INACTIVATION OF TRYPSIN AND CHYMOTRYPSIN INHIBITORS ...

Res. Bult., No. (130), Food Sci. & Agric. Res. Center, King Saud Univ., pp. (5-17) 2004
INACTIVATION OF TRYPSIN AND
CHYMOTRYPSIN INHIBITORS IN FENUGREEK
(Trigonella foenumgraecum L.) DEFATTED
SEED FLOUR
Salah A. Al-Maiman
ABSTRACT
Trigonella foenumgraecum L. defatted flour was assayed for
trypsin and α-chymotrypsin inhibitor activites. The trypsin and αchymotrypsin
inhibitors activities of fenugreek were 45.84 and 7.16
unit/mg protein, respectively. Heating the extract in autoclave (120 C)
for 80 min destroyed 53% of the trypsin inhibitor activity, whereas
only 20 % of the activity of this inhibitor destroyed by microwave
conventional heating for 8 min.
α-Chymotrypsin inhibitor activity in fenugreek defatted flours
was much more heat labile compared to trypsin inhibitors activity.
Autoclave (for 100 min) and microwave (8 min) heating inactivated
30% and 100% of α-chymotrypsin inhibitors activity, respectively.
Key words: Trypsin, chymotrypsin, fenugreek, inhibitors, autoclave,
microwave.
_____________________________________________________________
Dept. of Food Science and Nutrition, College of Food Sciences and Agriculture,
King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia
Email: smaiman@ksu.edu.sa
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INTRODUCTION
Fenugreek (Trigonella foenumgraecum), one of many
condiments known to mankind, has been cultivated for a very long
time. It has been popularly used in western Asia, northern India,
Africa and Mediterranean region as a food and medicine. The foliage
of fenugreek is commonly eaten as leafy vegetable. Its powder is
considered a good antiseptic and employed for improving skin texture.
Seeds of fenugreek are being consumed to facilitate lactation also.
The nutritional importance of food legumes as an economic
source of proteins has been recognized throughout the world, It
contains 26.2% protein, 5.8% fat and 44.1 % carbohydrate. (Patil et al.
1997.; Borget, 1992 and Geil and Anderson,1994 ). Consuming grain
legumes is more economically directed instead of converting them
into animal proteins. Legumes have been considered as leading
candidates in protein supply to the malnourished areas of the world
(Gent, 1994, Muehlbauer and Kaiser, 1994 and Oram and Agcaoili,
1994). Certain antinutritional factors, which negatively affected
protein digestibility have been reported as proteinase inhibitors
especially trypsin and α-chymotrypsin inhibitors (El-Mahdyl,1981 and
Ismail et al.1995) . These inhibitors stimulate pancreatic juice
secretion and cause pancreatic hypertrophy and growth inhibition
(Liener and Kakade 1980).
Legume proteins are difficult to digest because of the presence
of trypsin inhibitors (Mossor et al.1984). These naturally occurring
proteins have the property of combining with and inactivating trypsin
(Wilson, 1981).
Removal of the undesirable components from the dry legume
seeds is essential for improving their nutritional qualities. To achieve
this, several processing methods such as germination, soaking and
cooking have been used (Ismail et al.1995 and Barampama and Simart
1994). Furthermore, non conventional method such as the use of
microwave heating (Sakla et al., 1988; Snyder et al.1991; and Sakac et
al. 1996) and Irradiation (Ghazy et al. 1992 and Abu-Tarboush 1998)
have been investigated. Rajko et al. (1997) investigated the role of
microwave energy in reducing enzyme activity in whole soybeans.
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He indicated that, the characteristic feature of microwave heat is that
it ensures homogeneous operation in whole volumes of substance,
great penetrating depth, and selective absorption. The objective of this
study was to inactivate of trypsin and α-chymotrypsin inhibitors in
fenugreek seeds by autoclave and conventional microwave heating.
Material
MATERIALS AND METHODS
Fenugreek seeds were brought from the local market in
Algasseem area, 330 Km North West of Riyadh, Saudi Arabia. Seeds
were milled to pass through a 0.355 mm sieve, using an ultracentrifugal
mill (Resh type 2MI, F., Kurt Retsh GmbH and Co.,
Germany).
Sample Defatting
El-Tinay et al., (1988) method was used for the defatting of
fenugreek seeds. Sample was mixed with n-hexane (1:10 w/v) and
stirred for 2 hrs at room temperature, and then the mixture was left
overnight. In the next day, the mixture was filtered and the residue
was collected and dried for 12 hrs at room temperature. The process
was repeated two more times to extract the complete oil from the
sample. After milling and sieving (60 mesh), the defatted fenugreek
flour was kept in the refrigerator for further analysis.
Treatments
Samples were placed in autoclavable Petri dishes in one layer
then autoclaved (autoclave model No. TV30UL, Memmert, Germany)
and microwaved (microwave Model No. R-9H56 with an output
power of 850 W and a frequency of 2450 MHz equipped with
temperature sensor, Sharp Corp., Japan) at 120°C and 130°C,
respectively for different times ( 0, 20, 40, 80 and 100 min. for the
autoclave treatment and 0, 2, 4, 8 and 10 min. for the microwave
treatments).
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Trypsin Inhibitor Activity Assay of Fenugreek
Trypsin inhibitor activity determination of autoclaved and
microwaved fenugreek of defatted flour was conducted according to
the method of Kakade et al. (1969) using Nα-benzoyl-DL-arginine pnitroanilide
(Sigma Chemical Co., St. Louis, MO) at a concentration
of 30 mg/100 mL as a synthetic substrate. One gram of the defatted
flour was extracted with 15 mL of citrate buffer (pH 4.6), stirred for 2
h at room temperature (25 °C), and then centrifuged at 4500 rpm for
20 min. The reaction mixture consisted of 0.1 mL of the extract, 0.9
mL of distilled water, 7 mL of the synthetic substrate, and 1 mL of
trypsin type III (Sigma). Trypsin type III from bovine pancreas at
concentration of 4 mg/100 mL of 0.001 M HCI was used in the
reaction mixture. The absorbance was recorded at 410 nm, and the
inhibitor activity was calculated using a control sample of trypsin.
α-Α-chymotrypsin Inhibitor Activity Assay of Fenugreek
The method of Kakade et al. (1970) was employed for
determining α-chymotrypsin inhibitor activity using bovine pancreas,
type II α-chymotrypsin (Sigma), and 1% casein (BDH Chemicals,
Poole, U.K.) as substrate. One α-chymotrypsin unit (CU) was
arbitrarily defined as an increase of 0.01 absorbance unit at conditions
described in this method, and the α-chymotrypsin Inhibitor activity
was defined as the number of α-chymotrypsin units inhibited (CUI).
Statistical Analysis
Data from three replications for each time treatment
combinations were analyzed using analysis of variance and Fisher
LSD of the SAS program (Statistical Analysis Systems Institutes,
1995).
RESULTS AND DISCUSSION
Trypsin inhibitor activity in fenugreek defatted seed flour was
45.84 units/mg protein (table 1). This value is considered to be high if
compared to 26 unit/mg protein of defatted soy protein (Al-Kahtani
1995). Also it is higher than karkade defatted seed flour which was 41
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Table 1. Effect 1 of autoclave (120°C) and microwave(130°C)
heating on trypsin and α-chymotrypsin inhibitors of
fenugreek (Trigonella foenumgraecum L.) defatted seed
flour.
Autoclave
(120°)
Treatment
(min)
Microwave
(130°)
0 0
20 2
40 4
60 6
80 8
100 10
Trypsin Inhibitor
( unit /mg protein) 2
Autoclave
45.84 a
± 0.44
35.46 b
± 0.35
33.56 b
±1.49
30.40 c
±0.27
23.92 d
±2.73
22.28 d
±0.93
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Microwave
45.84 a
±0.44
45.71 a
±0.29
45.10 a
±0.70
38.47 b
±1.44
37.27 b
±0.65
37.02 b
±1.11
1 Means ± SD (n=3) means in column with unlike superscripts differ significantly (P≤ 0.05).
α-Chymotrypsin
inhbitor
( unit / mg protein)
Autoclave
7.16 a
±0.35
3.24 b
±0.16
2.77b c
±0.22
2.60 c
±0.08
2.47 cd
±0.05
2.03 d
±0.12
Microwave
7.16 a
±.0.35
2.16 b
±0.20
1.18 c
±0.04
0.98 c
±0.15
0 d
±0.00
0 d
±0.00
2 One unit of trypsin and α-chymotrypsin inhibitory was defined as that which reduces the activity of trypsin and α-chymotrypsin
by one unit.

unit/mg protein (Abu Traboush and Ahmad 1996). On the other
hand, the trypsin inhibitor value obtained in this study was lower than
that of the different defatted soy protein products which ranged from
60.8 to 107.5 unit/mg (Kakade et al. 1969). These differences in the
activity of trypsin inhibitor in the different studies of defatted soy
protein flour might be due to different chemical used like buffer that
have been used for extraction, substrate, type of seeds as well as
environmental factors (Ikeda and Kussano, 1983). El-Morsi et al.
(1991) were able to decrease trypsin inhibitor in fenugreek seeds by
up to 84% using germination for 120 huors. Al-Housein and Abu-
Tarboush (1997) reported that using BAPA as a substrate could
increase the trypsin inhibitor activity values by 25-30% compared to
using casein as a substrate. Inactivation of trypsin inhibitor activity
has been attributed to the destruction of disulfides amino group which
could have been affected by radiation (Lee, 1962 ).
El Morsi, 1982 has reported the inactivation of trypsin
inhibitors which could also be due to the destruction of lysine.
Fenugreek defatted seed flour contained around 7.16 unit/ mg protein
α-chymotrypsin inhibitor activity. This value is lower than that
reported for defatted soy protein flour (57.20 unit/mg protein) and
karkade defatted seed flour (21.8 unit/mg protein). However, it was
was higher than that reported by Al-Housein and Abu-Tarboush
(1997) for Al-Ban (Al-Yassar) seed protein.
Table 1 and figures 1 and 2 show the effect of autoclave and
microwave heating on fenugreek defatted flour. Activity of trypsin
inhibitor decreased as the heat-time for autoclave increased. Although
autoclave heating for 100 min caused more destruction of trypsin
inhibitor (55%) compared to 80 min heating by autoclave (53%), the
difference in destruction was not significant (table 1 and Figure 1).
The extracts of defatted flour, protein concentrate, and protein isolate
of soybean (Moringa peregrina), when boiled for 5 hrs at 95°C
retained 18.5% and 9.3% of the original trypsin inhibitor activities,
respectively (Al-Kahtani,1995). Koeppe et al. (1985) found that 20
% of the original amaranth trypsin inhibitor activity remained at the
end of 7 hrs heating at 100° C. It seems that trypsin inhibitor of fenugreek
is more resistant to heat than the trypsin inhibitor in Moringa
peregrine and amaranth.
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Activity (%)
100
80
60
40
20
0
1 2 3 4 5 6
Treatment
Figure-1: Trypsin inhibitor activity (%) after treatment with
microwave (130° C) and autoclave(120 °C) heating with
treatment 1= no heating; 2= 20 min autoclave and 2 min
microwave;3=40 min autoclave and 4 min microwave;
4=60 min autoclave and 6 min microwave; 5=80 min
autoclave and 8 min microwave and 6=100 min autoclave
and 10 minute microwave.
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Autoclave
Microwave

Activity (%)
100
80
60
40
20
0
1 2 3 4 5 6
Treatment
Figure-2. α-Chymotrypsin inhibitor activity (%) after treatment with
microwave (130° C) and autoclave(120 °C) heating with
treatment 1= no heating; 2= 20 min autoclave and 2 min
microwave;3=40 min autoclave and 4 min microwave;
4=60 min autoclave and 6 min microwave; 5=80 min
autoclave and 8 min microwave and 6=100 min autoclave
and 10 minute microwave.
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Autoclave
microwave

Microwave conventional heating had less effect on trypsin
inhibitor of fenugreek compared to autoclave heating (Table 1).
Destruction of trypsin inhibitor of fenugreek defatted flour by
microwave increased by time; however significant effect was noticed
only after 6 min of heating. At this time, heating time only only 20 %
of the trypsin inhibitor was destroyed (Figure 1).
α-Chymotrypsin inhibitor activity of fenugreek defatted flour
was less (7.16 units/ mg protein) than that of the trypsin inhibitor
activity (Table 1). This results agreed with the finding of Singh and
Jambunathan (1981) reported considerable differences in the levels of
trypsin and α-chymotrypsin inhibitors between two chickpea cultivars.
Abu-Tarboush and Ahmed (1996) reported also higher trypsin
inhibitor activities in karkade and soybean defatted flour than their αchymotrypsin
inhibitor.
Autoclave treatment reduced α-chymotrypsin inhibitor activity
in fenugreek defatted flour and destruction increased significantly
with the increase of heating time. However, no significant differences
were noticed among the heating times of 40, 60 and 80 min (Table 1).
The α-chymotrypsin inhibitor activity retained about 30 % of its
original activity after heating for 100 min (Figure 2). The gradual loss
in inhibitory activity of α-chymotrypsin was achieved by microwave
conventional heating with time (Table 1).
α-Chymotrypsin inhibitor activity inactivated completely by heating
with microwave after 8 min (Table 1 and Figure 2). The αchymotrypsin
inhibitor of fenugreek was more heat labile than trypsin
inhibitor and microwave conventional heating was more effective than
autoclave in inactivating the α-chymotrypsin inhibitor. Rajko et al.
(1997) mentioned that the use of microwave energy is more efficient
than the traditional heating treatment. They also added that the
characteristics feature of microwave heat is that it ensures
homogeneous operation in whole volume of substance, great
penetrating depth, and selective absorption.
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