseâ
0.5 l by the high DH end-product. In fact, the slopes of
the three curves were respectively, 2995, 8919 and
12 460. The three curves, when continued until the axis,
intersected at a point near the 1=V0 -axis indicating that
the inhibition was of a competitive–uncompetitive type.
3.3. Effect of peptide fractions on the inhibition ratio
The observed differences in the inhibition ratio could
be related to the size and the amount of the peptideÕs
fractions responsible for the most substantial inhibition
of Neutraseâ 0.5 l. A high DH end-product was first
separated into three fractions, noted as I, II and III
(<0.5, 0.5–2.4 and >2.4 kDa), by gel filtration on
Sephadex G-25 gel. Each fraction was then tested on
Neutraseâ 0.5 l inhibition during the hydrolysis. Data
showed that fractions containing small peptides are
more inhibitory than those containing medium or higher
ones. Therefore, the more the degree of hydrolysis in-
creases, generating a higher amount of low molecular
weight peptides, the more important the inhibition ratio
is (Table 1).
3.4. SE-HPLC analysis
For a better characterization of molecular weight of
10000
the different peptide fractions, size exclusion-high per-
8000
formance liquid chromatography (SE-HPLC) was used.
6000
4000 The SE-HPLC patterns obtained for the high DH end-
2000
product of hydrolysis and the derived fractions I, II and
0
-0.1
-2000
0.1 0.3
III are presented in Fig. 4(a)–(d) respectively. These
figures reveal that the peptide size distribution of the
high DH end-product was different from those of the
derivative fractions. To give a more precise interpreta-
tion, the peptide size distribution of the high DH end-
0.4
using four range fractions: 0–1 kDa (fraction 1), 1–2
kDa (fraction 2), 2–3 kDa (fraction 3) and >3 kDa
(fraction 4) (Table 2).
In this study, it is apparent that fraction I is mainly
composed of fraction 3 (2–3 kDa) whereas the entire
high DH end-product is mostly composed of fraction 1
(0–1 kDa). The peptides size distribution of fraction II is
 R. Kammoun et al. / Bioresource Technology 90 (2003) 249–254 253

Fig. 4. Size exclusion-high performance liquid chromatography elution profiles of the entire (a) and the derivative fraction of Neutraseâ 0.5 l in-
hibitor eluted from G-25 gel filtration column: fraction I (b), fraction II (c) and fraction III (d).

Table 2
Peptide percentage, inhibition ratio and molecular weight distribution of entire end-product and different inhibitor fractions
Fraction Weight ratio (%) Inhibition Chromatographic area of fractions in %a
ratio10 min (%)
Fraction 1 Fraction 2 Fraction 3 Fraction 4
(0–1 kDa) (1–2 kDa) (2–3 kDa) (>3 kDa)
Y X
Entire 100 45.5 ± 1.25 53.2 ± 8.2 25.4 ± 1.4 11.8 ± 0.6 9.4 ± 0.2
I 33 29.2 ± 0.7 11.9 ± 1.8 16.2 ± 0.9 42 ± 2.3 29.9 ± 0.7
II 30 46.2 ± 1.25 57.7 ± 8.9 39 ± 2.1 ND 2.6 ± 0.1
III 37 67.7 ± 1.7 88.7 ± 13.7 3.5 ± 0.2 2.7 ± 0.2 5.1 ± 0.1
The most important peptides percentages are in bold.
a
Means values and standard errors for three chromatograms.

composed mainly of fraction 1 and 2 (0–2 kDa) while Table 3

that of fraction III is largely made up of fraction 1. Correlations between Inhibition ratio10 min (%) and the percentage of
peptides matter contained in one or more fractions of the Neutraseâ
0.5 l inhibitors (the entire product and the derivatives fractions)
Chromatogram Sign of slope Correlation
3.5. Statistical treatments of results
fraction coefficient (r)
1 + 0.98a
The effect of inhibitor is directly related to peptide
2 ) 0.41
distribution and therefore linked indirectly to peptide 3 ) 0.92
fractions. The correlations between the Inhibition 4 ) 0.76
ratio10 min and the peptide percentages of each fraction
1+2 + 0.78
(Y versus X in Table 2), according to the least square 1+3 + 0.93
method (Carnahan et al., 1990), were calculated (Table 1+4 + 0.99a
3). A significant correlation (P < 0:05), with a standard 3+2 ) 0.99a
deviation r ¼ 0:98, was obtained between Inhibition 3+4 ) 0.78
ratio10 min and the percentage of peptides contained in Combinations were made between fractions that give a correlation
fraction 1 of all fractions. It is noted that fraction 1 coefficient higher than 0.9.
a
Correlations are significant at P < 0:05.
contained peptides of up to eight amino acid residues. A
positive correlation was also obtained with the peptide
percentages of fraction 3. The correlation was lower again significant (P < 0:05) and greater than 0.9 with
with fraction 4 (r ¼ 0:76) and became insignificant fractions 1 + 4 or 3 + 2. The higher the percentage of
(P > 0:05) with fraction 2ðr ¼ 0:41Þ. In the Table 3, we peptides of fractions 1 and 4 is, the more significant the
also combined some fractions that gave correlation co- Inhibition ratio10 min is (slope+). Inversely, the lower the
efficients higher than 0.9 when compared with all other percentage of peptides of fractions 3 and 2 is, the more
fractions. In that case, the standard deviations were significant the Inhibition ratio10 min is (slope)).
254 R. Kammoun et al. / Bioresource Technology 90 (2003) 249–254
From Table 3, we established that even if the low
molecular weight peptides exhibited the primary inhib-
itory effect, the high molecular weight fractions still
exhibit a secondary role on the inhibition process.
4. Conclusions
The study of Neutraseâ inhibition by end-product
displayed high sensitivity towards hydrolysis degree for
values less than 7.5%. SE-HPLC analysis showed that
molar weight distribution of fractions has an important
effect on the inhibition process. The entire high DH
hydrolysis end-product and fraction II are mostly
composed of relatively medium molecular weight pep-
tides (<2 kDa) while fractions III and I are essentially
formed of peptides less than 1 kDa and >2 kDa, re-
spectively.
All peptides of size less than 3 kDa contribute to the
inhibition process but those of less than 1 kDa have the
main role. Inhibition is limited when the reaction me-
dium contains more peptides of medium molecular
weight (1–2 kDa). This study represents an attempt at
understanding Neutraseâ inhibition that will allow us to
resolve this problem. Consequently, we suggest using a
two-step process. In the first step, a less specific hydro-
lytic enzyme (such as Alcalaseâ ) could be used in order
to produce a fraction composed of peptides of less than
3 kDa. In the second step, this fraction could be hy-
drolyzed by Neutraseâ using a continuous membrane
reactor to remove peptides of molecular weight less than
1 kDa.
Acknowledgements
This work was supported by funds from the Tunisian
Government (‘‘Contrat-Programme CBS-LMI’’). We
thank Mr. Hedi Aouissaoui and Miss Najla Masmoudi
for their technical assistance with chromatographic
analysis. Thanks are also due to Mrs. Hamdi Guebsi
and Moncef Affes for critically reading the manuscript.
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