CHAPTER III - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/24969/9/09_chapter 3.pdf · 2.9....
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CCHHAAPPTTEERR –– IIIIII FFUUNNCCTTIIOONNAALL PPRROOPPEERRTTIIEESS OOFF PPRROOTTEEIINN CCOONNCCEENNTTRRAATTEE
1. Introduction
The production of plant protein concentrates (PCs) is of phenomenal interest to food
industry because of the increasing applications of plant proteins in food especially in
developing countries (Akintayo et al., 1998; Sanchez-Vioque et al., 1999). To improve the
nutritional quality of the product or for economic reasons, the use of plant PCs in food as
functional ingredient is indeed extensive. For example, whey PCs (Jayaprakasha &
Brueckner, 1999) and soybean PCs (Qi et al., 1997) have been widely used as food foaming,
emulsifying, water binding and viscosity ingredients. However, these applications in the food
trade are almost restricted to protein from legumes (Chau et al., 1997; Qi et al., 1997;
Sanchez-Vioque et al., 1999) and cereals (Prakash, 1996; Jayaprakasha & Brueckner, 1999),
whereas other plant proteins are seldom used. People in the Far East and Asia Pacific have a
long tradition of consuming seaweeds as an integral part of their diet. In the western
countries, the principal uses of seaweeds are as sources of phycocolloids, thickening and
gelling agents for various industrial applications including, foods (Darcy-Vrillon, 1993;
Mabeau & Fleurence, 1993; Abbott, 1996).
Recently in France, seaweeds have been approved for use as vegetable and
condiments (Mabeau, 1989). Therefore, seaweeds are becoming a valuable vegetable (fresh
or dried) and an important food ingredient in human diet nowadays, even in the western
world. The nutritional potential of seaweeds as food protein sources differs according to
species (Fleurence et al., 1999). Seaweeds belonging to the Rhodophyta possess high levels
of proteins (10–30% DW) (Darcy-Vrillon, 1993; Mabeau & Fleurence, 1993) comparable to
those of edible land vegetables. In some red seaweeds, such as Palmaria palmata (L.) Kuntze
(dulse) and Porphyra tenera Kjellman (nori), the protein contents are 35 and 47% DW,
respectively (Fujiwara-Arasaki et al., 1984). These levels are even comparable to that of the
soybeans (35% DW).
However, only a couple of studies have been undertaken on the quality of seaweed
protein (Dam et al., 1986; Ito & Hori, 1989; Amano & Noda, 1990; Fleurence et al., 1999)
owing to the difficulties of extraction and preparation of seaweed PCs. The extraction of
seaweed protein by classical procedures is encumbered by the presence of large amounts of
cell wall polysaccharides, such as the alginates of the brown seaweeds or the carrageenans of
some red seaweeds. The high content of neutral polysaccharides (e.g. xylans and cellulose) in
some red and green seaweeds can also limit the protein accessibility (Fleurence et al., 1999).
These anionic and neutral polysaccharides are the chief encumberance during the extraction
and purification of seaweed protein (Ochiai et al., 1987; Ito & Hori, 1989; Jordan & Vilter,
1991; Fleurence et al., 1995).
The extraction procedures for seaweed proteins described in the literature are mainly
concerned with the extraction of specific seaweed enzymes such as proteases (Kadokami et
al., 1990), peroxides (Sheffield et al., 1993) or carboxylases (Hilditch et al., 1991). In
comparison, very little information about the extraction of the total protein fraction from
seaweed is available (Fleurence et al., 1995). After comparing with different classical and
enzymatic procedures (e.g. using aqueous polymer two-phase system, polysaccharidases, or
Tris HCl buffer), Fleurence et al., (1995) reported that the highest yield of seaweed PCs was
obtained by the use of NaOH and 2-mercaptoethanol after an initial aqueous extraction. The
inadequacy of bioassay techniques, including protein efficiency ratio (PER), for evaluating
protein quality has been recognized (Pellett & Young, 1980; Madi, 1993). Biological indices
like net protein ratio (NPR), nitrogen balance (NB), true protein digestibility (TD), biological
value (BV), net protein utilization (NPU) as well as utilizable protein (UP), which are widely
used in nutritional studies (Kalra & Jood, 1998; Wong & Cheung, 1998), are recommended
by the FAO/WHO (1991) for evaluating protein quality.
2. Materials methods
2.1. Sample preparation
Kappaphycus alvaerezii was collected from Port Okha, West coast of India in
September 2005. The fresh plants were sun dried followed by thorough washing with
distilled water to remove epiphytes. This clean seaweed was then oven dried at 60 oC for 16 h
to a constant weight. The dried samples were pulverized to obtain uniformly sized particles.
The milled seaweed sample was then stored in airtight plastic bags in a desiccator at room
temperature (25 oC) prior to extraction of the protein concentrate (PC).
2.2. Extraction of protein concentrate
Kappaphycus alvaerezii PC was extracted as described by Fleurence et al., (1995)
with slight modifications. In brief, seaweed powder was suspended in distilled water (1:20
w/v) to induce cell lysis by osmotic shock in order to facilitate subsequent protein extraction.
The suspension was then gently stirred overnight at 35◦C, which is the temperature found to
be optimal for seaweed protein solubility (Dua et al., 1993). After incubation, the suspension
was centrifuged at 10 000 × g and 4◦C for 20 min. The supernatant was collected and the
pellet was re-suspended in distilled water in the presence of 0.5% (v/v) 2-mercaptoethanol
(Venkataraman & Shivashankar, 1979). The mixture was then adjusted to pH 12 with 1 M
NaOH and gently stirred at room temperature (25◦C) for 2 h before centrifugation at the same
conditions mentioned above. The supernatant was collected and combined with the previous
supernatant. The combined supernatant was stirred at 0–4◦C and adjusted to pH 7 before
precipitation with solid ammonium sulphate. The extraction procedure mentioned above was
repeated five times on the residue.
2.3. Precipitation of seaweed protein concentrate
The seaweed PC was precipitated from the supernatant by slowly adding solid
ammonium sulphate with stirring until 85% saturation (60 g.100 ml-1) was attained
(Rosenberg, 1996). The mixture was then allowed to stand for 30 min at 15◦C before
decanting followed by centrifugation at the same conditions mentioned before. The pellet
(PCs) obtained was dialysed against distilled water until the total dissolved solutes (TDS)
(mg.l−1) of the dialysate was similar to that of the distilled water. The retentates containing
seaweed PC was then freeze-dried and stored in an air-tight bag in a desiccator before the
biological evaluation of the protein quality was performed.
2.4. Protein Content and Yield Determination
The percentage of total protein present in the seaweed PC was calculated by
multiplying percentage nitrogen estimated by Kjeldahl method (Wathelet, 1999) using KEL
PLUS-KES 20L Digestion unit attached to a KEL PLUS-CLASSIC DX Distillation unit (M/s
PELICAN Equipments, Chennai, India) by the factor 6.25. The Kjeldahl Digestion System
was used to digest the protein, with a setting of 2.0, 0.2, and 3.6 for alkali, delay, and steam,
respectively, to the determine nitrogen content of the protein samples. Protein yields were
calculated as
Weight (g) of PC × % protein content of PC
Yield (%) = -------------------------------------------------------------------------- × 100
Dry weight of seaweed (10 g) × % protein content of seaweed
2.5. Nitrogen Solubility
Nitrogen solubility (NS) was determined by the method of Bera & Mukherjee (1989).
Samples (100 mg each) were dispersed in 5 ml of distilled water and NaCl solutions at
varying concentration (0.1, 0.5, or 1.0 M). The pH was adjusted to 2.0, 4.0, 6.0, 8.0, 10.0,
and 12.0 using 1.0, 0.1, or 0.01 N HCl or NaOH. Samples were shaken at 145 rpm for 30 min
at room temperature and then centrifuged at 4000 x g for 30 min. Nitrogen contents of the
supernatant (NS) were determined by the Kjeldahl method, and percentage nitrogen
solubility was calculated as follows:
Nitrogen in the supernatant (mg)
NS (%) = ---------------------------------------------- × 100
Total nitrogen in 100 mg PC
2.6. Water holding and fat absorption capacities (WHC and FAC)
WHC and FAC were determined using the method of Carcea-Benecini (1986), with
slight modification. Briefly, 1.0 g of protein sample was dissolved in 10 ml distilled water,
after the mixture was thoroughly stirred and samples were centrifuged at 2000g for 30 min.
After the centrifugation, the amount of added distilled water resulting in the supernatant
liquid in the test tube was recorded. WHC (grams of water per gram of sample) was
calculated as WHC = (W2 / W1)/W0, where W0 is the weight of the dry sample (g), W1 is
the weight of the tube plus the dry sample (g), and W2 is the weight of the tube plus the
sediment (g). Triplicate samples were analyzed for each sample. For FAC, 1.0 g of sample
was weighed into centrifuge tubes that were pre-weighed and thoroughly mixed with 5 ml of
sunflower oil. The protein–oil mixture was centrifuged (3000g for 30 min). Immediately after
centrifugation, the supernatant was removed meticulously, and the tubes were weighed. FAC
(grams of oil per gram of protein) was calculated as FAC = (F2 / F1)/F0, where F0 is the
weight of the dry sample (g), F1 is the weight of the tube plus the dry sample (g), and F2 is
the weight of the tube plus the sediment (g). The samples were analyzed in triplicate.
2.7. Apparent viscosity
Apparent viscosity of the protein concentrate was determined with a Brookefield
viscometer (Synchrolectric Viscometer, Stoughton, MASS 02072) using Spindle No. 3 at 30
rpm. Different concentrations (1, 2, 4, 6 and 8 mg.ml-1) of the protein concentrate were
dissolved in distilled water for viscometeric measurements (at room temperature, pH 7).
2.8. Emulsifying and surface active properties
Emulsifying activity was measured using a modified method of Cooper &
Goldenberg (1987). In this method, hydrocarbon or oil was added to aqueous phase
containing the protein concentrate [hydrocarbon : protein concentrate (10 mg.ml-1) in a ratio
of 3:2, v/v)] and agitated vigorously for 2 min on a cyclo-mixer. The oil, emulsion and
aqueous layers were measured at different time intervals and an emulsification index (E) was
calculated as follows
Volume of the emulsion layer
Emulsifying index (E) = ------------------------------------------ × 100
Total Volume of the mixture
The emulsification index was noted with respect to time (15, 30, 90, 210, 390, 720
min) and was represented accordingly, i.e. the emulsification index after 15, 30, 90, 210, 390
and 720 minutes was represented as E15, E 30, E90, E210, E390 and E720 respectively. The
surface tension of 0.1 and 0.5% (w/v) protein concentrate was determined using a
Dataphysics Dynamic Contact Angle Meter and Tensiometer (DCAT 21), Dataphysics
Instruments GmbH, Germany using Wilhelmy plate (PT 11) made of platinum-iridium.
2.9. Foaming Capacity and Foam Stability
A modified method of Nath & Narasinga Rao (1981) was used to ascertain the
foaming capacity of the protein concentrate. A 100 ml solution of the protein concentrate (20
µg.ml-1) was whipped at low speed on a vortex mixture for 5 min in a 250 ml measuring
cylinder, and the foam volume was recorded after 30 seconds. The volume of foam was
recorded after 30, 60 and 90 minutes of standing at room temperature. The volume increase
was expressed as percent foam capacity as a function of time up to a period of 120 min. The
foaming properties were also determined as a function of pH.
2.10. DSC thermal characteristics
Differential scanning calorimetry (DSC) was performed using a Mettler Toledo Star
SW 7.01. According to the procedure of Meng and Ma (2001), with slight modifications. The
protein sample (5 mg) was dissolved in 1 ml of 0.06 M phosphate buffer (pH 7.0) containing
0.10 M NaCl. A 45 µl of protein solution was hermetically sealed in a stainless steel pan. The
sample was heated from 0 to 300°C at a rate of 10 °C. min-1, and the thermal properties were
referenced against another pan containing 45 íL of buffer without protein. The denaturation
peak temperature (Td) and enthalpy (H) were calculated by a thermal analysis software
program. The temperature at which denaturation started, known as the onset denaturation
temperature “T onset”, was calculated by taking the intercept of the baseline and the
extrapolated maximum slope of the peak. The peak denaturation temperature “T peak” was
considered to be the temperature at maximum heat flow. The enthalpy of thermal
denaturation was calculated from the area of the endothermic peak.
2.11. Thermal measurements
Thermo gravimetric measurements (TGA) were carried on a Mettler Toledo TGA
system, Greifensee, Switzerland with protein concentrate (5 mg) using a temperature
program in an air atmosphere from 0 to 500C at a rate of 10°C.min-1. The temperature at
which slope of the weight loss versus temperature curve starts to increase was considered as
the temperature of initiation of the degradation phenomenon.
2.12. Determination of average particle size and specific surface area
The protein concentrate of K. alvarezii particle size distribution of the solution was
measured using a laser diffraction instrument (Mastersizer Hydro 2000S (A), version 2.00.
Malvern Instruments, Ltd., U.K).The prepared solution were diluted 500-fold using buffer
solution to avoid multiple scattering effects prior to analysis.
2.13. Scanning electron microscopy (SEM)
Sample preparation for SEM was as described by Feng (2000) with a slight
modification. Samples prepared at 8% w/w protein in the presence of 10, 20, 30 and 40 mM
of NaCl. Samples were fixed in 2.5% glutaraldehyde. Fixed samples were dehydrated
through a series of ethanol solutions of increasing concentration (25, 50, 75, 95, and 100%,
v/v) for 30 min each. Dehydrated samples were stored in desiccator until further use. Dried
samples were mounted on aluminum specimen stubs and coated with gold using a Sputter
Coater (Polaran model No-SC7620 U.K). The samples were examined at 12000x using SEM
(Oxford instrument model No-7353 U.K) at an accelerated voltage of 15 keV with secondary
electron detector (SE1).
2.14. FT-IR Spectroscopy
The lyophilized protein concentrate of K. alvarezii was ground with potassium
bromide at a 1/100 ratio (w/ w). This protein concentrate was pressed at high pressure into a
KBr pellet. The spectral analysis was carried out using NXR FT-IR module (Thermo electron
corporation USA).The FT-IR spectra of sample was recorded in the 4000-400 cm-1 region at
room temperature.
2.15. FT-Raman Spectroscopy
FT-Raman spectra were recorded on a NXR FT-Raman module (Thermo electron
corporation USA). Spectral resolution was set at 4 cm-1 and laser power at 1437 mW, and
frequency calibration of the instrument was undertaken using the sulfur line at 217 cm-1. All
data presented are based on 128 co-added spectra. The original spectra in the 0-4000 cm-1
region were baseline corrected and normalized using the phenylalanine peak near 1005 cm-1
(Hilditch et al., 1991). The results obtained here were shown as the tentative assignment of
the major bands in the spectra to vibrational motions of various side chains or peptide
backbone, which was compared to FT-Raman spectra reported in literature. Spectral data
within this region were smoothened with the Savitsky-Golay five-point algorithm and
deconvoluted using a nonlinear least-squares curve-fitting subroutine with Gaussian type
functions. The percentage of each secondary structure component (-helix, -sheet, -turn,
and random coil) was determined as the corresponding fitting peak area contained in the
fitting range. Peak intensities and secondary structure components are expressed as the
average of the replicate spectra with a typical coefficient of variation of 10%.
3. Results and Discussion
3.1. Protein Content and Yield of K. alvarezii
Using ammonium sulfate precipitation technique, 7.81 2.42 % protein concentrate
could be obtained from K. alvarezii. This protein concentrate (PC) contained 62.3 1.62 %
total protein which showed good solubility at acidic and alkaline pH as well as in the
presence of salts (discussed later). Prakash & Narasinga (1986) obtained similar results. S.
hemiphyllum yields 9.50 2.13% protein concentrate containing 85.0 1.06 % total protein
(Kahing &Wong 2001). The yield of PC obtained using K. alvarezii was slightly lower than
those of S. hemiphyllum, with less total PC. The low yield recorded for PC obtained using K.
alvarezii is because of the fact that this seaweed is a carragenophytic seaweed.
3.2. Nitrogen solubility
The effect of pH and salt concentration on nitrogen solubility of this protein
concentrate is depicted in (Figure 1). The minimum nitrogen solubility was evident at pH 4.5,
which was 18.5% for fenugreek protein concentrate (Nazar & Tinay, 2007). But minimum
solubility of nitrogen of K. alvarezii was recorded in water at pH 4 (33.72 1.23 %), and
these values were notable enough. The predominant proteins of K. alvarezii PC likely to have
isoelectric pH around this value. No much differences in nitrogen solubility values were
noticed at pH 8 and 10. The PC showed only an incremental increase in solubility from pH
8–12 in water as well as in NaCl concentrations. However, at pH 12, K. alvarezii PC reached
58.72 1.68 % solubility at 0.5 M NaCl concentration, which was comparatively lower than
the one reported for fenugreek PC i.e. 86.3% at pH 10 (Nazar & Tinay, 2007). High protein
solubility, in both the acid and alkaline pH is a core characteristic in food formulation, as
reported by Idouraine et al., (1991). Seena & Sridhar (2005) reported that, at highly acidic
and alkaline pH, the protein acquires net positive and negative charges, respectively, which
favour the repulsion of molecules and thereby increase the solubility of the protein. Solubility
is a physico-chemical property of a protein that crucially affects its functional properties as
manifested in foods, mainly emulsifying, foaming, and gel forming abilities (Sikorski, 2001).
3.3. Water holding and fat absorption capacities (WHC and FAC)
The water-holding capacity of K. alvarezii protein concentrate was 2.223 0.039 ml
water.g-1 of protein. This value is lower than that reported for the protein concentrate of
Egyptian fenugreek (3.52 ml water.g-1 of protein) containing 35.8% crude protein (Abdelaal
et al., 1986). The water-holding capacity is a critical property of proteins in viscous foods,
e.g. soups, dough, custards and baked products, because these are supposed to imbibe water
without dissolution of protein, thereby providing body, thickening and viscosity (Adeyeye et
al., 1994; Seena & Sridhar, 2005).
Fat absorption capacity of K. alvarezii PC was 1.29 0.201 ml oil.g-1 of protein,
which is lower than that reported by Nazar et al., (2007) for fenugreek (Trigonella foenum
graecum) protein concentrate (1.56 oil.g-1). The mechanism of fat/oil absorption capacity was
explained by Kinsella (1979) as a physical entrapment of oil. Fat/oil absorption capacity is a
pivotal determinant of flavor retention. Fat emulsion capacity and stability are important
attributes of additives for the stabilization of fat emulsions. Chau & Cheung (1997) reported
that surface area and hydrophobicity improve oil absorption capacity. Thus, K. alvarezii PC
had good water and oil holding capacity.
3.4. Apparent viscosity
An apparent visocity of 42.33 0.21 cps or (0.04233 0.00021 Pas) could be
obtained using 1 mg.ml-1 of protein concentrate. Further increase in the concentration of PC
caused a proportional increase in the viscosity. At 8 mg.ml-1, an apparent viscosity of 55.67
0.25 cps could be obtained (Table 1). In general, low apparent viscosity is observed in
proteins when their molecular mass is reduced by proteolysis. Tsumura et al., (2005) reported
functional properties of soy protein hydrolysates, wherein they reported an apparent viscosity
of approximately 10-40 mPa for reduced--conglycinin hydrolysate (RCH) and reduced-
glycinin hydrolysate (RGH) of the same by selective proteolysis. Prakash & Narasinga
(1988) studied structural similarities among the high molecular weight protein fractions of
oilseeds and reported an intrinsic viscosity of 4.9 and 3.0 for glycin and -globulin
respectively. Lower viscosity of protein suspension before heating is desirable during
pumping and piping, and higher viscosity and gel formation after heating is desirable for the
thickening of soup, and production of sausage and meat analog (Yu et al., 2007). Speiciene
et al., (2007) studied the effect of chitosan on the properties of emulsions stabilized by whey
proteins, wherein they reported an apparent viscosity of 0.005 to 0.01 Pas in the presence of
shear stress. In the present study, the K. alvarezii PC obtained had a higher apparent visocity
than the above reported whey proteins.
3.5. Emulsifying and surface active property
K. alvarezii protein concentrate efficiently emulsified aliphatic and aromatic
hydrocarbons such as kerosene, xylene, carbon tetrachloride and hexane and oils such as
silicone oil, paraffin oil, groundnut oil, cotton seed oil, jatropha oil, cedar wood oil, jojoba
oil, sunflower oil and olive oil. The emulsification indices of K. alvarezii PC with different
hydrocarbons and oils are shown in (Table 2). The maximum emulsification indices was
observed with cedar wood oil (99.67 0.58), jatropha oil (99.33 1.15) and olive oil (99.00
1.73). This PC showed good emulsifying activity with groundnut oil (77 1.00), cotton
seed oil (75.68 0.58) and xylene (73.33 1.58) after 15 min. Formation of stable emulsions
was observed using cedar wood oil (E720 = 75.33 2.08), olive oil (E720 = 54.33 1.16) and
jatropha oil (E720 = 53.67 1.53) at 10 mg.ml-1 concentration. It has been reported that the
hydrophobic lipid portion in emulsan is responsible for its emulsifying action (Ashtaputre &
Shah, 1995). The emulsion stability of the PC was time dependent i.e. emulsification stability
gradually decreased with increasing time interval.
A surface tension of 72.05 0.04 mN.m-1 was recorded for distilled water. Surface
tension values of 50.10 0.03 and 44.02 0.03 mN.m-1 were recorded using 0.1 and 0.5%
protein concentrate. Thus it could be concluded that the protein concentrate was surface
active and hence could decrease the surface tension of distilled water. Several reports are
available on the use of several biological products of plant and bacterial origin. In fact, plant
root mucilages contained powerful surfactants that would alter the interaction of soil solids
with water and ions, and the rates of microbial processes (Reed et al., 2003). The emulsifying
activity of acacia gums depended to a great extent on the nature and concentration of the
protein present in it (Dickinson et al., 1990). According to Dickinson et al., (1991), a high
percentage of hydrophobic amino acids in the protein moiety were favorable for
emulsification. In the present study, the amount of protein present in the concentrate may be
accountable for the formation of stable emulsions.
3.6. Foaming Capacity and Foam Stability
The foaming capacity (FC) and stability of K. alvarezii protein concentrate is shown
in (Figure 2 & 3). This property is pH-dependent. The lowest FC (28.67 3.06 %) was
obtained at pH 6.0. The highest forming capacity (53.33 2.309) were noted at pH 4.0. The
foaming capacity of K. alvarezii protein concentrate was comparatively lower than the
fenugreek protein concentrate i.e. 89.5 % (Nazar et al., 2007).The basic requirements for a
protein to be a good foaming agent are the ability to adsorb rapidly at the air-water interface
during bubbling; and the ability to undergo rapid conformational changes at the interface
(Fidantsi & Doxastakis, 2001). Studies on effect of pH on foam stability revealed that the
foaming stability decreased with increasing time. Maximum foaming ability (45.33 1.16)
was recorded at pH 2 at 30 min.
3.7. DSC thermal characteristics
Figure 4 shows typical DSC thermograms of K. alvarezii protein concentrate in
0.06M phosphate buffer (pH 7.0). The protein sample was heated from 0 to 300 °C at a rate
of 10 °C.min-1. The PC exhibited two observable endothermic peaks of which the major
endothermic peak temperature (Td) was recorded at about 109.25°C and the minor one at
108.52 °C (Tm). The enthalpy of the thermal denaturation was H - 5.3 J.mg-1.
3.8. Thermal measurements
The TGA curve showed weight loss during the heating process. The percentage
weight loss of the protein concentrate was 82.22% for 3.8 mg of the sample.
3.9. Determination of average particle size and specific surface area
The Figure 5 shows relative refractive index (RI) of the K. alvarezii PC and it
was 1.330, i.e. the ratio of the refractive index of emulsion particles (1.570) to that of the
dispersion medium (distilled water). The absorbance value of the emulsion particles was
1.00. The average particle size of 99.98 % of the particles in the emulsion was below 10.00
m. The specific surface area was 1.9558 m2.g-1. Singh et al., (2003) have been reported a
mixture of milk proteins and k-carrageenan to have RI of 1.095 and an absorbance value of
0.001.
3.10. Scanning electron microscopy (SEM)
Samples were prepared using sodium chloride (NaCl) and calcium chloride (CaCl2)
solutions. Ions affect protein conformation by electrostatic interactions with the charged
groups and the protein polar groups or by hydrophobic interactions between protein
molecules (Damodaran & Kinsella, 1982).A gel like morphology of the protein concentrate
could be observed in the presence of sodium chloride (Figure 6). Gels with less
homogeneous and more compact microstructure could be observed in presence of low salt
concentration (10 µM and 20 µM of NaCl). On increasing the salt concentration (30 µM and
40 µM NaCl), the gels appeared more aggregated with particulate structure, owing to protein
aggregation observed at high ionic strengths. It has been reported that formation of fine or
particulate gels depends on pH and ionic strength. Aggregated polypeptide chains are formed
close to the protein pI and or at high ionic strengths, whereas far from the pI (very low or
high pH), fine polypeptide chains are formed (Harwalkar & Kalab, 1985; Van Kleef, 1986;
Heertje &Van Kleef, 1986; Stading & Hermansson, 1991). The neutral salts that favor
salting-out at concentrations above 0.15M and near the pI such as NaCl stabilize the protein
by reinforcing hydrophobic interactions among molecules and decreasing its solubility
(Damodaran & Kinsella, 1982; Foegeding et al., 1995). Other proteins (milk whey proteins)
also form opaque and coagulate-type gels of low hardness with a high degree of syneresis at
NaCl concentrations above 0.2 M (McClements et al., 1993).
Unlike NaCl, the gels formed using several concentrations of CaCl2 (10 µM-40 µM)
presented a finer and tighter structure (Figure 7). High CaCl2 concentration promoted a gel
matrix formed by chains of protein threads. Ca2+ establishes bridges with the protein at
alkaline pH. However, at acidic pH, the Ca2+ ion competes with the H+ for the same binding
centers; hence, it would not establish bridges with the protein as at alkaline pH (Kroll, 1984).
Ca2+ can interact with water, thus modifying the aqueous surroundings of the protein,
increasing the protein aggregation. CaCl2, a divalent salt, is an exception in the salting-out
effect of the Hofmeister series. Combined with -lactoglobulin, this salt forms particulate
gels at an ionic strength 0.15 M (Foegeding et al., 1995).
3.11. FT-IR spectroscopy
FT-IR spectra provide information about the structural composition of proteins. The
spectrum of the PC (Figure 8) exhibited a band at 616 cm-1 which could be due the presence
of phosphate group (Bahy, 2005). A stretching band at 704 cm-1, revealed out of plane N-H
bending (Jung, 2000). The strong absorption bands present in 924 cm-1 region (3,6-anhydro-
D-galactose) and in the 848 cm-1 region (D-galactose-4-sulphate) were characteristic of
carrageenan (Pereira & Mesquita, 2003). Since this protein isolate belongs to a
carrageenophyte, it is obvious that this phycocolloid would be present as a contaminant. 1039
cm-1 could be assigned to (C>O) stretching (Sigee et al., 2002). The presence of histidine
was revealed by the stretch at 1403 cm-1 (Gregoriou et al., 1995). The band at 1647 cm-1
indicated stands for the -helix, where the amide I is typically in the range of 1648-1658 cm-
1. 2356 cm-1 showed symmetric stretch of CH2 present in the protein, while, 2923 cm-1
showed the asymmetric stretching of –C-H (CH2) (Guillen & Cabo, 1997). The stretch at
3146 cm-1 was also indicative of the C—H stretch of histidine imidazole (Puustinen et al.,
1997).
3.12. FT-Raman spectroscopy
The investigation of the structural properties of K. alvarezii protein concentrate has
not been addressed yet. In the present study, FT-Raman spectroscopy proved to be a valuable
tool which allowed the direct monitoring and thorough spectral analysis of K. alvarezii
protein concentrate (Figure 9, Table 3). The protein concentrate exhibited a shoulder band at
486.23 cm-1 indicating the presence of carotenoids. The band at 512.93 cm-1 vividly
represented the presence of gauche- gauche- gauche conformation. Moreover it also indicated
cystine, cystein and methionine (S-S stretching), whose structural information could be
interpreted as conformation of heterogeneity of cystine residues (Li-Chan, 1996). The band
located at 860.14 cm-1 corresponding to tyrosine (Try) doublet band, were useful for
monitoring the microenvironment around the tyrosine. The protein concentrate spectrum also
indicated bands assigned to the following groups: symmetric CCC stretch various CCC
stretches at 867.59 cm-1, amide III (random coil) at 953.06 cm-1 and anti-symmetric CCC
stretch at 1085.84 cm-1 (Sarkardei & Howell, 2007). The intensity of the Raman 998.33 cm-1
indicated the presence of phenylalanine (Howell et al., 2001). The peak at 1562.15 cm-1
displayed information about the microenvironment of the tryptophan (Trp) residues, while,
the peak located at 1101.78 cm-1 was sensitive to conformational changes of the polypeptide
back bone (Supawan et al., 2006).
Based on the above mentioned studies, it is stated that, the protein concentrate (PC) of
Kappaphycus alvarezii obtained using ammonium sulfate precipitation possessed a variety of
properties. A yield of 7.81 2.42 % of PC containing 62.3 1.62 % total protein was
obtained. Minimum nitrogen solubility (33.72 1.23 %) was observed at pH 4.0, while
maximum nitrogen solubility (58.72 1.68 %) was observed at pH 12 which was in the
presence of 0.5 M NaCl. Measurement of emulsifying and foaming properties of protein
concentrate showed that they were largely affected by time interval and pH levels. The
maximum emulsification index 99.67 0.58 was noted for cedar wood oil after 15 seconds,
whereas most stable emulsion was recorded for jatropha oil after 720 min (E720 = 53.67
1.53).The maximum foaming ability (53.33 2.309 %) of the PC was recorded at pH 4.0.
The PC recorded high fat absorption capacity (1.29 0.201 ml oil.g-1) with water absorption
capacity (2.223 0.039 ml H2O.g-1). DSC analysis showed that thermal transitions occurred
at about 109.25 °C at neutral pH. The apparent viscosity increased with increasing
concentration of PC. SEM study showed that gels formed at 10µM NaCl were composed of
less homogeneous and more compact microstructure, while the same appeared more
aggregated with particulate structure at high concentration of NaCl (40 µM). However the
gels formed using several concentrations of CaCl2 (10.00 µM – 40.00 µM), presented a finer
and tighter structure. The composition of PC investigated through FTIR indicated the
absorption band at 1647 cm–1 (-helix of the secondary structure) and the band at 3146 cm–1
was indicative of C-H stretching of histidine imidazole. FT Raman spectral band at 860.14
cm–1 was ascribed to tyrosine (Tyr) doublet, while the band at 953.06 cm–1 indicated the
stretch of amide III (random coil). The peak at 1562.15 cm–1 displayed information about
tryptophan (Try) residues, and the one located at 1101.78 cm–1 was sensitive to
conformational changes of the polypeptide backbone.
The analytical data on crude protein content of K. alvarezii, suggests their high potential as a
cheap source of alternative protein for human consumption. This protein was found to be
more soluble at acidic and alkaline pHs than near neutral pH. Emulsifying and foaming
properties for the concentrate were comparable to many reports, indicating an important role
in food systems, such as salads and ice cream. The good protein concentrate solubility could
be of use for the production of beverages. It may also be used as supplement to enhance the
low nitrogen content of traditional food such as cereals and tubers.
Table 1
Apparent viscosity K. alvarezii PC at room temperature.
Concentration
(mg.ml-1)
Viscosity
(cps)
1 42.33 0.21
2 44.33 0.30
4 48.00 0.26
6 48.33 0.31
8 55.67 0.25
Table 2
Stability of emulsions formed by the protein isolate (10 mg.ml-1) using different oils / hydrocarbons.
tOils /Hydrocarbons
Emulsification index
E15 E30 E90 E210 E390 E720
Silicone oil 56.67 1.16 51.67 0.57 50.43 0.58 50.33 0.58 49.67 0.48 49.00 1.00
Paraffin Oil 68.33 1.16 64.33 3.06 58.33 0.58 52.33 2.52 52.67 0.58 50.33 1.16
Kerosene 64.00 2.65 49.67 0.58 46.67 0.58 43.33 1.53 40.33 0.58 40.67 1.16
Groundnut oil 77.00 1.00 72.67 1.53 68.00 1.73 55.33 1.53 49.00 1.73 39.67 0.58
Cotton seed oil 75.67 0.58 73.67 1.53 70.67 0.58 66.33 1.53 58.33 1.16 42.67 1.53
Jatropha oil 99.33 1.15 83.00 2.00 80.00 1.73 67.67 1.53 60.00 1.00 53.67 1.53
Cedar wood oil 99.67 0.58 91.00 1.00 90.00 2.00 81.33 1.16 75.00 2.65 75.33 2.08
Jojoba oil 57.67 1.53 52.67 1.53 46.67 0.58 41.00 1.53 40.33 0.58 40.33 1.52
Sunflower oil 64.33 1.16 62.00 1.00 52.67 0.58 45.67 1.00 40.67 0.58 41.33 0.58
Olive oil 99.00 1.73 91.33 1.16 82.33 2.08 71.67 1.16 65.33 2.88 54.33 1.16
Xylene 73.33 1.58 61.33 1.53 60.67 1.16 61.00 1.00 54.33 1.16 53.33 1.53
Carbon tetrachloride 64.00 2.65 63.33 1.53 60.00 1.00 57.33 0.58 51.33 0.58 50.67 0.58
Hexane 52.67 2.52 49.00 1.00 43.00 1.00 42.00 1.00 39.67 0.58 39.67 0.58
Table 3
Tentative assignments of major bands in the FT-Raman spectra of K. alvarezii protein
concentrate.
Band assignment Wavenumber (cm-1)
Carotenoid 486.23
S-S stretching 512.93
Tyrosine (Try) doublet 860.14
Symmetric CCC stretch various CCC stretches 867.59
Amide III (random coil) 953.06
Phenylalanine 998.33
Anti-symmetric CCC stretch 1085.84
Polypeptide back bone 1101.78
Tryptophan (Trp) residues 1562.15
0
10
20
30
40
50
60
70
2 4 6 8 10 12pH
Nitr
ogen
sol
ubili
ty (
%)
KAPI in water 0.1 M NaCl 0.5 M NaCl
Figure 1. Effect of pH and NaCl concentration on nitrogen solubility of K. alvarezii protein concentrate
0
10
20
30
40
50
60
2 4 6 8 10pH
Foa
m c
apac
ity (
%)
.
Figure 2. Effect of pH on foaming capacity of K.alvarezii protein concentrate
0
10
20
30
40
50
2 4 6 8 10pH
Foa
m s
tabi
lity
(%)
30 min 60 min 90 min
Figure 3. Effect of pH and different time interval on foaming stability of
K. alvarezii protein concentrate.
Figure 4. Typical DSC thermogram of K.alvarezii protein concentrate.
Figure 6. SEM images of K. alvarezii protein concentrate in the presence of different
concentrations of NaCl (a) 10M (b) 20 M (c) 30 M and (d) 40M.
(a) (b)
(d) (c)
Figure 5. Amount of the intensity peak for K. alvarezii protein concentrate particle size
distribution.
(a) (b)
(c) (d)
Figure 7. SEM images of K. alvarezii protein concentrate in the presence of different
concentrations of CaCl2 (a) 10 M (b) 20 M (c) 30 M and (d) 40 M.
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