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204 Acta Chim. Slov. 2024, 71, 204–214
Isleroglu and Olgun: Eects of the Extraction Conditions on Functional ...
DOI: 10.17344/acsi.2023.8576
Scientic paper
Eects of the Extraction Conditions on Functional
and Structural Characteristics of Proteins from
Fenugreek Seeds
Hilal Isleroglu* and Gamze Nur Olgun
Tokat Gaziosmanpasa University, Faculty of Engineering and Architecture, Food Engineering Dept., 60150, Tokat, Turkey
* Corresponding author: E-mail: hilal.isleroglu@gop.edu.tr
Phone: +903562521616 (2888); Fax: +903562521729
Received: 12-06-2023
Abstract
e study aims to optimize the extraction process and characterize the proteins found in fenugreek seeds. e water and
oil holding capacities, coagulated protein content, foaming, and emulsication properties of the isolated proteins were
investigated under all extraction conditions. Also, solubility, molecular weights, structural and thermal properties were
determined. In the extraction processes carried out at dierent pH (pH 6.0–12.0) and solid:solvent ratios (20–60 g/L), it
was determined that the highest extraction yield (94.3 ± 0.3%) was achieved when the pH was 11.47 and the solid-solvent
ratio was 34.50 g/L. ree distinct bands (46, 59, and 80 kDa) in the range of 22–175 kDa were determined for the fenu-
greek seed protein isolate obtained under optimum extraction conditions. Protein secondary structures were determined
using Fourier Transform Infrared (FT-IR) spectra and it was determined that β-sheet structures were highly present. In
addition, denaturation temperature and denaturation enthalpy were calculated as ~119 °C and 28 mJ/g, respectively.
Keywords: Protein isolate, fenugreek seeds, extraction, secondary structure, emulsifying properties
1. Introduction
Proteins are crucial macronutrients in human nutri-
tion, and historically, they have been primarily obtained
from animal sources. However, with the increasing popu-
lation in recent years, the availability of animal protein
sources has been decreasing. As a result, there is now an
increasing demand for alternative protein sources, such as
plant-based proteins. Plant-based proteins are becoming
more popular due to their health benets and their ability
to promote physical function.1,2 Although animal sources
contain high-quality proteins, they contain high levels of
components such as cholesterol and saturated fatty acids,
which cause diseases such as cardiovascular diseases and
cancer when consumed frequently. Diets containing plant-
based proteins are known to prevent cardiovascular dis-
eases, hypertension, obesity, and some types of cancer.3 In
addition to increasing awareness of healthy nutrition, in-
creasing sustainability concerns regarding food supply al-
so increases consumers’ tendency to prefer plant-based
proteins. Furthermore, the fact that plant proteins, pre-
ferred by special consumer groups such as vegans and veg-
etarians, are cheaper and have a wide variety of sources,
has made plants an alternative protein source for their use
in food applications.4,5
Although plant-based proteins have many advan-
tages, plant protein sources contain non-nutritive compo-
nents (tannins, phytic acid, trypsin inhibitors, oligosaccha-
rides, etc.), show weaker amino acid diversity than animal
proteins, and their digestibility is not good. In addition,
the fact that the functional properties of dierent protein
isolates obtained from a wide variety of plant sources have
not been well established limits their use in food formula-
tions.6 However, knowing the physico-chemical properties
that aect the use of plant proteins in food formulations
is very important in terms of improving the quality prop-
erties of the product. e physico-chemical properties of
proteins are dened as the physical and chemical prop-
erties that aect the behavior of proteins in foods during
production, storage, preparation, and consumption. Solu-
bility, gelling, emulsication, foam formation, water and
oil holding capacity, viscosity and lm formation are some
of the common physico-chemical properties of proteins.
In addition to the structural properties of the proteins
such as amino acid composition, surface hydrophobicity
and hydrophobic/hydrophilic ratio, the extraction meth-
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Isleroglu and Olgun: Eects of the Extraction Conditions on Functional ...
od and the parameters used in their production are also
parameters that aect the physico-chemical properties of
proteins.7,8
Extraction of plant-based proteins, like other pro-
teins, is generally carried out by dissolving the material
in a medium far from the isoelectric point and then pre-
cipitating the soluble proteins at the isoelectric point.9
Alkaline extraction, which provides high protein yield, is
generally used in the extraction of plant proteins. With the
increase in the pH value of the solvent medium, acidic and
neutral amino acids become ionized, and thus the solu-
bility of proteins increases. More than 90% protein yield
can be obtained with the alkaline extraction method.10 Al-
though high yields are obtained with alkaline extraction,
the digestibility of the protein is aected because the struc-
ture of lysine and cysteine is disrupted, which negatively
aects the overall quality of the protein.11 erefore, it is
necessary to determine the alkaline conditions specic to
that protein source that will improve or not aect the phys-
icochemical properties of the protein. In addition, alkali
concentration as well as other parameters such as solid:-
solvent ratio, extraction time, and temperature should be
optimized for maximum protein yield and preservation
of physicochemical properties.10 To identify new protein
sources and gain application areas, it is necessary to char-
acterize the obtained proteins. For this reason, in recent
years, studies on the optimization of alkaline extraction
conditions of plant-based proteins in terms of protein
yield and physico-chemical properties of isolated proteins
have been published in the literature.2,12–16
Fenugreek (Trigonella foenum graecum), known to
have many health benets, is an annual herbaceous plant
in the legume family. Fenugreek, which has a widespread
area in the world, diers from other legumes with its
appearance and dierent smell. e protein content of
fenugreek seeds has been reported to be in the range of
25–38%. e proteins in fenugreek seeds consist of albu-
min, globulin, glutelin, and prolamins. In a study where
the our obtained from fenugreek seeds was used in dif-
ferent proportions instead of wheat our, it was report-
ed that the protein content of products such as bread,
biscuits, noodles, and pasta increased signicantly, and
there was an improvement in their sensory and rheo-
logical properties.17 erefore, fenugreek seeds, which
have high nutritional value, are thought to be a potential
protein source.
In this study, the eects of dierent solid:solvent ra-
tios and pH levels on the extraction yield of the proteins in
fenugreek seeds were determined, and the conditions that
ensure the highest extraction yield were optimized using
response surface methodology. e eects of the extrac-
tion conditions on the functional properties namely water
holding capacity, oil holding capacity, coagulated protein
content, foam capacity, foam stability, emulsion activity,
emulsion stability, and emulsion capacity of the isolated
proteins were investigated. Additionally, the structural
and thermal properties and molecular weight patterns of
fenugreek seed protein isolates obtained under optimum
extraction conditions were determined.
2. Materials and Methods
2. 1. Material
Aer removing the foreign substances in the fenu-
greek seeds purchased from a local market, the seeds were
powdered using a household grinder. e powdered seed
samples were passed through a 630 µm sieve, and defatting
was applied to the under-sieve samples using hexane. To
remove the residual hexane, the samples were le to dry
at 50 °C for 12 hours and the obtained defatted fenugreek
seeds samples were used for protein extraction.
2. 2. Chemicals
H2SO4 (CAS#: 7664-93-9), HCl (CAS#: 7647-01-1),
NaOH (CAS#: 1310-73-2), Brilliant Blue G-250 (CAS#:
6104-58-1) and Na2HPO4 dibasic dihydrate (CAS#:
10028-24-7) were obtained from Sigma-Aldrich, Germa-
ny. Boric acid (CAS#: 1043-35-3), methanol (CAS#: 67-
56-1) and H3PO4 (CAS#: 7664-38-2) were obtained from
Merck KGaA, Germany. Hexane (CAS#: 110-54-3) and
citric acid monohydrate (CAS#: 5949-29-1) were provided
by Tekkim Chemicals, Turkey. Kjeldahl tablets (Kjeltabs
ST, AA 09) were obtained from Gerhardt, Germany. Tashi-
ro indicator (CAS#: 64-17-5) was obtained from Riedel-de
Haën™, Germany. Biuret Reagent (CB2145) was obtained
from ChemBio, Turkey. Sodium phosphate dibasic (CAS#:
151-21-3) was obtained from BioBasic, Canada.
2. 3. Extraction Process and Isolation of the
Proteins
Protein extraction from the defatted fenugreek seeds
was carried out by mixing (at 750 rpm for 4 hours) the
suspensions prepared in dierent solid:solvent ratios with
distilled water as a solvent at dierent pH values. To op-
timize the extraction process, pH value (pH 6.0–12.0)
and solid-solvent ratio (20–60 g/L) were chosen as in-
dependent variables, and a 'Central Composite Design'
was carried out (Table 1). e samples were centrifuged
at 6000 rpm for 15 minutes at the end of the extraction
process. Extraction yield was calculated by proportioning
the amount of protein in the supernatant phase (extract)
to the protein amount of the initial powdered seed sample
(Eq. 1). During the study, the protein contents of the sam-
ples were determined by Kjeldahl method18 in all protein
isolates and powder samples, and by Bradford method19 in
supernatant phases.
Extraction yield (%) = [Protein amount of extract (g) /
Protein amount of fenugreek seeds (g)] × 100 (1)
206 Acta Chim. Slov. 2024, 71, 204–214
Isleroglu and Olgun: Eects of the Extraction Conditions on Functional ...
In the optimization process, the extraction yield was
used as a response and the conditions providing the high-
est extraction yield were determined with a desirability
function approach. e model used for regression analysis
is given in Eq. 2.
(2)
where, β0, βi, βii and βij are the coecients, X is the inde-
pendent variable and k is the number of independent var-
iables.
Aer the extraction process, the pH values of the ex-
tracts were adjusted to 4.0 and incubated at room temper-
ature for 6 hours. Aer the incubation, the samples were
centrifuged at 9000 rpm for 60 minutes, the supernatant
was removed, and the precipitate was washed three times
(5 minutes at 6000 rpm) using distilled water. e washed
precipitates were then collected and lyophilized for 72
hours (Christ Alpha 1–4 LSC Plus, Germany). Powder
protein isolates obtained as a result of the lyophilization
were stored in sealed tubes at –18 °C until the analyses. By
determining the amount of protein remaining in the su-
pernatant phase at the end of precipitation, the average
recovery in the precipitation process was calculated as
93.75 ± 0.55%.
2. 4. Characterization of the Protein Isolates
2. 4. 1. Coagulated Protein
e percentage of the coagulated protein in the
samples was determined using the method described by
Kramer and Kwee.20 For this purpose, 0.2 g protein iso-
late was dissolved with 10 ml of citrate-phosphate solution
(pH 7.0) at a concentration of 0.025 M and centrifuged.
Biuret reagent was added to the supernatant phase and the
solution was kept in the dark for 30 minutes. e solution
was then incubated at 100 °C for 15 minutes and cooled
to room temperature. Aer the cooling, the heating pro-
cess was applied once again. e coagulated protein (%)
was calculated using the absorbances of the samples before
heating (A1) and aer heating (A2) at 540 nm (Eq. 3).
Coagulated protein (%) = [(A1–A2) / (A1)] × 100 (3)
2. 4. 2. Water and Oil Holding Capacity
Water holding capacity and oil holding capacity were
determined by modifying the method of Vinayashree and
Vasu. 10 Aer vortexing 250 mg of protein isolate with 15
ml of distilled water, it was kept at room temperature for 1
hour. en, it was centrifuged at 3000 rpm for 20 minutes,
the supernatant phase was removed, and the remaining
sample was weighed. e water holding capacity is calcu-
lated in the g water/g sample. To determine the oil holding
capacity, olive oil was used instead of water, and the oil
holding capacity was expressed in g oil/g sample.
2. 4. 3. Foaming Capacity and Foam Stability
e foaming capacity and foam stability of the pro-
tein isolates were determined by the method proposed by
Timilsena et al.21 Aqueous solutions of the protein isolate
at a concentration of 20 g/L were homogenized with a ho-
mogenizer (Ultra-Turrax IKA T-18 Basic, USA) at 10000
rpm for 5 minutes. Total volumes before homogenization
(V0) and aer homogenization (V1) were measured, and
foaming capacity (%) was calculated using Eq. 4.
Foaming capacity (%) = [(V1–V0) / (V0)] × 100 (4)
e foam stability was calculated using Eq. 5 by de-
termining the total volume (V2) of the homogenized sam-
ple aer it was kept at room temperature for 1 hour.
Foam stability (%) = [(V2–V0) / (V1–V0)] × 100 (5)
2. 4. 4. Emulsifying Properties
Emulsion activity and emulsion stability of the pro-
tein isolates were determined using the turbidity method
modied by Feyzi et al.22 First, 22.5 mg of the sample was
weighed into a 15 mL tube, 4.5 mL of phosphate buer
solution (pH 7.0) was added, and the sample was vor-
texed for 1 minute. Sunower oil (1.5 mL) was added to
this mixture and homogenized at 22000 rpm for 2 min-
utes. To determine the emulsion stability, immediately af-
ter the homogenization (t=0), 250 µL emulsion was mixed
with 50 mL sodium dodecyl sulfate at a concentration of
1 g/L, and the absorbance of this mixture at 500 nm was
recorded (A0). Similarly, the same process was applied to
the initial emulsion that was kept at room temperature for
15 minutes (t=15) and its absorbance was recorded (A15).
Emulsion stability (min) was calculated using Eq. 6.
Emulsion stability (min) = [A0 / (A0–A15)] × t (6)
Emulsion activity (m2/g) was determined using Eq. 7.
Emulsion activity (m2/g) = (2T × D) / (Φ × C)
= (2 × 2.303 × A0 × D) / (Φ × C × L) (7)
where, T is the turbidity (T= 2.303xA0/L), D is the dilution
factor (200), Φ is the emulsion oil volume fraction (g oil/g
sample), C is the protein concentration in the solution
(0.005 g/ml), L is the cuvette path length (10–2 m).
e method given by Neto et al.23 was modied to
determine the emulsion capacity. First, an equal volume
of sunower oil was added to the protein isolate solutions
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prepared at a concentration of 1.0 % (w/v), and an emul-
sion was formed by homogenizing with ultra-turrax (7200
rpm, 2 min). ese emulsions were then centrifuged at
3250 rpm for 2 minutes. e total height of the emulsion in
the tube before centrifugation was expressed as H0 (cm),
the height of the emulsied layer of the centrifuged emul-
sion was expressed as H1 (cm), and the emulsion capacity
was calculated using Eq. 8.
Emulsion capacity (%) = [H1 / H0] × 100 (8)
2. 4. 5. Protein Solubility
e solubility of the protein isolates (g/L) obtained
under optimum extraction conditions was determined by
the method reported by Feyzi et al.22 e pH values of the
protein isolate solutions prepared with distilled water at
a concentration of 15 g/L were adjusted to values in the
range of 2.0–12.0 using HCl or NaOH. Aer agitating the
samples for 30 minutes at room temperature, they were
centrifuged at 6000 rpm for 15 minutes to determine the
protein content in the supernatant phase.
2. 4. 6. Sodium Dodecyl Sulfate-Polyacrylamide
Gel Electrophoresis (SDS-PAGE)
e SDS-PAGE method24 was used to determine the
molecular weights of proteins obtained under optimum
extraction conditions. In the study, 12% gel was used and
10 µg and 50 µg of samples were loaded. Samples were run
under 200 V voltage for 50 min and the gel was stained
with Comassie Brillant Blue R-250.
2. 4. 7. Fourier Transform Infrared (FT-IR)
Spectroscopy
Structural properties of protein isolates obtained un-
der optimum conditions were determined using a Fourier
Transform Infrared (FT-IR) Spectrometer (Perkin Elmer
400, USA). Diamond ATR method was used in the anal-
ysis and measurements were made in the spectrum range
of 4000–400 cm–1. Considering the Amide I region (1600–
1700 cm–1) in the FT-IR spectra, the protein secondary
structures of the protein isolates were determined by de-
convolution of the peaks and curve tting using the Peakt
v4.12 package program (Systat Soware, USA).
2. 4. 8. ermal Properties
Denaturation temperature (Td, °C) and denaturation
enthalpy (ΔHd, mJ/g) of the protein isolates obtained un-
der optimum conditions were determined using a Dier-
ential Scanning Calorimetry (DSC) (Perkin Elmer DSC
8000, USA). Analyses were carried out in a nitrogen envi-
ronment, in the temperature range of 20–200 °C and at a
heating rate of 5 °C/minute.
2. 5. Statistical Analysis
e one-sample t-test and 'Univariate Variance Anal-
ysis, Duncan post hoc' test were performed using the SPSS
21.0 soware package. Regression analysis, contour plots,
and optimization processes were performed using Design
Expert 7.0 (Stat-Ease, Inc., USA) soware to determine the
eects of all process variables.
3. Results and Discussion
3. 1. Extraction Process
e experimental design used for the extraction
process of the proteins found in fenugreek seeds and the
extraction yields are given in Table 1. According to the
results, the highest extraction yield (93.13 ± 1.36%) was
obtained under the condition that the solid-solvent ratio
was 20 g/L and the pH value was 12.0. e lowest extrac-
tion yield was determined when the solid-solvent ratio
was 60 g/L and the pH value was 6.0 (Table 1). Lower
extraction yields were observed at all pHs when the sol-
id-solvent ratio was the highest (60 g/L). e decrease in
protein extraction yields when the solid-solvent ratio is
high can be explained by the fact that non-protein com-
pounds (gum, mucilage, etc.) in the extraction medium
make protein extraction dicult. It is thought that pro-
tein extraction yields increase by providing a more ef-
fective mixing process at low solid-solvent ratios (20–40
g/L) and increasing the solid-solvent contact surface.
It was determined that the pH value chosen as another
independent variable in the protein extraction process
also aects the extraction yield. e extraction yields
increased with increasing pH values in all solid-solvent
ratios (Table 1). is situation is associated with the in-
creased solubility of the proteins in fenugreek seeds at
high pH values. Similarly, Feyzi et al.25 reported that the
solubility of fenugreek seed proteins increased in an alka-
line environment (pH 9.25). Jarpa-Parra et al.26 reported
that the extraction yield and purity of the obtained pro-
teins increased by using pH values ≥9.0 in protein extrac-
tion from lentils. Gao et al.27 carried out protein extrac-
tion from yellow peas, which belong to the legume family
as fenugreek seeds, and found that the protein extraction
yield increased with increasing pH.
To optimize the extraction process, the extraction
yield was chosen as the response, and a second-order poly-
nomial model was constructed. According to the ANOVA
results given in Table 2, the developed model was found
to be statistically signicant (p < 0.05), and the lack of t
was found to be statistically insignicant (p > 0.05). e
linear and quadratic eects of the solid-solvent ratio and
pH on extraction yield were determined to be statistically
signicant (p < 0.05). On the other hand, it was observed
that the solid-solvent ratio-pH interaction did not have a
statistically signicant eect on the extraction yield (p >
208 Acta Chim. Slov. 2024, 71, 204–214
Isleroglu and Olgun: Eects of the Extraction Conditions on Functional ...
0.05) (Table 2). e model equation, written in terms of
the real values of the factors, is given in Eq. 9.
Table 2. ANOVA table and statistical parameters
Source Degrees Sum of Mean F Value p – Value
of freedom squares square
Model 5 3055.79 611.16 417.80 < 0.0001
X1 1 378.55 378.55 258.79 < 0.0001
X2 1 2041.64 2041.64 1395.72 < 0.0001
X1X 2 1 5.00 5.00 3.42 0.1069
X12 1 114.18 114.18 78.06 < 0.0001
X22 1 286.98 286.98 196.19 < 0.0001
Residual 7 10.24 1.46
Lack of Fit 3 7.62 2.54 3.88 0.1118
Pure Error 4 2.62 0.66
Total 12 3066.03
R2: 0.9967, adj- R2: 0.9943, adequate precision: 64.234,
PRESS: 60.10, C.V. (%): 1.58
X1: solid:solvent ratio (g/L), X2: pH, adj- R2: adjusted R2, PRESS:
predicted residual error sum of squares, C.V. (%): coecient of var-
iation
(9)
e conditions at which maximum protein extrac-
tion yield was achieved were determined by the desirabili-
ty (d) function approach. e scale of the desirability func-
tion ranges from the completely unacceptable response
(d=0) to the response corresponding to the target value
(d=1), and the value of d increases as the desirability of the
dependent variable increases. e response surface con-
tour plots of the predicted desirability values and the ex-
traction yields (%) are shown in Fig. 1a and Fig. 1b, respec-
tively. As seen in Fig. 1, the conditions where the maximum
desirability value (d=1) was obtained (maximum extrac-
tion yield) were selected as the optimum extraction condi-
tions. e solid:solvent ratio was 34.5 g/L and pH was
11.47, and the predicted extraction yield was 94.08% at the
optimum conditions (Fig. 1b). For the experimental vali-
dation, the extraction process was performed in triplicate
under the predicted optimum conditions, and no statisti-
cally signicant dierence (p>0.05) was determined be-
tween the experimental extraction yield (94.29 ± 0.26%)
and the predicted one.
Fig 1. Counter plots of (a) desirability values and (b) predicted ex-
traction yields (%)
3. 2. Characterization of the Protein Isolates
e functional properties of the protein isolates ob-
tained under dierent extraction conditions are given in
Table 3. e foaming properties of the fenugreek seeds
protein isolates were determined by measuring the foam-
ing capacity and foam stability. While foaming capacity is
dened by the increase in the volume of the solution in the
foaming process, foam stability is dened as the ability to
keep the air in the foams formed.28 e foaming capacity
of fenugreek seed protein isolates was determined between
10.67 and 18.00%, and the foam stability was determined
between 51.92 and 69.67%. As a result of extractions per-
Table 1. Experimental design and extraction yields (%)
Experi- Solid:solvent pH Extraction yield
ment no ratio (g/L) (X1) (X2) (%)
1 40 9.0 84.72 (± 0.68)
2 20 6.0 58.62 (± 1.36)
3 60 9.0 68.30 (± 0.72)
4 40 9.0 83.76 (± 0.41)
5 40 9.0 85.96 (± 0.27)
6 20 12.0 93.13 (± 1.36)
7 60 12.0 80.12 (± 0.63)
8 40 12.0 91.71 (± 0.54)
9 40 9.0 85.00 (± 0.54)
10 40 9.0 84.43 (± 0.81)
11 40 6.0 54.52 (± 1.08)
12 20 9.0 85.46 (± 1.90)
13 60 6.0 41.14 (± 0.27)
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Isleroglu and Olgun: Eects of the Extraction Conditions on Functional ...
formed at pH 12.0, it was determined that the highest
foaming capacity and foam stability values were obtained
(p < 0.05). Also, it was determined that the highest values
were obtained in the extractions performed at a medium
level (40 g/L) solid:solvent ratio (Table 3). Dierences in
the extraction conditions applied when obtaining the pro-
tein isolate caused the formation of dierent protein struc-
tures and fractions, aecting the foaming properties.29 e
foaming capacity of the protein isolate obtained under op-
timum conditions was determined as 19.00 ± 1.00%, and
the foam stability was determined as 74.13 ± 2.16%.
e water and oil holding capacities are dened as the
amount of water or oil absorbed per unit of protein, and the
leakage of substances such as water or oil from the products
can be prevented because of these properties of proteins
during storage of the food. In addition, the oil holding ca-
pacity is important in terms of keeping the oil-soluble avor
substances and the texture of the product. e water and/
or oil holding capacity of protein isolates is related to the
number of polar or nonpolar amino acids in the structure,
surface hydrophobicity and conformation of the proteins.28
e water holding capacities of fenugreek seed protein iso-
lates varied between 2.04 and 2.73 g/g. It was determined
that the highest water holding capacity values belonged to
the samples extracted at pH 12.0 (Table 3). In other studies,
the water holding capacity value for fenugreek seed protein
concentrate was 1.56 g/g17 and for fenugreek seed protein
isolate 2.70 g/g 25. Liu et al.30 characterized the axseed pro-
tein isolates and reached water holding capacity values in
the range of 0.83–1.05 g/g. Kaur and Ghosal31 reported the
water holding capacity of protein isolate obtained from de-
fatted sunower meal as 2.00 g/g, and Yancheshmeh et al.32
reported the water retention capacity of the protein isolate
obtained from vetch seed as 2.01 g/g. When compared to
the studies conducted in the literature, it was observed that
the determined water holding capacity value of 2.64 ± 0.04
g/g of fenugreek seed protein isolate obtained under opti-
mum extraction conditions was higher than many plant-de-
rived protein isolates. It was determined that the oil holding
capacity values of fenugreek seed protein isolates varied be-
tween 1.46 and 2.10 g/g. It was observed that the highest oil
holding capacity values were obtained in the protein isolates
produced as a result of extractions performed at pH 12.0
(p<0.05) (Table 3). e oil holding capacity value obtained
under optimum conditions was determined to be 2.00 ±
0.01 g/g. El Nesri and El Tinay17 determined the oil hold-
ing capacity value of fenugreek seed protein concentrate as
1.56 g/g. Feyzi et al.25 determined the oil holding capacity
value of fenugreek seed protein isolate as 6.06 ± 0.28 g/g. It
is thought that dierent oil retention capacity values may
be related to the extraction conditions of the seeds and the
climate in which they are grown.25
e coagulated protein (%) refers to the protein
percentage of the total soluble protein that will coagulate
when heated to 100 °C. Since the uncoagulated protein is
in soluble form, it can leak out of the system, which is un-
desirable. However, non-coagulating proteins are advan-
tageous in forming viscous systems and increasing nutri-
tional value in liquid systems (e.g., in breakfast drinks). It
was determined that the coagulated protein values of fenu-
greek seed protein isolates varied between 3.01 and 4.96%.
A decrease in the coagulated protein values was observed
at increasing pH values during extraction (p < 0.05) (Table
3). Feyzi et al.25 determined the coagulated protein value
as 3.17% and emphasized that the proteins can be used in
the production of beverages with high nutritional value
and protein-added fruit juices due to their low coagulated
protein percentages.
Proteins can prevent agglomeration and creaming by
forming a layer around oil droplets at the water-oil inter-
face, that are immiscible and thermodynamically unsta-
ble due to their amphiphilic structure. e emulsication
properties of plant-derived proteins are of great importance
for their use in the food industry. Emulsion properties of
the proteins are aected by internal factors such as surface
charge, hydrophobicity, solubility, molecular size, exibility
of the lm formed, and external factors such as presence of
other substances in the environment, pH, ionic strength,
temperature, protein extraction methods and protein con-
centration.33 Emulsion capacity is dened as the maximum
amount of oil that can be emulsied by a certain amount of
protein and is expressed as a percentage.22 It was observed
Table 3. Functional properties of the protein isolates
pH Solid:solvent Foaming Foam Coagulated Water holding) Oil holding
ratio (g/L) capacity (%) stability (%) protein (%) capacity (g/g capacity (g/g)
6 20 11.50 ± 0.71c 52.80 ± 2.45d 4.81 ± 0.26a 2.13 ± 0.03d 1.57 ± 0.02d
40 11.50 ± 0.71c 68.06 ± 1.20ab 4.98 ± 0.26a 2.04 ± 0.01e 1.52 ± 0.03e
60 10.67 ± 0.58c 51.92 ± 2.72d 5.06 ± 0.13a 2.04 ± 0.03e 1.46 ± 0.04f
9 20 14.00 ± 1.00b 60.66 ± 2.60c 3.69 ± 0.13c 2.18 ± 0.02c 1.91 ± 0.02c
40 15.00 ± 1.00b 63.33 ± 4.71bc 4.02 ± 0.28bc 2.18 ± 0.03c 1.98 ± 0.02b
60 14.00 ± 0.00b 61.25 ± 1.77c 4.15 ± 0.18b 2.19 ± 0.01c 1.89 ± 0.01c
12 20 17.00 ± 0.00a 64.29 ± 0.00abc 3.07 ± 0.27d 2.66 ± 0.02b 2.07 ± 0.04a
40 18.00 ± 0.00a 69.67 ± 1.30a 3.08 ± 0.21d 2.65 ± 0.04b 2.09 ± 0.03a
60 17.00 ± 0.00a 69.44 ± 3.93a 3.16 ± 0.18d 2.73 ± 0.02a 2.10 ± 0.02a
a–g Mean values given dierent letters in the same column are statistically dierent from each other (p < 0.05).
210 Acta Chim. Slov. 2024, 71, 204–214
Isleroglu and Olgun: Eects of the Extraction Conditions on Functional ...
that the emulsion capacities of the fenugreek seed protein
isolates varied between 18.30 and 26.00% (Fig. 2a). It was
determined that the emulsion capacities of the obtained
fenugreek seed protein isolates were higher when extract-
ed at higher pH values (p < 0.05) (Fig. 2a). e emulsion
capacity of fenugreek seed protein isolates obtained under
optimum conditions was determined as 26.52 ± 0.26%.
Emulsion activity is dened as the maximum emulsion sur-
face area per unit protein measured spectrophotometrical-
ly based on turbidity.34 It was determined that the emulsion
activities of fenugreek seed protein isolates ranged between
75.82 and 80.95 m2/g (Fig 2b). Furthermore, the emulsion
activity of fenugreek seed protein isolates obtained under
optimum conditions was determined as 78.21 ± 0.28 m2/g.
Emulsion stability was determined based on the change
in turbidity over time. e emulsion stability of fenugreek
seed protein was observed to vary between 23.65 and 28.06
minutes (Fig. 2c). e emulsion stability of fenugreek seed
protein isolates obtained under optimum conditions was
determined as 28.73 ± 0.35 minutes.
Proteins exhibit maximum solubility in highly acid-
ic or basic conditions far from the isoelectric point. e
results obtained in the study showed that the solubility
properties of the fenugreek seed protein isolate obtained
under optimum extraction conditions comply with this
phenomenon. As seen in Fig. 3a, solubility values follow
a characteristic U-shaped curve in the pH range of 2–12.
While the solubility values of the samples ranged between
0.27 and 8.46 g/L, the lowest solubility was observed at
pH 4.0. is can be explained by the fact that fenugreek
seed proteins have an isoelectric point in the pH range of
4.0–4.5.22 Since an equilibrium occurs between negatively
and positively charged ions at the isoelectric point, the net
charge becomes zero. us, as the electrostatic repulsion
forces decrease, proteins lose their solubility and collapse
as a result of the hydrophobic interactions. On the other
hand, the electrostatic repulsion force that occurs between
the charged ions in acidic and alkaline conditions far from
the isoelectric point, which may be dierent for each pro-
tein, ensures the dissolution of the proteins.35 When the
solubilities at high pH values were examined, it was seen
that the highest values were obtained at pH 11.0 and pH
12.0 (Fig. 3a). e better solubility of the fenugreek seed
proteins at high pH values can be explained by the inhibi-
tion of the formation of protein aggregates by the repulsive
force of a larger number of negatively charged ions.25 Sim-
ilar results for some plant-derived proteins in the litera-
ture have been obtained for soy protein isolate, Moringa
oleifera seed protein isolate, bitter melon protein isolate,
axseed protein isolate, and chickpea protein isolate.36–40
e molecular weight distribution of the fenugreek seed
protein isolate was determined to be between ~175 kDa
and ~22 kDa, and 10 bands with molecular weights of ap-
proximately 175, 159, 80, 59, 46, 38, 31, 27, 23, and 22 kDa
were detected. However, 3 distinct bands were observed.
ese three most prominent bands were detected as ~80,
59, and 46 kDa (Fig. 3b). e bands between 22 and 70
kDa are found to be associated with globulins, specically
legumins and vicilins, which constitute the primary pro-
tein constituents in legumes.41 ese proteins were fur-
ther fractionated into distinct subunits: β-legumin was
observed at approximately 22 kDa, while α-legumin was
observed at around 40 kDa.42 Hence, the bands obtained
at ~38 and 46 kDa could be associated with α-legumin for
the fenugreek seed protein isolate. Moreover, the visible
bands at ~22 and 23 kDa could be associated with the pres-
ence of β-legumin. e bands ranging from 50 to 80 kDa
have been attributed to vicilin and covicilin. Two of the
predominant bands observed at ~59 and 80 kDa could be
ascribed to the polypeptide constituents of vicilin and con-
vicilin.43,44
Fig 2. Emulsifying properties of fenugreek protein isolates (a) emul-
sifying capacity, (b) emulsifying activity, (c) emulsifying stability
211
Acta Chim. Slov. 2024, 71, 204–214
Isleroglu and Olgun: Eects of the Extraction Conditions on Functional ...
Fig. 4 shows the FT-IR spectra of the fenugreek seed
protein isolates obtained under optimum conditions. As
seen in Fig 4, the Amide I band is observed at the wave
number of 1600–1700 cm–1. e region between wave
numbers of 1480–1585 cm–1 is dened as the Amide II
region, and around 40–60% N–H bending vibration and
around 18–40% C–N stretching vibration are observed in
this region. It was observed that the peaks obtained at the
Fig 3. Characteristics of the protein isolates produced at optimum extraction conditions (a) solubility, (b) SDS-PAGE image
(a) (b)
Fig 4. FT-IR spectra of the fenugreek seed protein isolate a) original spectrum, b) deconvolution in the Amide I region
212 Acta Chim. Slov. 2024, 71, 204–214
Isleroglu and Olgun: Eects of the Extraction Conditions on Functional ...
wave numbers of 1447, 1515, and 1532 cm–1 for the ob-
tained protein isolate were located in the Amide II region
(Fig. 4a). e detection of Amide I and Amide II bands is
considered an absolute indicator of the presence of protein
structure.45 e Amide III region is observed at the wave
numbers between 1200–1400 cm–1 and indicates the exist-
ence of interactions between protein and other macromol-
ecules such as carbohydrates. e presence of this region
in proteins occurs depending on the side ring structure.
C–N stretching vibrations and N–H bending vibrations
are observed in this region.46 It was determined that the
peaks obtained at the wave numbers of 1240–1394 cm–1
for the fenugreek seed protein isolates were in the Amide
III region (Fig. 4a). Secondary structures of the fenugreek
seed protein isolates were determined using the peaks in
the Amide I region (1600–1700 cm–1) in the FT-IR spec-
trum. ere are α-helix, β-sheet, random coil, or β-turn
conformations in the Amide I band of proteins.47 It has
been reported that the β-sheet structure was observed in
the wave numbers 1612–1640 cm–1 and 1689–1695 cm–1.48
Furthermore, the α-helix was observed at 1651–1660 cm–1
49,50, the random coil conformation was observed at 1641–
1650 cm–1 49 and the β-turn was observed at 1661–1688
cm–1 50. In the FT-IR spectra, 13 peaks were observed in
the Amide I region, and the ratio of the fractions in the
protein secondary structure was determined by deconvo-
lution of the peaks in the Amide I region (Fig. 4b). As a
result of the analysis, 38.69% of the secondary structure is
β-sheet, 18.96% is α-helix, 10.39% is random coil, 26.76%
is β-turn and 5.20% is side ring. e high presence of the
β-sheet structures indicates that protein isolates have high
thermal stability.50 To determine the thermal properties
of the fenugreek seed protein isolates, denaturation tem-
peratures (Td) and denaturation enthalpies (ΔHd) were de-
termined using Dierential Scanning Calorimetry (DSC).
An endothermic peak was observed indicating that energy
was required for denaturation to occur, and the denatura-
tion temperature of the fenugreek seed protein isolate was
118.85 °C. In the literature, denaturation temperatures
of 91 °C for cowpea protein isolate51, 95 °C for axseed
protein isolate52, 103 °C for quince seed protein isolate53
and 105 °C for fenugreek seed protein isolate have been re-
ported25. e denaturation enthalpy value (ΔHd) of the ob-
tained protein isolate was also calculated and determined
as 28 mJ/g.
4. Conclusion
e study aimed to extract proteins from fenugreek
seeds using the alkaline extraction process at dierent pH
values (pH 6.0–12.0) and solid:solvent ratios (20–60 g/L),
and to determine the optimum conditions for the high-
est extraction yield. e optimum extraction conditions
were determined as pH 11.47 and solid:solvent ratio 34.5
g/L, and an extraction yield of 94.3% was achieved under
these conditions. e protein isolates obtained under dif-
ferent extraction conditions have various properties, in-
cluding water holding capacity of ~2.0–2.7 g/g, oil holding
capacity of ~1.5–2.1 g/g, coagulated protein content of
~3.0–5.0%, foam capacity of ~11.0–18.0%, foam stability
of ~%52.0–70.0, emulsion stability of ~24.0–28.0 minutes,
emulsion activity of ~76.0–81.0 m2/g, and emulsion capac-
ity of ~18.3–26.0%. Solubility properties showed that the
fenugreek seeds protein isolate was soluble both acidic and
basic conditions, which makes it a good candidate for both
types of drinks. e study also included secondary struc-
ture analysis and thermal property determination, which
revealed the thermal stability of the protein isolates. As a
result, the extraction process was optimized to achieve the
highest extraction yield, which distinguishes it from other
studies in the literature. e study also examined how each
extraction condition aected the characteristics of protein
isolates. In this study, unlike other studies in the litera-
ture, the extraction process was optimized to provide the
highest extraction eciency and it was revealed how each
extraction condition aected the characteristics of protein
isolates. e results showed that the functional properties
of protein isolates obtained under dierent extraction con-
ditions are competitive or even better than other plant-de-
rived proteins in the literature. Based on these ndings,
fenugreek seed protein isolate, produced under optimum
extraction conditions, may be an excellent alternative
plant-based protein for several food applications. Its func-
tional, structural, and thermal properties make it suitable
for use in dierent formulations in many food processes.
Acknowledgments
is study was nancially supported by Tokat Gazi-
osmanpasa University Scientic Research Projects Com-
mittee (Project No: 2022/114).
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Povzetek
Namen študije je optimizacija postopka ekstrakcije in karakterizacija beljakovin, ki jih najdemo v semenih triplata. Zmo-
gljivosti zadrževanja vode in olja, vsebnost koaguliranih proteinov, penjenje in emulgiranje izoliranih proteinov so bile
raziskane pri vseh pogojih ekstrakcije. Določene so bile tudi topnost, molekulske mase, strukturne in toplotne lastnosti.
Pri ekstrakcijskih postopkih, izvedenih pri različnih pH (pH 6,0–12,0) in razmerjih trdna snov:topilo (20–60 g/L), je bilo
ugotovljeno, da je bil največji izkoristek ekstrakcije (94,3 ± 0,3 %) dosežen pri pH 11,47 in razmerju med trdno snovjo
in topilom 34,50 g/L. Določeni so bili trije različni pasovi (46, 59 in 80 kDa) v območju 22–175 kDa za proteinski izolat
semena triplata, pridobljenega pri optimalnih pogojih ekstrakcije. Sekundarne strukture proteinov so bile določene z
uporabo Fourierove transformacijske infrardeče spektroskopije (FT-IR) in ugotovljeno je bilo, da so bile dobro zastopane
β-planarne strukture. Poleg tega sta bili izračunani temperatura in entalpija denaturacije kot ~119 °C, oziroma 28 mJ/g.
