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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