Food

Chemistry
Food Chemistry 106 (2007) 1166–1174
www.elsevier.com/locate/foodchem

Preparation and characterization of a protein hydrolysate

from an oilseed flour mixture
C. Radha, Parigi Ramesh Kumar, V. Prakash *
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore 570 020, India

Received 16 February 2007; received in revised form 13 June 2007; accepted 18 July 2007

Abstract

A novel protein hydrolysate was prepared from the mixture of oilseed flours (soybean, sesame and peanut) and determined physico-
chemical & functional properties along with comparison of individual oilseed flour hydrolysate of soybean. Mixed flour obtained from
oilseed flours viz. soybean, sesame and peanut by using calculated amounts in the ratio of 1.1:1.7:0.7, respectively was used as a starting
raw material having balanced amino acid profile. Protein hydrolysates were prepared from mixed flour and soybean flour by a double
enzyme treatment method to a level of 40% degree of hydrolysis. The dried protein hydrolysate prepared from the mixed flour had 72%
crude protein. This protein was characterized by gel filtration chromatography and SDS-PAGE. Comparison of the amino acid profile of
the protein hydrolysate from mixed flours and soyabean flours showed a significant increase in the former one with respect to amino acid
contents usually deficient of single oilseed flour hydrolysate. The product is creamish yellow in colour and had a solubility of >90% over
a wide pH range of 2–10. The mixed flour protein hydrolysate showed better functional attributes such as foaming, as compared to that
from soybean flour alone.
Ó 2007 Elsevier Ltd. All rights reserved.

Keywords: Mixed oilseed flour; Protein hydrolysate; Balanced essential amino acid contents; Functional properties; Foam capacity; Foam stability

1. Introduction the protein hydrolysates should be rich in low molecular

weight peptides, especially di- and tripeptides, with as little
Protein hydrolysates have been used, since 1940s for the as possible free amino acids, for qualifying to have a high
nutritional management of individuals who cannot digest nutritional and therapeutic values (Vijayalakshmi, Lemi-
protein (Cuthbertson, 1950). Food protein hydrolysates eux, & Amoit, 1986). On the other hand, large molecular
have a wide range of applications as ingredients in the weight peptides (more than 20 amino acid residues) are pre-
areas of nutrition, food industry, health care and cosmetics sumed to be associated with the improvement of function-
and it was well reported (Bautista et al., 2000; Clemente, ality of hydrolysates. Thus, irrespective of the use of the
2000; Frokjaer, 1994; Giese, 1994; Weir, 1986). Protein protein hydrolysates, it is always important to characterize
hydrolysates possess a number of functional properties, them on the basis of their peptide size (Gauthier, Vachon,
which make them more attractive as a protein source in & Savoie, 1986). Application of proteinases is often an
human nutrition, both in products for general and special attractive means for obtaining better functional properties
medical use (Sven Frokjaer, 1994). From a nutritional of food proteins, without impairing their nutritional value.
point of view, the demand for protein or truly for amino Enzymatic hydrolysis can produce hydrolysates with well
acids can be fulfilled equally well by an intake of free amino defined peptide profiles and extensive review of enzymatic
acid, protein hydrolysate or intact protein. For dietary use, protein hydrolysates in human nutrition was reported in
literature (Clemente, 2000).
*
Corresponding author. Tel.: +91 821 2517760; fax: +91 821 2516308. Oilseeds are attracting increasing interest as a source of
E-mail address: prakash@cftri.com (V. Prakash). edible proteins. Physico-chemical and functional properties

0308-8146/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2007.07.063
 C. Radha et al. / Food Chemistry 106 (2007) 1166–1174 1167

of oilseed proteins such as soybean, peanut (groundnut) these proteins could find applications in the food industry
and sesame have been well reviewed (Kinsella, Damoda- if appropriate hydrolysis processes are applied. One of the
ran, & German, 1985; Prakash & Narasinga Rao, 1986). most efficient means of increasing protein solubility as
These oilseeds contain about 20–25% protein and the pro- well as improving the functional properties of oilseed pro-
tein content of defatted meals from dehulled oilseeds teins is to subject them to enzymatic hydrolysis (Delvalle,
depends on the seed and ranges between 35% and 60% 1981; Kabirulla & Wills, 1981; Shen, 1992; Were, Hetti-
and proteins from the defatted cake can be extracted in arachchy, & Kalapathy, 1997). By means of protease
water or dilute salt solutions. The amino acid composition hydrolysis, functional properties of oilseed flour can be
(expressed as milligrams/gram crude protein) of these three improved (Hrckova, Rusnakova, & Zemanovic, 2002;
oil seed proteins (soybean, peanut and sesame) comprises Taha & Ibrahim, 2002). It has been observed that the
of total amino acids (TAA) of about 936, 945 and 947, extent of proteolytic degradation of food proteins affects
respectively and in that total essential amino acids (TEAA) the functional properties of the hydrolysates (Babiker,
constitutes about 365, 349 and 373 which essentially about 2000; Hettiarachchy & Kalapathy, 1998; Kristinsson &
39%, 37% and 39%, respectively (Bodwell & Hopkins, Rasco, 2000; Quaglia & Orban, 1987). It is possible, lar-
1985). gely depending on enzyme specificity and the degree of
Soy beans are the most abundant protein meal and the hydrolysis (DH) achieved, to generate hydrolysate prod-
approximate protein content of whole oilseed and defatted ucts with either enhanced or reduced functionality, e.g.,
soy flour is about 43% and 52%, respectively. The total solubility, emulsification, foaming and gelation properties.
essential amino acid composition of soy flour constitutes (FitzGerald & O’Cuinn, 2006). Structural modification of
41.18 g amino acid/16 g nitrogen and individually these proteins is expected to alter their functional properties for
amino acid contents (His, Ile, Leu, Lys, Met, Cys, Phe, use as food ingredients and for rendering proteins, most
Tyr, Thr, Try and Val) are 2.6, 4.8, 6.5, 5.7, 1.34, 1.45, usable in formulating nutritional food products (Hamada,
4.72, 3.4, 4.27, 1.8 and 4.6, respectively and aspartic and 1994).One of the important physico-chemical and func-
glutamic acids which are acidic amino acids constitutes tional property of protein hydrolysates is their solubility
11.26 and 17.18, respectively (Campbell, Kraut, Yackel, over a wide range of pH, temperature, nitrogen concen-
& Yang, 1985). tration and ionic conditions (Adler-Nissen, 1986; Kester
Sesame seed contains about 22–25% protein and defat- & Richardson, 1984). The degradation of the protein into
ted sesame meal contains 40–50% protein content. This oil- peptides generally renders the product more soluble espe-
seed is very important as a protein source and the amino cially at the isoelectric point (Chobert et al., 1996). Lot of
acid composition of the sesame seeds is unique and unusual work has been carried out in the field of functionality of
among the oilseed proteins, due to its high content of sul- individual oilseed protein products & enzymatic food pro-
phur-containing amino acids (methionine and cysteine) tein hydrolysates and it has been well reviewed (Moure
and low content of lysine (Johnson, Suleiman, & Lusas, et al., 2006; FitzGerald & O’Cuinn, 2006; Raksakulthai
1979). The total essential amino acid composition of ses- & Haard, 2003). But until now, studies pertaining to mix-
ame flour constitutes 37.4 g amino acid/16 g nitrogen and ture of oilseed flour protein hydrolysate has not been car-
individually these amino acid contents (His, Ile, Leu, Lys, ried out and so we have taken up this study to mix the
Met, Cys, Phe, Tyr, Thr, Try and Val) are 2.4, 3.9, 6.7, oilseed flours in specific and strategic ratio in preparing
2.6, 2.5, 1.5 4.5, 3.7, 3.4, 1.5 and 4.7, respectively & aspartic protein hydrolysate which is rich in desired amino acids
and glutamic acids constitutes 8.2 and 16.2, respectively and properties.
(Kinsella & Mohite, 1985). The aim of the research work was to prepare a protein
Defatted peanut flour contains 47–55% high quality pro- hydrolysate which contains all the essential amino acids.
tein which is deficient in both lysine and methionine but This report describes the composition, amino acid profile,
with other high essential amino acid content (Basha & Pan- molecular weight distribution and functional properties
choly, 1982; USDA-NAL, 2005). The total essential amino of a protein hydrolysate prepared from the enzymatic
acid composition of peanut flour constitutes 34.1 g amino hydrolysis of a mixed flour that consisted of soybean, ses-
acid/16 g nitrogen and individually these amino acid con- ame and peanut flours.
tents (His, Ile, Leu, Lys, Met, Cys, Phe, Tyr, Thr, Try
and Val) are 2.3, 3.2, 6.4, 3.0, 0.9, 1.0, 4.7, 3.7, 2.6, 1.0 2. Materials and methods
and 5.3, respectively and aspartic and glutamic acids con-
stitutes 11.26 and 17.18, respectively (Rhee, 1985) and 2.1. Materials
lends itself being used in many food applications (Prin-
yawiwatkul, Beuchat, & McWatters, 1993). Soybean, peanut and sesame seeds were purchased
In recent times, exhaustive compilation of the most from the local market. Food grade enzyme protease P
interesting techno-functional properties of several prod- ‘‘Amano” 6 (having not less than 60,000 u/g proteolytic
ucts (meals, concentrates and isolates) obtained from oil- activity) was purchased from M/s. Amano Pharmaceutical
seeds on various operational conditions is well reviewed Co. Ltd., Nagoya, Japan. Papain having not less than
(Moure, Sineiro, Dominguez & Parajo, 2006). However, 20,000 tyrosine units (TU)/mg was purchased from
1168 C. Radha et al. / Food Chemistry 106 (2007) 1166–1174

EnzoChem Laboratories. Sodium dodecyl sulphate (SDS), One set of mixed flour was subjected to similar treatment,
2,4,6-trinitrobenzene sulphonic acid (TNBS), L-leucine, but without the addition of enzyme (untreated flour) in
tricine, and acrylamide were obtained from Sigma Chem- order to determine the extent modification due to proteol-
ical Company, St. Louis, MO, USA. All other reagents ysis/processing conditions. The degree of hydrolysis was
used were of Analar (AR) grade from Qualigens and E. determined spectrophotometrically by the trinitrobenzene
Merck, Mumbai, India. sulphonic acid method, as described by Adler-Nissen
(1979).
2.2. Methods
2.2.4. Analysis
2.2.1. Preparation of defatted flours Moisture content was determined by using a Sartorius
Soybean, peanut and sesame seeds were cleaned, MA-30 Moisture meter (Fischer General Scientific (SEA)
graded in a grading machine to remove stones and impu- Pvt. Ltd, Singapore) at 130 °C to reach a constant weight.
rities. Water was sprayed on the seeds to raise the mois- Total nitrogen content was determined according to the
ture level by 2%. The conditioned seeds were dried in Kjeldahl procedure (AOAC, 1990).
an electrically heated roaster at 50–55 °C. The dehulling
was done by passing through a plate type mill (Model 2.2.5. Molecular weight distribution
A 453, Chandra Manufacturing Co., Chennai, India) with Molecular weight distribution in the protein hydroly-
an attached air blower. The dehulled seeds were equili- sates was determined by SDS-PAGE, according to the pro-
brated at 20% moisture and passed through a flaking cedure of Laemmli (1970), as modified by Schagger and
machine (Model J #6725, Kvarnmaskiner, Malmo, Swe- Von Jagow (1987), using 15% Tricine gels. The electropho-
den) maintaining a drum clearance of 0.3–0.5 mm to resis was run at 50 mA in 1.00 mm thick gels. The gels were
obtain flakes of 0.3mm thickness and dried to 5% mois- stained with Coomassie blue. The approximate molecular
ture level. The dried flakes were defatted by repeated weight of the hydrolysate was determined using low molec-
extractions with n-hexane, vacuum dried to remove sol- ular weight standards obtained from Sigma Chemical com-
vent and ground, passed through 60 mesh sieve. The fat pany, St. Louis, MO, USA.
content was determined by Soxhlet extraction method The molecular weight distribution of the samples was
(AOAC, 2000). The defatted flakes were dried and pow- determined by gel filtration using a Fast Protein Liquid
dered in a Quadrumat mill (Brabender, Quadrumat Chromatography system, equipped with a Superdex-75
Senior, Duisburg, Germany). The fractions obtained using HR 10/30 column from Amersham Pharmacia, Uppasala,
standard sieves which have pore size below 100 l were Sweden. A sample volume of 100 ll was loaded and the
used as defatted flours in this study. eluent used was 20 mM phosphate buffer, (pH 7.6 contain-
ing 0.2 M sodium chloride) at a flow rate of 0.5 ml/min.
2.2.2. Preparation of the raw material Elution was monitored at 280 nm.
Amino acid composition of the three defatted flours
(soybean, sesame and peanut) were determined using 2.2.6. Amino acid analysis
HPLC (Shimadzu- LC-10A, Japan). The flours were mixed Amino acid composition was determined by HPLC(Shi-
at a ratio of 1.1:1.7:0.7 for soybean, sesame and peanut, madzu Model LC-10A, Japan) using the method of
respectively, to get a mixture having a balanced amino acid Bidlingmeyer, Cohen, and Tarvin (1984). Samples were
profile. This mixed flour was used for the preparation of hydrolysed using 6 N HCl (1% w/v) phenol vapour at
protein hydrolysate. 110 °C for 24 h under vacuum. Protein hydrolysates were
treated with phenyl isothiocyanate to form phenylthiocarb-
2.2.3. Preparation of protein hydrolysate amyl derivatives of the amino acids, which were then ana-
To prepare protein hydrolysate, 10 g of mixed flour was lyzed by C18 reverse phase column.
suspended in 100 mL of distilled water and pH was
adjusted to 7.6 using 1 N sodium hydroxide (NaOH) and 2.2.7. Scanning electron microscopy
stirred for 1 h for the extraction of total protein at room Scanning electron microscopic (SEM) studies of the
temperature. The slurry was hydrolysed using 0.3–1% w/ flour and hydrolysates were carried out using Scanning
w of fungal protease at 40–45 °C for 2 h. After incubation Electron Microscope (LEO 435 VP, Cambridge, UK).
at 45 °C for 2 h, the temperature was raised to 50–55 °C Before loading the sample into the system, it was coated
and the hydrolysis was continued with the addition of with gold using Poloron SEM coating system E-5000.
0.3–1% w/w of papain for another 90 min. The enzyme Average coating time was 2–3 min. Thickness of the coat-
was inactivated by keeping the mixture in boiling water ing was 200–300 nm, which was calculated using the for-
bath for 10 min. The slurry was cooled to room tempera- mula: T = 7.5 It, where I = current in mA, t = time in
ture and the insoluble carbohydrate rich fraction was minutes, T = thickness in Å. The coated sample was loaded
removed by centrifugation at 6000g for 30 min. The clari- on the system and the image was viewed under 20 kV
fied protein hydrolysate was freeze-dried. Soy protein potential using secondary electron image. The image was
hydrolysate was also prepared under identical conditions. captured using 35 mm Ricoh Camera.
 C. Radha et al. / Food Chemistry 106 (2007) 1166–1174 1169

2.2.8. Nitrogen solubility 3.2. Chemical composition

Solubilities of the untreated sample, soy protein hydro-
lysate and mixed flour hydrolysate were determined Proximate analysis of the mixed flour hydrolysate dem-
according to the method of Chobert, Sitohy, and Whitaker onstrated a high content of crude protein (72.2%), which is
(1988). Samples were dispersed in distilled water (1% w/v) comparable with the protein content of soy protein hydro-
and the pH of the solution was adjusted to 10 with HCl or lysate (69%). The moisture contents were 11.6% and 9.4%
NaOH of high normality to limit dilution. After a 30 min for mixed flour hydrolysate and soy protein hydrolysate
equilibrium period at room temperature and readjustment respectively. The degree of hydrolysis of the hydrolysate
of the pH, if necessary, the samples were centrifuged at was 40% and for soy protein hydrolysate was 32–35%.
10,000g using a Hitachi 55P-2 Automatic Preparative
Ultracentrifuge. The solubility was determined by measur- 3.3. Sodium dodecyl sulphate-polyacrylamide gel
ing the amount of Kjeldahl nitrogen in the supernatant. electrophoresis (SDS-PAGE)
The nitrogen solubility (NS) was calculated according to
the formula: SDS-PAGE of untreated flour, soy protein hydrolysate
Nitrogen content in the supernatant and mixed flour hydrolysate are shown in Fig. 1. The
NSð%Þ ¼  100 results show that hydrolysates with >35% degree of hydro-
Total Nitrogen content in the sample
lysis are mainly composed of low molecular weight proteins
and peptides. In the absence of enzyme treatment, in
2.2.9. Foaming properties untreated flour the protein had a low mobility because of
Foam capacity (FC) and foam stability (FS) were deter- the high molecular size of protein (lane 1). The protein
mined according to the methods described by Lawhon, has a high mobility after the enzyme treatment because
Cater, and Mattil (1972) and Kinsella (1976) as simplified of the proteolytic degradation, and the presence of larger
by Booma and Prakash (1990). A 2% aqueous dispersion number of lower molecular weight protein bands in the
of the protein sample was mixed thoroughly in a blender mixed flour hydrolysate (lane 2), compared to the soy pro-
and whipped for 3 min at high speed. The contents were tein hydrolysate alone (lane 3) and this may be due to the
immediately transferred into a 250 mL graduated measur- presence of other proteins (peanut and sesame) in the
ing cylinder, along with foam. The volume of foam was mixed flour.
recorded after 30 s and FC was calculated as the increase
in volume (mL) of the protein dispersion upon mixing
and expressed as percentage increase in volume. FS was
determined by measuring the fall in volume of foam after
1 2 3 4 MW
30 min of standing.

2.2.10. Statistical analysis

Data and Statistical analysis were performed using Sci-
entific Graphic and analysis Computer software OriginPro
(version 7) supplied by Origin Lab Corporation, North- 16,950
ampton, MA, USA and data was expressed as 14,440
Mean ± standard deviation of three experiments.

3. Results and discussion 8,160

3.1. Preparation of protein hydrolysates

6,210
The double enzyme method of protein hydrolysis
yielded 40% degree of hydrolysis. For obtaining an exten-
sive hydrolysate, a mixture of exo and endo peptidases is
often used. In general, single enzymes may not provide
2,510
an extensive hydrolysate in a reasonable period of time
(Lahl & Barun, 1994). Here the oilseed proteins were
hydrolysed using fungal protease and papain sequentially.
Fungal protease is a well known non-specific endoprotease
broadly used in food research for the generation of protein Fig. 1. SDS-PAGE of untreated mixed flour (lane 1), mixed flour
hydrolysate (lane 2), soy protein hydrolysate (lane 3) and molecular
hydrolysates (Babiker, 2000). Starting with this enzyme
weight standards (lane 4 – Molecular weight ranges from 2510 to
pre-digestion of the protein is achieved increasing the num- 16,950 Da-peptide molecular weight marker (MWSDS17S) from Sigma
ber of C-terminal sites for the action of papain which is an Chemical Company, USA). The positions and molecular masses of
endoproteinase. standards are indicated on the right.
1170 C. Radha et al. / Food Chemistry 106 (2007) 1166–1174

3.4. Size exclusion chromatography totally excluded from the gel and hence a molecular weight
of larger than 70,000. Fig. 2c shows elution profile of soy
Size exclusion chromatography was used to obtain protein hydrolysate and in Fig. 2d elution profile of mixed
quantitative data on the size distribution after the enzyme flour hydrolysate is shown. During the enzyme digestion,
treatment using standard kit for the column in the range both in the case of soy protein hydrolysate and in mixed
of molecular weight of 3000 to 70,000 Da and the results flour hydrolysate, increasing proportions of peaks of lower
are shown in Fig. 2. In Fig. 2a is shown elution profile of molecular weight peptides and proteins were found (Fig. 2c
standard proteins. The standard proteins are Ovalbumin and d). As we can seen from the Fig. 2d, which represents
(43,000 Da); Myoglobin (17,600); Ribonuclease (13,700) mixed flour hydrolysate, the second and third peaks fall in
and Aprotinin (6500 Da) which are represented by peak the range of 17.6–13.7 kDa and all other peaks are 6.5 kDa
2, 3, 4 and 5, respectively and peak 1 represents undefined and lower in molecular weight. These results suggest that
aggregates. From Fig. 2b, it is clear that the untreated flour proteins and peptides of lower molecular weights are
is composed of high molecular weight proteins that eluted formed in mixed flour hydrolysate. The peak 1 represent
with the void volume. This corresponds to a material undefined aggregates and before ovalbumin standard peak
this peak emerges out and this data suggest that undefined
aggregates are eluting earlier in mixed flour hydrolysate.
These undefined aggregates are formed during enzymatic
2.0
2
protein hydrolysis process which are similar to plastein
4 type reaction but not necessarily plastein. In mixed flour
1.5 3
hydrolysate, the peak is a very heterogeneous one showing
1.0
1 both the unhydrolyzed protein probably and also the
0.5
5
hydrolysed fractions (Fig. 2d).
Since in a typical hydrolysate, the range is very large
0.0
including the unhydrolysed protein and the small peptides
0 10 20 30 40
and perhaps the unhydrolysed protein has not penetrated
2.0 the gel as there are many times polymers can form depend-
1.5
ing upon the conditions of the hydrolysis such as undefined
aggregates or plastein type reactions. This is not uncom-
1.0 mon in protein hydrolysates. Thus the most evident change
Absorbance at 280 nm

0.5 observed in the hydrolysate with respect to the untreated

protein is the reduction in the molecular weight of the pro-
0.0 tein, as a consequence of protease action. Doucet, Gauthi-
0 10 20 30 40 er, Otter, and Foegeding (2003) studied enzyme – induced
2.0 gelation of extensively hydrolyzed whey proteins by Alca-
1.5
lase and compared with plastein reaction and characterized
the interactions and their results suggest that gelation seems
1.0 to be caused by physical aggregation, mainly via hydropho-
0.5 bic interactions with hydrogen bonding and electrostatic
interactions playing a minor role. Their data on SDS-
0.0
PAGE also indicated electrophoretic gels showed aggre-
0 10 20 30 40
gates not entering the gel and suggests that some aggregates
2.0 survived heating in the presence of SDS even though it is
1.5 considered as one of the most vigorous treatments to dis-
rupt physical aggregation and concluded that the presence
1.0
of a small amount of high molecular weight material is in
0.5 accordance with other investigations that established phys-
ical aggregation as the main mechanism involved. The
0.0
results of Andrews and Alichanidis (1990) suggests that
0 10 20 30 40
the enzyme was acting as a template for hydrophobic aggre-
Elution Volume (ml)
gation of peptides. Lozano and Combes (1991) in their
Fig. 2. Size exclusion chromatogram of FPLC Elution profile of (a) study also observed small changes in size-exclusion chroma-
standard proteins (The standard proteins are Ovalbumin (43,000 Da-peak tography profiles during the plastein type reaction. In the
2); Myoglobin (17,600-peak 3); Ribonuclease (13,700-peak 3); Aprotinin course of hydrolysis of sodium caseinate with pancreatic
(6500 Da-peak 4)). The peak 1 represents undefined aggregates. (b)
proteinases aggregates were formed and the aggregates
untreated mixed flour (c) soy protein hydrolysate and (d) mixed flour
hydrolysate in 20 mM phosphate buffer (pH 7.6) containing 0.2 M sodium had properties almost identical to plasteins (Lorenzen &
chloride. Superdex 75 HR 10/30 column (optimal separation range 3000– Schlimme, 1991). Our data also suggests that formation
70,000 Da) was used. of undefined aggregates in mixed flour hydrolysate.
 C. Radha et al. / Food Chemistry 106 (2007) 1166–1174 1171

These results may be due to the splitting of peptide

bonds as a result of series of simultaneous reactions. In
the initial stage, the enzyme cleaves the soluble protein
and is adsorbed on to the insoluble protein aggregates.
The soluble proteins are hydrolysed more quickly than
the compact insoluble protein aggregates. The later gets
solubilized slowly by the action or the adsorbed enzyme
in the subsequent stages of the reaction (Paraado, Millan,
Hernandez-Pinzon, Bautista, & Machado, 1993).

3.5. Amino acid composition

The essential amino acid composition of mixed flour,

soy protein hydrolysate and mixed flour hydrolysate are
shown in Table 1, along with the essential amino acid com-
position according to the FAO requirement. It is clear that
the mixed flour hydrolysate contains all the essential amino
acids in good proportion as compared to the soy protein
hydrolysate. It is also comparable with the FAO require-
ment of amino acids. The results in Table 1 indicate that
the amino acid composition of the mixed flour hydrolysate
closely resembles that of the protein from which it is pre-
pared, except for threonine and leucine, which probably
reflects the low solubility of these amino acids. If a large
number of insoluble peptides had been generated as prod-
uct, and lost during the clarification step, the amino acid
composition of the hydrolysate would not have coincided
with that of the mixed flour. The results show that the
mixed flour hydrolysate is well balanced in its essential
amino acid contents.

3.6. Scanning electron microscopy

Scanning Electron Microscopy was used to examine the Fig. 3. Scanning electron microscopic picture of (a) untreated flour and
micro structural changes of proteins after the enzymatic (b) mixed flour hydrolysate. LEO 435 VP, Cambridge model Scanning
hydrolysis. Fig. 3a and b shows the SEM pictures of Electron Microscope was used. For untreated mixed flour (a), the scale is
untreated mixed flour and mixed flour hydrolysate, respec- (bar length represents) 10 lm; working distance 13 mm; magnification 1 K
X; HT 20 K; For mixed flour hydrolysate (b), the scale is (bar length
tively. The data shows that the protein has degraded into represents)10 lm; working distance 13 mm; magnification 500 X; HT
small fragments after the enzyme treatment. Also there is 20 K.

Table 1 a reduction in the particle size of the flour after hydrolysis

Essential Amino acid composition of mixed flour, mixed flour hydrolysate,
(Fig. 3b) compared to untreated mixed flour (Fig. 3a).
soy protein hydrolysate and FAO requirement
Average particle size of the flour is in the range of 18.06–
Amino Mixed Mixed flour Soy protein FAO*
15.24 l whereas that of the hydrolysate is 7.35–5.33 l.
acid flour (g/ hydrolysate (g/ hydrolysate requirement
100 g) 100 g) (g/100 g) (g/100 g) Since normally SEM results are empirical what we see here
is the decrease in protein aggregates.
Threonine 3.6 ± 0.16 2.1 ± 0.05 1.9 ± 0.05 3.0
Tyrosine 3.5 ± 0.02 3.0 ± 0.03 1.5 ± 0.03 2.8
Valine 4.6 ± 0.07 3.8 ± 0.06 2.3 ± 0.02 4.2 3.7. Nitrogen solubility
Methionine 1.7 ± 0.05 1.5 ± 0.02 0.6 ± 0.01 2.2
Isoleucine 3.8 ± 0.12 2.9 ± 0.06 2.1 ± 0.03 4.2 Solubility characteristics of protein are among the most
Leucine 7.0 ± 0.23 5.5 ± 0.07 3.8 ± 0.17 4.8
important functional properties since many functional per-
Phenyl 4.0 ± 0.04 3.6 ± 0.04 2.6 ± 0.06 2.8
alanine formances of proteins depend upon their capacity to go
Lysine 4.3 ± 0.17 3.5 ± 0.08 2.7 ± 0.04 4.2 into solution initially. Fig. 4 shows the nitrogen solubility
Values are mean ± SD of three experiments. curve of untreated flour, soy protein hydrolysate and mixed
*
Recommended dietary allowances, 10th edition, Edited by National flour hydrolysate over a pH range of 2–10. As shown in the
Research Council, P-57, 1989. figure, the solubility of the hydrolysates is enhanced
1172 C. Radha et al. / Food Chemistry 106 (2007) 1166–1174

100 in above two amino acids, defatted peanut flour contains

b 47–55% high quality protein with other high essential
amino acid content (Basha & Pancholy, 1982; USDA-
NAL, 2005) and lends itself being used in many food appli-
80 c
cations (Prinyawiwatkul et al., 1993). The development of a
peanut protein concentrate (PPC) from defatted peanut
Percent nitrogen solubility

flour would also provide the food industry with a new high
60 protein food ingredient for product formulation requiring
high emulsifying capacity and this could be a good source
of protein fortification for a variety of food products for
40 protein deficient consumers in developing countries. (Yu,
Ahmedna, & Goktepe, 2007). The mixed flour compensates
for the limiting amino acids of these oil seeds (Table 1). The
main advantage of enzyme hydrolysis over acid and alkali
20
hydrolysis is that the nutritive quality of the protein
a remains practically the same as that of the starting protein
without altering the amino acid profile.
0
2 4 6 8 10
3.8. Foam capacity (FC) and foam stability(FS)
pH

Fig. 4. Nitrogen solubility profile of the flour in water as a function of pH Foam capacities and foam stabilities of the mixed flour
(ranging from 2.0 to 10.0) before and after hydrolysis. The sample hydrolysate, mixed flour, soybean flour, sesame flour and
concentration was 1% (w/v). (a) untreated mixed flour, (b) mixed flour peanut flour are shown in Table 2. The foam capacity
hydrolysate, and (c) soy protein hydrolysate.
and foam stability of mixed flour hydrolysate were
122 ± 5% and 90 ± 3 mL, respectively. The results shows
considerably at pH 4–5, as compared to that of the intact a significant increase in the foaming capacity of the mixed
proteins. The solubility pattern of mixed flour hydrolysate flour hydrolysate, as compared to the respective flour and
is in agreement with that of soy protein hydrolysate. How- individual flours. The foam capacity and foam stability val-
ever, the nitrogen solubility of mixed flour hydrolysate at ues of the individual flours (soy, peanut and sesame)were
all pH values was higher than that of the untreated flour. lesser than that of the mixed flour hydrolysate. Peanut flour
The nitrogen solubilities of mixed flour at pH 2.0, 4.0, shows lesser foam capacity and foam stability (40 ± 2%
6.0, 8.0 and 10.0 were 70%, 8%, 50%, 75% and 80%, respec- and 10 ± 1 mL) compared to foam capacities and foam sta-
tively, while those of mixed flour hydrolysate were 94%, bilities of soy flour and sesame flour. Proteins in disper-
90%, 92%, 94% and 94%, respectively. sions cause a lowering of the surface tension at the water
It has been suggested that an increase in the solubility of air interface thus creating foaming capacity (Surowke &
protein hydrolysate over that of the original protein is due Fik, 1994). Foaming capacity is also determined by molec-
to the reduction of its secondary structure, and also due to ular flexibility and physico-chemical properties (hydropho-
the enzymatic release of smaller polypeptide units from the bicity, net charge and charge distribution, hydrodynamic
protein (Mahmoud, 1994; Phillips & Beuchat, 1981; Tur- properties) of proteins and to form efficiently (i.e. to pos-
geon, Gauthier, & Paquin, 1992). Both the hydrolysed sam- sess high foamability), a protein needs to adsorb rapidly
ple showed a similar pattern of nitrogen solubility values, during the transient stage of foam formation (Gonzalez-
which could be due to the fact that the specificity of the Perez, Vereijken, Van Koningsveld, Gruppen, & Voragen,
enzyme used was the same in both the cases. Nitrogen sol- 2005; Graham & Philips, 1976; Martin, Grolle, Bos, Cohen
ubilities of these hydrolysates were pH independent over Stuart, & van Vilet, 2002). These results suggest an increase
the range studied. Such behaviour is explained by the fact in surface activity, probably due to the initially greater
that smaller, more hydrophilic, and more solvated polypep-
tide units are produced as a consequence of enzymatic Table 2
Foam capacities and foam stabilities of the mixed flour protein hydro-
hydrolysis. Hence protein aggregates are no longer formed lysate, mixed flour and individual oilseed flours
even at the isoelectric pH (Cheftel, Cuq, & Lorient, 1985).
Protein Foam Capacity (FC) Foam Stability (FS)
This is an important feature, which could increase the use (%) (mL)
of the hydrolysates in many food and nonfood applications.
Mixed flour protein 122 ± 5 90 ± 3
Oilseed proteins do not exhibit similar amino acid pat- hydrolysate
terns. For example, soybeans are deficient in methionine Mixed flour 42 ± 2 34 ± 2
but are a good source of lysine, sesame is deficient in lysine Soy flour 56 ± 3 42 ± 2
but a good source of sulfur-containing amino acids, peanut Peanut flour 40 ± 2 10 ± 1
is deficient in both these amino acids (Amaya, Young, Sesame flour 52 ± 3 16 ± 2
Mixon, & Norden, 1977). Eventhough peanut is deficient Values are mean ± SD of three experiments.
 C. Radha et al. / Food Chemistry 106 (2007) 1166–1174 1173

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4. Conclusion
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