ARTICLE IN PRESS
LWT 40 (2007) 1520–1526
www.elsevier.com/locate/lwt
Some physicochemical and functional properties of alfalfa
soluble leaf proteins
B.P. Lamsala,, R.G. Koegelb, S. Gunasekaranc
a
Grain Science and Industry Department, Kansas State University, Manhattan, KS 66506, USA
b
US Dairy Forage Research Center, 1925 Linden Dr. West, Madison, WI 53706, USA
c
Biological Systems Engineering Department, UW-Madison, 460 Henry Mall, Madison, WI 53706, USA
Received 7 August 2006; received in revised form 12 November 2006; accepted 16 November 2006
Abstract
Important physicochemical and functional properties of soluble leaf proteins (SLPs) from alfalfa herbage are presented. Subunits molecular
weight (MW) distribution, denaturation temperature, and functional properties like, emulsification, foaming, and solubility are discussed. SLP
concentrates were prepared by acid precipitation, and ultrafiltration of clarified alfalfa juice. The MW of major soluble protein component
ribulose 1,5, bisphosphate carboxylase/oxygenase was estimated to be around 490 kDa. Denaturation temperature of soluble proteins was
observed to be around 70–75 1C. Most of the functional properties were affected by concentrate preparation. Acid-precipitated SLP
concentrate showed lowest emulsifying properties and nitrogen solubility. Heat stability of emulsions was good. Foam overrun for SLP
concentrate depended on pH and was stable around protein’s isoelectric point. Stress relaxation tests on 7/100 g SLP gels indicated that they
were softer gels and relaxed faster compared to 13/100 g WPI gels. SLP preparations showed encouraging functional properties.
Published by Elsevier Ltd. on behalf of Swiss Society of Food Science and Technology.
Keywords: Soluble leaf proteins; Functional properties; Stress relaxation
1. Introduction
Several novel sources, like leaf, have been suggested to
meet the ever-increasing world demand for proteins. They
are the most abundant and renewable proteins in nature.
Leaf proteins contain one of the purest and highly nutritive
components: rubisco (ribulose 1, 5-bisphosphate carboxylase). Rubisco, also called Fraction-I protein, accounts for
up to 30–70/100 g of soluble leaf proteins (SLP) (Douillard
& de Mathan, 1994) and plays a part in photosynthesis. It
is believed that rubisco could be the main product when
‘cracking’ leaves into protein, fiber, vitamins, pigments and
other products. SLP concentrates have commonly been
prepared by differential heating (55–60 1C) of expressed
leaf juice followed by centrifugation. The clear brown
supernatant containing soluble proteins could be heatcoagulated to yield a light-colored bland protein concentrate, more than 90/100 g protein on dry basis. The range of
Corresponding author. Tel.: +1 785 532 2875; fax: +1 785 532 7010.
E-mail address: [email protected] (B.P. Lamsal).
applications in the food industry for such heat-coagulated
concentrate is limited by its low solubility. Precipitation of
protein through pH reduction (pH 3.5) at low temperature
(2 1C) was investigated as an alternative to heat-coagulation (Miller, de Fremery, & Kohler, 1975). Raising the
pH to 7.0 redissolved most of the protein precipitated at
pH 3.5.
The proteins functional properties and applications are
affected by their purification and/or concentration methods. Food properties that may be influenced by proteins
include water holding capacity, emulsification, foaming,
viscosity, gelation, and texture (Giese, 1994). Protein
influences these properties via two characteristics: water
solubility and hydrodynamic properties. Solubility is the
result of surface-active properties of a protein, which
affects foaming, emulsification, and water and fat binding
properties among others. Hydrodynamic properties influence viscosity, gelation, thickening, and texturization.
Water solubility of protein is the result of protein amino
acid composition and distribution, molecular flexibility,
and shape and size, whereas the hydrodynamic properties
0023-6438/$30.00 Published by Elsevier Ltd. on behalf of Swiss Society of Food Science and Technology.
doi:10.1016/j.lwt.2006.11.010
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B.P. Lamsal et al. / LWT 40 (2007) 1520–1526
are due to shape and size of the protein (Hall, 1996), thus,
affecting food systems during preparation, processing,
storage and consumption.
The amino acid composition and structure of SLP,
notably rubisco, has been investigated. Rubisco is a
globular protein and in higher plants having molecular
weight (MW) close to 550 kDa and a general structure of
L8S8-eight large subunits with MWs around 55 kDa and
eight small subunits with MWs close to 12.5 kDa. Four
dimers of the large subunit constitute a core of eight large
subunits with a four-fold axis of symmetry and a barrel-like
general shape (Barbeau & Kinsella, 1988; Douillard & de
Mathan, 1994). The denaturation temperature of rubisco is
76.2 1C and its heat of denaturation enthalpy is 6.3 kcal/kg
(Tomimatsu, 1980). Macromolecular properties and subunit interactions (Hood, Cheng, Koch, & Brunner, 1981;
Noguchi, Maekawa, Fujimoto, Satake, & Sakakibara,
1978; Tomimatsu, 1980) of alfalfa leaf extract and purified
rubisco have been reported.
The functional properties of SLP, e.g., solubility, water
and fat absorption, gelation, emulsification, foaming and
whipping are comparable to those of standard proteins
(Barbeau, 1990; Knuckles & Kohler, 1982; Sheen, 1991;
Wang & Kinsella, 1976). Betschart (1974) reported that the
solubility of acid-precipitated leaf protein was markedly
influenced by pH. Lower solubility of heat-precipitated
(80 1C) proteins was attributed to irreversible effects on the
physical state of the protein upon heating, e.g., denaturation. Miller et al. (1975) reported that the solubility of
protein decreased rapidly, from about 90/100 g to 15/100 g,
as the precipitation and wash temperature increased from 2
to 25 1C. Apart from solubility, other functional properties
of alfalfa leaf proteins (Wang & Kinsella, 1976) and that of
soluble protein fraction (Knuckles & Kohler, 1982) have
been studied. Knuckles and Kohler (1982) reported that
the emulsion activity and emulsion stability (ES) of alfalfa
protein concentrate and soy protein isolate were similar.
Emulsion capacity of the SLP concentrate at 0.5, 1, and
2/100 g protein was greater than 700, 521 and 246 mL of oil/g.
Such decrease in emulsifying capacity with concentration was
also observed by Wang and Kinsella (1976) for acidprecipitated alfalfa proteins. In all these studies, the SLP
was prepared by acid precipitation at its isoelectric point.
We believe that if the proteins retained their native
structure during concentration their physicochemical and
functional properties will be different. Accordingly, we
prepared SLP by different membrane concentration
method (Lamsal, Koegel, & Gunasekaran, 2005). Therefore, the objectives of this study were to investigate
important physicochemical properties of SLP from alfalfa
herbage extract and to evaluate some functional properties
of SLP concentrates prepared by different methods.
2. Materials and methods
Clarified alfalfa juice (CAJ) preparation: CAJ was
prepared as described in Lamsal, Koegel, and Boettcher
1521
(2003). Briefly, 4–5-week-old alfalfa herbage was fieldharvested and macerated in a modified Gehl Corporation,
West Bend, WI hammer mill, 660 mm dia 510 mm wide
(Koegel, Straub, & Boettcher, 2000) and then pressed in a
multi-cone press (Chase, 2000). CAJ was then prepared by
removing green proteins from pressed alfalfa juice after
heating (55 1C) and centrifuging (3066g, Super-D-Canter
P660; Sharples, Warminster, PA). Physicochemical properties of SLP were determined with CAJ as described below.
SLP physicochemical properties: SLP properties evaluated were protein MW distribution with sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE),
and protein denaturation profile with differential scanning
calorimetry (DSC). SDS-PAGE was run with a 10/100 g
polyacrylamide resolving gel and 4/100 g stacking gel
following Tong, Barbano, and Jordan (1989) with some
minor modifications. Low-range standard MW markers,
ranging from 107 to 20.2 kDa, were used (Bio-Rad
laboratories, Hercules, CA). DSC profiling was carried
out by placing 10–15 mg CAJ at different concentrations in
hermetically sealed aluminum cups (TA Instruments,
model no. 991100.901, New Castle, DE). The sample was
first heated to 90 1C at 5 1C/min, and then cooled back to
25 1C. It was reheated to 90 1C at the same rate. This
second-heating profile served as a baseline reference for the
sample and minor differences between the first and second
heating were attributed to protein denaturation.
2.1. SLP functional properties
SLP concentrates preparation: Acid-precipitated and
ultrafiltered SLP concentrates were prepared from CAJ
as described in Lamsal et al. (2005). Briefly, CAJ, at pH
6.0, was adjusted to pH 3.5 with addition of 1 mol/L HCl
to precipitate SLP, centrifuged at 1200g, 4 1C for 10 min
(Beckman, model J2-21, Palo Alto, CA) and washed three
times with pH-adjusted distilled water. One-half of the
washed precipitate at pH 3.5 was freeze-dried and denoted
AP. Other half of the precipitate was redissolved at pH 7.0
with the addition of 0.1 mol/L NaOH, centrifuged at
37,000g for 20 min and freeze-dried. The resolublilized
concentrate was denoted RP. Part of CAJ prepared was
concentrated in a custom-made rotary ultrafiltration device
(Lamsal & Koegel, 2005) using 10 kDa MW cut-off
(MWCO) membrane (PBGC membrane, Millipore Co.,
Bedford, MA). The filter was operated at a feed pressure of
242 kPa, membrane clearance of 5 mm and rotor speed of
16.7 rev/s. The retentate was then freeze-dried (Freezemobile 35ES, Virtis Inc., Gardiner, NY) and named UFR.
Protein content of the sample was determined as described
later. Dry basis true protein (TP) content of SLP
concentrates were 72/100 g, for AP, 63/100 g for RP, and
35/100 g for UFR.
Nitrogen solubility: SLP solubility profile over the pH
range of 2–9 was evaluated as per Knuckles and Kohler
(1982). About 400 mg of AP, RP, or UFR was added to
30 mL of distilled water. After the pH of solution was
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B.P. Lamsal et al. / LWT 40 (2007) 1520–1526
adjusted to the desired value with 1 mol/L NaOH or 1 mol/L
HCl, it was shaken for 30 min at room temperature
(model no. 3520, Labline Instruments Inc., Melrose Park,
IL). Samples were then centrifuged at 37,000g for 20 min at
4 1C (Beckman, model J2-21, Palo Alto, CA). Total
nitrogen (TN) was determined on decant samples as
described in later section and expressed as the percent
nitrogen solubilized. The averages of duplicate samples are
reported.
Emulsification properties: Emulsification activity (EA),
ES, and emulsification capacity (EC) of SLP were tested
following Wang and Kinsella (1976) and Knuckles and
Kohler (1982) with reduced sample size to be within
blender capacity. The blending/mixing for all the emulsion
works was done with a laboratory blender (51BL31,
Waring Laboratory, Torrington, CT) with different bowls.
To evaluate EA and ES, 2.1 g of SLP concentrate was
added to 30 mL of distilled water in a 250-mL blender bowl
and dispersed at 300 rev/s for 30 s. About 30 mL of
vegetable cooking oil was then added and blended at
367 rev/s for 1 min. The emulsion was then poured into
four 15-mL centrifuge tubes. For EA, two of the tubes were
centrifuged at 1300g for 5 min (Beckman, model J2-21,
Palo Alto, CA) and total height and emulsion layer height
recorded. For ES, the remaining two tubes were first heated
in an 80 1C water bath for 30 min before centrifuging at
1300g for 5 min. The total height and emulsion layer height
were recorded. EA and ES were calculated as the ratio of
emulsion layer height to the total height. EC was evaluated
with 0.5 g of SLP concentrate by adding 25 mL of distilled
water in the 250-mL blender bowl. It was then dispersed
for 30 s at 300 rev/s. About 25 mL of cooking oil was then
added and blended at 367 rev/s for 1 min. Cooking oil was
also filled in a glass buret. As blending continued, oil was
added directly into the blender approximately at the rate of
1 mL/s until the phase inversion occurred. The phase
inversion was characterized by a sudden decrease in the
current load on the blender monitored with a clip-on
ammeter (RS-3, Amprobe Instrument, Lynbrook, NY).
The EC of the protein was expressed as the total volume of
oil emulsified per gram of the protein in the sample.
Whippability and foam stability: Concentrate RP was
tested per Knuckles and Kohler (1982) at 2/100 g protein
concentration. Effect of pH on foam stability was studied
at pH values of 4, 7, and 10. About 60 mL solution at
desired pH was whipped for 2 min in a Waring blender at
300 rev/s. The foam was immediately poured into a 1000mL graduated cylinder and the total volume and drain
volume were noted at 0, 15, 30, 60, 80 and 120 min. The
foaming capacity (or % overrun) was calculated as
Viscoelastic properties of SLP gels: Heat-set SLP gels
were prepared as described in Lamsal et al. (2005). SLP
solution was prepared by vacuum-evaporating CAJ.
Standing gels were prepared in stainless steel molds with
7/100 g SLP and 13/100 g whey protein isolate solutions
(WPI, 90/100 g protein, Davisco Foods International, Eden
Prairie, MN) by heating at 90 1C for 1 h. The WPI gel was
used for comparison. Gels were cooled overnight at 4 1C
and warmed to room temperature for testing. Stress
relaxation test in compression was carried out with gels
10 mm dia 6 mm long under constant applied strains
using a universal testing machine (Synergie 200, MTS
Corporation, Cary, NC). The applied strains of 0.05, 0.08,
0.12, and 0.2 mm/mm under crosshead speed of 0.5 mm/s
were maintained up to 5 min and the stress decay with time
was recorded. Stress-time data were analyzed using
linearization technique (Peleg, 1979; Tang, Tung, & Zeng,
1998). First, stress value at any time, s(t), was normalized
with respect to the instantaneous stress at time zero, s(0).
The ratio of time and normalized stress was fitted against
time to produce a linear plot as per Eq. (1), slope of which
is the constant k2 and the intercept k1. Application of this
relation assumes linearity of the data. Second, the
equilibrium stress in the samples at infinite time, se, was
estimated as per Eq. (2), and the initial decay rate of stress
ratio was obtained using Eq. (3):
% volume increase
Physicochemical properties of CAJ: The dry matter
content of CAJ ranged from 2/100 to 13/100 g and the
average was 7.2/100 g, with standard deviation (SD) of
0.02. The average TP content of CAJ was 10.2/100 g dry
basis with SD of 0.05. The lipid content of CAJ determined
as per Bligh and Dyer (1959), averaged at 4.2/100 g dry
¼ 100 ðvolume after whipping 60 mLÞ=60 mL.
Foam stability was reported as foam volume standing
after a holding time. The averages of duplicate measurements were reported.
sð0Þt
¼ k1 þ k2 t,
sð0Þ sðtÞ
1
se ¼ sð0Þ 1 ,
k2
1 dsðtÞ
1
¼ .
sð0Þ dt t¼0
k1
(1)
(2)
(3)
Protein content and moisture content determination: The
protein content of CAJ was determined with a combustiontype TN analyzer (FP-2000, LECO Corporation, St. Joseph,
MI). TN and non-protein nitrogen (NPN) as protein soluble
in 0.2 g/mL trichloroacetic acid (TCA) were determined and
TP content was obtained by multiplying the difference by
6.25 (Eakin & Singh, 1978). Dry matter or total solid in
sample was determined as per Lamsal et al. (2003) by heating
in a forced-draft oven (Isotemp, model 825F, Fisher
Scientific, Dubuque, IA).
Statistical analysis: Means were compared with GLM
procedure in SAS statistical software (SAS Institute, Inc.,
Cary, NC) at po0.05, where appropriate.
3. Results and discussion
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107 kDa
90 kDa
0.015
0.010
0.005
0.000
-0.005
-0.010
-0.015
-0.020
-0.025
-0.030
Exo down
40
50
60
70
80
90
100
Temperature, °C
Fig. 2. Differential scanning calorimeter profiles for soluble leaf proteins
as 0.5/100 g (B), 0.8/100 g (+), and 1.8/100 g ( ) protein clarified alfalfa
juice.
conformational changes that produce endothermic peaks.
Heating weakens the forces that hold protein tertiary and
quaternary structures in place, i.e., hydrogen bonds,
hydrophobic interactions, van der Waals forces and
exposes buried hydrophobic sites. The exposed hydrophobic patches come together and form aggregates. DSC
analysis of protein measures the net changes between
endothermic denaturation process and the exothermic
process of aggregation (Farkas & Mohacsi-Farkas, 1996).
Tomimatsu (1980) obtained single endothermic DSC peak
at 76.2 1C for purified alfalfa rubisco at 0.11/100 g
concentration. Our CAJ was a mixture of proteins along
with other solubles; thus we observed a number of peaks in
DSC profile. Two peaks are distinct for 0.5/100 g (around
68 and 77 1C), and 0.8/100 g (around 70 and 75 1C) protein
CAJ, whereas three peaks (at around 58, 65, and 75 1C) are
prominent for 1.8/100 g protein CAJ. The leftward shift in
peak temperature is consistent with SLP protein gelling
behavior (Lamsal et al., 2005), which seemed to contradict
Farkas and Mohacsi-Farkas (1996). They reported that soy
protein isolates became stable towards heat when the water
content was lower.
3.1. SLP functional properties
49.6 kDa
35 kDa
27.8 kDa
20.2 kDa
Lanes
0.020
Heat flow, mW
basis, with SD of 0.02. Such wide variations in protein and
dry matter reflect on the climatic conditions at the time of
alfalfa harvest (Telek & Martin, 1983). The CAJ dry matter
and protein values were determined for preparations from
alfalfa harvest of summer and fall of 2000–2003. Rain,
morning dew, late fall harvest, etc. influenced these values
and are presented here to show the variation possible.
MW distribution: The SLP from alfalfa herbage juice
contains a mixture of proteins, MW of which ranges
approximately from 6 kDa to about 550 kDa (Barbeau &
Kinsella, 1988). Fig. 1 shows an SDS-PAGE for nonclarified and clarified alfalfa juice. Nonclarified juice shows
protein bands well above 107 kDa and below 20.2 kDa
range and in between. CAJ had prominent protein bands at
around 49, and 30 kDa and well below 20 kDa range,
around 10–12 kDa range. The bands above 107 kDa, likely
the green protein fraction of the whole juice were missing
from CAJ. The bands at 49 kDa and around 12 kDa for
CAJ are SDS-dissociated subunits of rubisco (Barbeau &
Kinsella, 1988; Hood et al., 1981; Tomimatsu, 1980). Hood
et al. (1981) noted that the native protein structure of
alfalfa rubisco dissociates completely during SDS-PAGE
and large and small subunits MW were 52 and 12.5 kDa,
respectively. Other light protein bands seen between 35 and
28 kDa could either be the Fraction-II proteins or
proteolytic artifact. As alfalfa rubisco is composed of eight
large and eight small subunits (Barbeau & Kinsella, 1988),
our alfalfa rubisco MW could be estimated at around
488 kD, which again is within the published range
(Tomimatsu, 1980).
Denaturation temperature: Fig. 2 shows the DSC profiles
for CAJ at 0.5, 0.8, and 1.8/100 g protein content. The
highest peaks for 0.5, 0.8, and 1.8/100 g protein CAJ
concentrations were at 70, 68 and 65 1C, respectively,
showing the effect of protein concentration. But CAJ
protein denaturation had started earlier—66 1C for
0.5/100g, 62 1C for 0.8/100 g, and 50 1C for 1.8/100 g
protein CAJ samples. DSC profiling gives insight into the
response of a protein to the applied heat causing
1523
1
2
3
Fig. 1. Sodium dodecylsulfate polyacrylamide gel electrophoresis for
uncentrifuged (lane 2) and clarified alfalfa juice (lane 3) along with Biorad
standard protein markers (lane 1). Sample loading, 4 mL.
Nitrogen solubility: Fig. 3 shows nitrogen solubility
profiles in water for SLP concentrates. For a concentrate
UFR, the protein solubility was the least at pH 4.5, the
isoelectric point of the protein. There was no appreciable
difference in solubility for AP and RP below pH 4.5, but it
increased in the alkaline range. Higher protein solubilities
on either side of the isoelectric point have been reported for
leaf proteins of various preparations (Betschart, 1974;
Knuckles & Kohler, 1982; Ng, 1975; Wang & Kinsella,
1976). Solubility profiles were markedly affected by SLP
concentrate preparation method. Acid precipitated concentrate, AP, was least soluble over the pH range of 2–9
among the concentrates tested. RP, and UFR solubilities
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1524
Table 1
Emulsifying properties of SLP concentrate and egg white protein (EWP)
samples
120
Protein resolubilized, g/100g
100
SLP
concentrates
80
Emulsifying
Emulsifying
capacity (mL oil/ activity (%)
g protein DM)
Mean7SE
Mean7SE
Mean7SE
474714a
24277b
29274c
15972d
9271ab
7572c
8872b
9671a
6972bc
6371c
8172a
7472b
Emulsion
stability (%)
40
UFR
AP
RP
EWP
20
SE, standard error of mean; n ¼ 3. Means sharing same superscript letter
are not significantly different at po0:05.
60
0
210
2
3
4
5
6
7
8
9
180
Fig. 3. Protein solubility profiles for soluble leaf concentrates over the pH
range of 2.0–9.0. Symbols, diamonds, acid precipitated concentrates (AP);
squares, redissolved acid precipitated concentrates (RP); and triangles,
ultrafiltered concentrate (UFR). Concentrate AP was precipitated at pH
3.5; RP was redissolved at pH 7.0, and UFR was membrane concentrated
at pH 7.0, prior to freeze drying.
were higher, 97/100 g, and 90/100 g, respectively, at pH 9.0.
Both RP and UFR were at pH 7 prior to freeze-drying,
whereas AP was at pH 3.5 along with associated electric
charges. These charges may have played a role in solubility
of SLP concentrates at various pHs. SLP concentrates were
more soluble in alkaline range than in acidic range. Partial
hydrolysis could account for some of the enhanced
solubility at acid and alkaline pH values (Betschart,
1974). However, greater than 90/100 g% of the solubilized
protein at the pH range employed was TP, i.e., insoluble in
0.2 g/mL TCA, implying that the proteins were intact and
not smaller hydrolyzed peptides.
Emulsification properties: The EC, EA, and ES at pH 7
for SLP concentrates UFR, AP, RP, and egg white protein
(EWP) concentrate are shown in Table 1. AP showed
lowest EA, ES, and EC among SLP samples. For the
concentrates tested, EAs were consistently lower than ES,
which indicated strengthening and stabilizing of emulsion
due to heating at 80 1C prior to centrifugation. Wang and
Kinsella (1976) also reported stabilizing of unclarified
alfalfa leaf protein emulsion by heating. UFR concentrate
showed the highest EC, possibly owing to the fact that
most of the proteins would have been in native form,
whereas other two concentrates had pH-modified proteins.
It was interesting to observe that EWP had lower EC than
all SLP samples, although it had higher EA and ES. EC of
AP and UFR concentrates progressively decreased with
increase in protein concentration in dispersion (data not
shown). Decrease in EC with increase in protein concentration has been reported for alfalfa leaf protein concentrates, meat proteins, and soy protein isolates (Knuckles &
Kohler, 1982; Wang & Kinsella, 1976).
Foam volume, mL
pH
150
120
90
60
30
0
0
0.5
1
1.5
2
Elapsed time, h
Fig. 4. Standing foam volumes at different times for redissolved acid
precipitated concentrate (RP) at different pH and 2/100 g protein
concentration. Symbols, diamonds pH 4.5; squares pH 7, and triangles
pH 10. RP was prepared by precipitating alfalfa soluble leaf proteins at
pH 3.5 and redissolving at pH 7.0 before freeze drying.
Whippability and foam stability: Mean7standard errors
of foam overruns for 2/100 g SLP were 15571, 21871, and
23873% at pH 4.5, 7, and 10, respectively. These overruns
are lower than reported by (Knuckles & Kohler, 1982) for
spray-dried alfalfa protein concentrate at similar concentration. Foam stabilities with pH are shown in Fig. 4.
Although the initial volumes were higher for high-pH
foams, they were not stable compared to low-pH foams.
Foam volume practically reached zero by 1.5 h for pH 7
and pH 10 foams, whereas pH 4.5 foam was stable at
96 mL by 2 h. This is consistent with reported foaming
behavior of alfalfa SLP concentrates (Knuckles & Kohler,
1982), with foams most stable at pH near 4.5. The greater
stability of foams near protein’s isoelectric point is
generally attributed to zero net charge on proteins in the
interface, among other things, thus minimal electrostatic
repulsion (Wilde & Clark, 1996; Zayas, 1997). Decreased
electrostatic repulsion in the foam interface at protein
isoelectric point also causes packing of compact protein
molecules into the interface to a greater extent, thus,
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B.P. Lamsal et al. / LWT 40 (2007) 1520–1526
1525
Table 2
Stress relaxation and log-linear parameters for soluble leaf proteins (SLP), and whey protein isolate (WPI) gels at 0.5 mm/s crosshead speed
s1, kPa
s35 min , kPa
Log-linear model parameters: Eqs. (1)–(3)
Mean7SE2
Mean7SE
k2
Mean7SE
7/100 g SLP
0.05
0.08
0.12
0.20
4.670.1d
6.570.3c
8.370.2b
12.670.5a
0.5570.02d
0.7470.03c
0.9370.03b
1.1670.06a
1.1170a
1.1170a
1.1170a
1.0970b
13/100 g WPI, pH 7
0.05
0.08
0.12
0.20
3.070.1d
5.070.4c
7.770.1b
11.870.5a
1.870.1d
3.170.2c
4.770.1b
7.370.3a
Applied strain mm/mm
1/k1, s1
Mean7SE
0.1170.01c
0.1470.01b
0.1370.0b
0.1670.01a
2.3670.08a
2.4070.05a
2.4070.05a
2.4270.02a
0.01070a
0.01170b
0.01370a
0.01470a
Mean ratio s5 min/se
s4e , kPa Mean7SE
0.4770.01d
0.6570.02c
0.8170.03b
0.9970.05a
1.17ab
1.14b
1.14ab
1.17a
1.770.1d
2.970.2c
4.570.1b
6.970.3a
1.06a
1.06ab
1.05c
1.05bc
1—instantaneous stress at the start of loading; 2—standard error, n ¼ 324; 3—stress at the end of 5 min run; 4—theoretical equilibrium stress at t ¼ N.
Means sharing same superscript letter are not significantly different at po0:05.
forming stronger and stable films (German & Phillips,
1994). The thickness and rigidity of the protein films
adsorbed at the air-water interface was also attributed to
electrostatic intermolecular attractions at the isoelectric
point of proteins (Zayas, 1997). Electrostatic repulsion of
the protein surface film was not important in the isoelectric
point region (Zayas, 1997).
Viscoelastic properties of SLP gels: Table 2 summarizes
average initial stresses, equilibrium stresses and log-linear
parameters for 7/100 g SLP, and 13/100 g WPI gels at
different strain levels. Coefficient of determination (R2) for
stress–time data linearization as per Eq. (1) was greater
than 0.99 verifying independence of coefficients k1 and k2
to test duration (Peleg & Pollak, 1982). Also, the ratios of
actual equilibrium stresses at 5 min, s5 min to theoretical
equilibrium stresses, se (Eq. (2)) were p1.2 for SLP and
1.06 for WPI indicating that observed equilibrium stresses
reached closer to the theoretical ones. This lends credence
to 5-min test runs for estimating the short-term viscoelastic
properties of these gel samples. Initial stresses decay to an
equilibrium value increased slightly with applied strain for
SLP gels, but increased considerably for WPI gels. This is
also corroborated by almost constant rate of stress decay
(1/k1) for a given WPI sample, even though the initial
stresses increased with applied strains. This contrasts
decaying behavior of gellan gels reported by Tang et al.
(1998), for which equilibrium stresses decreased with
imposed strains. SLP gels relaxed 10-fold faster than WPI
gels as indicated by 1/k1 values. Thus, with comparable
initial stresses, WPI gels retained about 60% of initial
stress at all strain levels, whereas SLP gels retained only
about 9–12%. Dominant liquid-like behavior in SLP gel
was evident by its faster relaxation rates, as in a viscoelastic
entity Newtonian fluid relaxes instantly and Hookean body
never relaxes. This can also be inferred from the average
slope of the linearized plots (k2) for SLP and WPI gels,
which were about 1.1 and 2.4. For a short-term relaxation
experiment, it is fair to assume that for k241, the material
can be treated as solid on pertinent time scale (Peleg &
Pollak, 1982), for when k2 ¼ 1, se is zero (Eq. (2))
indicating liquid-like behavior.
Time-dependent gel characteristics are attributed to
release of hydraulic pressure induced within the gel during
loading (Peleg & Pollak, 1982; Tang et al., 1998). Rapid
compression of the polymer/water system during sudden
loading builds up hydraulic pressure, which causes polymer
network to fail if exceeded certain limit. At small strains
and compression rates, polymer network deforms without
breaking, but an internal hydraulic pressure develops due
to the resistance of the network to the liquid seepage.
Differential pressure between gel surface and the interior
would force the water held in gel matrix towards free
surfaces. This leads to release of some hydraulic pressure
and contribute to relaxation. The rate of stress relaxation
depends on the magnitude of capillary forces that hold the
water, the resistance to water seepage and the distance for
the water to flow (Tang et al., 1998). The first two factors
depend on gel network pore sizes and the third on the gel
dimensions. Since the gel dimensions were reasonably
constant in our tests, the pore size variation is chiefly
responsible for different relaxation rates in SLP and WPI
gels. While WPI produces fine-stranded clear gels at pH
away from isoelectric point (Turgeon & Beaulieu, 2001),
SLP gels were not clear that probably had bigger pores
resulting in different stress relaxation rates.
4. Summary and conclusion
Some physicochemical and functional properties of SLP
from alfalfa were evaluated. The MW of rubisco was
estimated to be around 490 kDa. Denaturation temperature of SLP in CAJ was observed to be around 70–75 1C
in DSC experiments. TP in CAJ was lost due to
time–temperature effect, most likely, through proteolytic
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B.P. Lamsal et al. / LWT 40 (2007) 1520–1526
activities. Increase in NPN value in the samples supported
these observations. Most of the functional properties were
affected by concentrate preparation. Acid-precipitated SLP
concentrate showed lowest emulsifying properties and
nitrogen solubility. Heat stability of emulsions was good.
Foam overrun for SLP concentrate depended on pH and
was stable around protein’s isoelectric point. Stress
relaxation tests on 7/100 g SLP gels indicated that they
were softer gels and relaxed faster compared to 13/100 g
WPI gels.
References
Barbeau, W. E. (1990). Functional properties of leaf proteins: Criteria
required in food application. Italian Journal of Food Science, 2(4),
213–225.
Barbeau, W. E., & Kinsella, J. E. (1988). Ribulose bisphosphate
carboxylase/oxygenase (rubisco) from green leaves—Potentials as a
food protein. Food Reviews International, 4(1), 93–127.
Betschart, A. A. (1974). Nitrogen solubility of alfalfa protein concentrate
as influenced by various factors. Journal of Food Science, 39(6),
1110–1115.
Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction
and purification. Canadian Journal of Biochemistry and Physiology,
37(8), 910–917.
Chase, K. B. (2000). Design and evaluation of a continuous press with a
multiple conical roll chamber. Masters thesis. Madison, WI: University
of Wisconsin-Madison.
Douillard, R., & de Mathan, O. (1994). Leaf proteins for food use:
Potential of RUBISCO. In B. J. Hudson (Ed.), New and developing
sources of food proteins. London: Chapman & Hall.
Eakin, D. E., & Singh, R. P. (1978). Alfalfa protein fractionation by
ultrafiltration. Journal of Food Science, 43(2), 544–552.
Farkas, J., & Mohacsi-Farkas, C. (1996). Application of differential
scanning calorimetry in food research and food quality assurance.
Journal of Thermal Analysis, 47, 1787–1803.
German, J. B., & Phillips, L. (1994). Protein interactions in foams:
Protein–gas phase interaction. In N. S. Hettiarachchi, & G. R. Ziegler
(Eds.), Protein functionality in food systems (pp. 181–208). New York:
Marcel Dekker Inc.
Giese, J. (1994). Proteins as ingredients: Types, functions, applications.
Food-Technology, 48(10), 49–54 56, 58, 60.
Hall, G. M. (1996). Basic concepts. In G. M. Hall (Ed.), Methods of testing
protein functionality (pp. 1–10). London, UK: Blackie Academic &
Professionals.
Hood, L. L., Cheng, S. G., Koch, U., & Brunner, J. R. (1981). Alfalfa
proteins: Isolation and partial characterization of the major component—Fraction I protein. Journal of Food Science, 46(6), 1843–1850.
Knuckles, B. E., & Kohler, G. O. (1982). Functional properties of edible
protein concentrates from alfalfa. Journal of Agricultural and Food
Chemistry, 30(4), 748–752.
Koegel, R. G., Straub, R. J., & Boettcher, M. E. (2000). In-field wet
fractionation of transgenic leguminous herbage. ASAE Paper No.
001040. Presented at 2000 ASAE Annual International Meeting, July
9–12, Milwaukee, WI.
Lamsal, B. P., & Koegel, R. G. (2005). Evaluation of a dynamic
ultrafiltration device in concentrating soluble alfalfa leaf proteins.
Transactions of the ASAE, 48(2), 691–701.
Lamsal, B. P., Koegel, R. G., & Boettcher, M. E. (2003). Separation of
protein fractions in alfalfa juice: Effects of some pre-treatment
methods. Transactions of the ASAE, 46(3), 715–720.
Lamsal, B. P., Koegel, R. G., & Gunasekaran, S. (2005). Gelation of
alfalfa soluble leaf proteins. Transactions of the ASAE, 48(6),
2229–2235.
Miller, R. E., de Fremery, D., & Kohler, G. O. (1975). Soluble protein
concentrate from alfalfa by low-tmperature acid precipitation. Journal
of Agricultural and Food Chemistry, 23(6), 1177–1179.
Ng, K. K. (1975). Recovery of proteins from plant juices of alfalfa, bromegrass, pea vine, and sorghum and studies on freeze-, roller-, and spraydried protein concentrates obtained from alfalfa juice. Masters thesis.
Madison, WI: University of Wisconsin-Madison.
Noguchi, H., Maekawa, T., Fujimoto, S., Satake, I., & Sakakibara, M.
(1978). Physico-chemical studies on Fraction 1 protein from alfalfa.
Agricultural and Biological Chemistry, 42(8), 1553–1558.
Peleg, M. (1979). Characterization of the stress relaxation curves of solid
foods. Journal of Food Science, 44(1), 277–281.
Peleg, M., & Pollak, N. (1982). The problem of equilibrium conditions in
stress relaxation analyses of solid foods. Journal of Texture Studies,
13(1), 1–11.
Sheen, S. J. (1991). Comparison of chemical and functional properties of
soluble leaf proteins from four plant species. Journal of Agricultural
and food Chemistry, 39(4), 681–685.
Tang, J., Tung, M. A., & Zeng, Y. (1998). Characterization of gellan
gels using stress relaxation. Journal of Food Engineering, 38(3),
279–295.
Telek, L., & Martin, F. W. (1983). Tropical plants for leaf protein
concentrates. In L. Telek, & H. D. Graham (Eds.), Leaf protein
concentrates (pp. 81–116). Westport, CT: AVI Publishing Company.
Tomimatsu, Y. (1980). Macromolecular properties and subunit interactions of ribulose 1,5-bisphosphate carboxylasae/oxygenase from
alfalfa. Biochimica et Biophysica Acta, 622, 85–93.
Tong, P. S., Barbano, D. M., & Jordan, W. K. (1989). Characterization of
proteinaceous membrane foulants from whey ultrafiltration. Journal of
Dairy Science, 72(6), 1435–1442.
Turgeon, S. L., & Beaulieu, M. (2001). Improvement and modification of
whey protein gel texture using polysaccharides. Food Hydrocolloids,
15(4–6), 583–591.
Wang, J. C., & Kinsella, J. E. (1976). Functional properties of
novel protein: Alfalfa leaf proteins. Journal of Food Science, 41(2),
286–292.
Wilde, P. J., & Clark, D. C. (1996). Foam formation and stability. In G.
M. Hall (Ed.), Methods of testing protein functionality (pp. 110–152).
Blackie Academic and Professional.
Zayas, J. F. (1997). Foaming properties of proteins. In Functionality of
proteins in food. Heidelberg, Berlin: Springer (pp. 260–304).