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1 PhD Scholar, ICAR-Central Institute of Agricultural Engineering, India
2 Scientist, ICAR-Central Institute of Agricultural Engineering, India
3Principal Scientist, ICAR-Central Institute of Agricultural Engineering, India
Submission: July 12, 2018; Published: October 11, 2018
*Corresponding author: Hima John PhD Scholar, ICAR-Central Institute of Agricultural Engineering, Bhopal, India.
Shekh Mukhtar Mansuri, Scientist, ICAR-Central Institute of Agricultural Engineering, Bhopal, India
How to cite this article: Hima J, Shekh M M, Saroj K G, Lalan K S. Rheological Properties and Particle Size Distribution of Soy Protein Isolate as Affected by
Drying Methods. Nutri Food Sci Int J. 2018; 7(5): 555721. DOI: 10.19080/NFSIJ.2018.07.555721.
Soy protein isolate (SPI) solution prepared by ultrafiltration was dried using spray, freeze and oven dryers. Rheological properties (storage and loss modulus, complex viscosity, loss tangent), protein solubility and particle size distribution (D4,3, D3,2 and span) of different isolate powders were compared with commercially available counterpart. Commercial SPI showed a shear thinning behavior while rest of the samples exhibited a shear thickening behavior. Commercial and oven dried SPI (60 °C for 48 hours) SPI shown almost constant loss tangent less than unity during oscillation test which indicated its solid or gel property. Commercial and oven dried SPI samples had comparable size distribution whereas freeze dried SPI shown higher peak and narrow distribution pattern. Commercial SPI had lesser uniformity compared to rest of the samples. Water solubility index of spray dried SPI samples were high compared to freeze and oven dried samples.
Keywords: Soy protein isolate; Spray dryer; Oven dryer; Particle size distribution; Storage and loss modulus; Protein solubility
Soybean is a rich and affordable source of plant protein and can be used as a protein supplement or ingredient in many food items. Soy protein isolate (SPI) is the most concentrated form of soy protein. Now a days, use of isolates in food industry is also increasing. Proteinsare used in food formulations to perform functional roles that are important for consumer food acceptance . Lusas & Riaz  reported that soy proteins mainly used as ingredients in formulated foods and substitute for other ingredients. Soy protein can replace many ingredients in food formulations in meat or dairy products without changing the taste and quality of foods. It can be used as emulsifiers, texture enhancers and as ingredients to increase or replace protein content in food products like bread, pastry products, beverages and meat . Numbers of investigations have been carried out to find out the variation in functional properties of SPIs under different processing conditions [4-6].
Rheological properties and particle size distribution of soy protein is equally important along with the functional properties it possesses. Rheological properties can be correlated with other flow behavior related functional properties. Similar studies have been conducted by [7,8] to incorporate different proteins in meat balls. Sabater  conducted an experimentto correlate flow characteristics of emulsions with sausage texture. Particle size
distribution as well as the uniformity of particle is a major factor contributing the properties and behavior of food formulations and beverages. Particle size affects the solubility as well as the texture of final food preparations. Soluble proteins are easier to incorporate as food ingredients and other properties, like gelation and emulsification. Shen  reported that solubility of soy protein products are highly dependent on the physico-chemical states of protein molecules, which are either favorably or adversely affected by heating, drying and other processing treatments during their manufacture and storage. Kinsella  reported that heating of protein reduces its solubility, the extent of which depends on the intensity and duration of the heat treatment and therefore the solubility is an indicator of protein denaturation.
Over the year’s ultrafiltration has received considerable interest for concentration of soy proteins as proteins are retained by the membrane while the oligosaccharides and minerals are removed as permeate. The products with improved properties are obtained through ultrafiltration and there also with no excessive use of chemicals . Different drying methods used to dry the protein solutions obtained through ultrafiltration influences the properties of protein isolate powder. Joshi et al.  reported that the drying method used for preparation of lupin protein isolate (LPI) can significantly affect the physicochemical properties,
which in turn adversely affect the functionality of proteins.
Variation of functional properties of SPI with different drying
methods is reported by Hu et al., .
An attempt has been made to investigate the effect of drying
methods on SPI properties. The objective of this study is to
gain insights to how the rheological properties, particle size
distribution and solubility characteristics of SPIs obtained through
ultrafiltration are varying according to the drying methods.
Defatted soy flour used as the starting material for extraction
of soy protein was purchased from local market. Commercially
available SPI was procured from Sonic Biochem Extractions Ltd.,
Indore (M.P), India.
Extraction of defatted soy flour was performed in purified
water at pH 9 (adjusted with 0.2 M NaOH) and at 50 ºC with a
solid/liquid ratio of 1/10, using a mechanical stirrer (Jyoti, model
JSI-555, India) for one and half hours. Solid-liquid separation was
performed in a centrifuge (Remi instruments-model K-70, India)
at 10kg for 20 minutes at 15 °C temperature . The supernatant
obtained was used as a feed to the ultrafiltration after prefiltration
through a micro filtration unit (Millipore). 10kDa hollow
fibre type ultrafiltration cartridge (GE Healthcare, model UFP-10-
C-4MA, USA) was used with maximum trans-membrane pressure
2kg/cm2 and a volume concentration ratio of 3.5 for getting a
concentrate with total soluble solids 20 °Brix.
Concentrated solution obtained from ultrafiltration was dried
using a freeze dryer (Bio Sync Technology, New Delhi), spray
dryer (Yamato mini spray dryer, ADL 31) and hot air over dryer
(Oric, reliable instruments, India).
For freeze drying protein solution was frozen for 12 hours
at -18 °C. Frozen samples then freeze dried at 45 °C compressor
temperature and 0.5mm vacuum pressure. Freeze dried samples
were grounded using a mortar and pestle and sieved through a
No. 100 mesh.
Laboratory scale spray dryer with double fluid nozzle
arrangement and co-current flow pattern was used for study.
Spray dryer was operated with a combination of three inlet
temperatures (170, 180 and 190 °C) &three atomizing air
pressure ranges (0.15-0.2, 0.2-0.25 and 0.25-0.3kg/cm2), while
outlet temperature was fixed at 85 °C. Obtained SPI powders were
stored under refrigerated condition for further analyses.
Laboratory scale hot air oven was used for drying SPI solution
obtained by ultrafiltration. Combinations of drying time (24, 36
and 48 hours) and temperatures (40, 50 and 60 °C) were set for
the study. Dried protein isolates were grounded using a mortar
and pestle and sieved through a No. 100 mesh.
Changes in rheological properties of SPI with different drying
methods were measured with a rheometer (Anton Paar, MCR51,
Austria (software: RHEOPLUS/32 V2.81)). Storage modulus(G’),
loss modulus (G”) andviscosity (η) were measured for ultrafiltered
spray dried, freeze dried and oven dried SPI. Obtained
results were compared with commercially available counterpart.
Parallel plate geometry (PP50-SN8586) with 50mm diameter was
used for testing the samples. About 3ml of protein dispersion (25%
w/v) was loaded onto the lower plate set at 25 °C. Gap between
upper and lower plate was set as 1mm. Rheological characteristics
of protein dispersion were measured using dynamic frequency
sweep test by comparing the dependence of G’ and G’’ of protein
isolate dispersion with angular frequency. Oscillation mode was
set with 5% shear rate and dynamic angular frequency starting
from 0.001 to 1000 1/s. 16 data points were measured with
mean point duration 10 seconds. All measurements are made in
Particle size distribution of ultra-filtered SPIs (spray dried,
oven dried and freeze dried) and commercially available SPI were determined using a particle size analyser (Mastersizer, Malvern
Inc., Worcestershire, United Kingdom) with wet feed attachment.
Samples were dispersed in deionised water (1% w/v). Particle
size analysis for average particle sizes (D[3,2] - Surface Area
Moment Mean - Sauter Mean Diameter and D[4,3] - Volume or
Mass Moment Mean - De BrouckereMean Diameter) and width of
the distribution or Relative Span Factor (RSF) were analyzed.
The, Mean diameters D[3,2] and D[4,3] are defined by:
Where ni is the number of particles of diameter di.
RSF is defined as:
Where: D(v,0.5), D(v,0.9) & D(v,0.1) are the particle diameters
where 50%, 90% and 10% volume distribution below this value
respectively. The obscuration of all samples was maintained
between 10 and 15%. Pump speed was set as 3000rpm and
beginning of test, samples were sonicated. All measurements are
made in triplicate.
WAC was determined according to the method of Beuchat 
with some modifications. Approximately 0.5g of SPI was taken in
50ml centrifuge tube and 6ml distilled water was added to that.
The tube was agitated on a vortex mixer for 1min at higher speed.
The sample left for 30min and centrifuged at 5000g for 20min.
WAC can be expressed as ml of water per gram of sample and was
calculated using the following formula:
WSI was determined according to the method of Morsy et al.
 with some modifications. 0.2gram SPI was mixed with 5ml
distilled water in a 50ml centrifuge tube and agitated in a vortex
mixer for 2 minutes. The solution was centrifuged for 15 minutes
at 3000rpm and the supernatant was separated. Water solubility
index was calculated as:
Triplicate runs were carried out for each experiment and the
data were subjected to statistical analysis. Statistical analysis
was done using SAS 9.3 software. One-way analysis of variance
(ANOVA) was performed to evaluate the significance in differences
(p<0.05) and LSD was used to determine whether the averages of
two sets of measurements were significantly different at P<0.05.
SPI samples variation in storage modulus (G’), loss modulus
(G”) and complex viscosity (η) with angular frequency is shown in
(Figure 1). G’ and G” showed similar kind of frequency dependence
for all different treated samples except for commercial sample.
Moduli of oven dried, and spray dried SPI samples showed more
frequency dependence [higher slope of log (G’) and log (G”) vs.
frequency] than that of the commercial SPI. Frequency sweep test
G’ and G’’ values of for various treatment samples at the beginning
were 30 to 3880 Pa and 11 to 187 Pa and at the end 33 to 6510 Pa
and 165 to 407 Pa respectively.
Normal control diet feed was prepared using soybean
(27%), whole maize flour (Zea mays) (59%), vegetable oil
(10%) and salt/vitamin mix (4%) was from Sigma-Aldrich Co.
Ltd., Poole Dorset, UK. Same ingredients were used for test
diet except Detarium senegalense seed oil was used wholly for
10% OSO feed and varying percentages (1.5%, 1.0%, and 0.5%)
were supplemented with commercially consumed vegetable oil
(VO) to make up the difference. Total oil composition was 10%
for all formulated feeds used in this study. The different feed
ingredients were thoroughly mixed, made into pellets for easy
handling by animals and oven dried to prevent the growth of
mold. Fully dried feeds were kept in previously labeled air tight
bags and were stored at 4 °C to prevent microbial spoilage.
Commercial SPI showed almost constant G’ with increase in
angular frequency whereas rest of the samples shown a gradual
increase (Figure 1(a)). The increase in G’ was irrespective of the
treatments and this shows the solid like characteristics of SPI
samples with increased angular frequency. Initially highest G’
value was obtained for commercial sample followed by different
spray dried and oven dried samples but at higher angular
frequency all the treatments shows G’ values almost in the
same range. G” of all samples increased with increase in angular
frequency irrespective of the treatment (Figure 1(b)). Commercial
SPI showed highest G” value but the rate of increase is less than
spray dried and oven dried. Hence commercial SPI shows more
liquid like properties with increase in angular frequency. Doublier
 reported that the frequency independence in G’ and G” is an
indication of a strong gel network, with no possibility of rupturing
the junction zones within the time-scale of experiments. The
strong frequency dependence of G’ and G” indicates that there
is no specific interaction between molecules. Large slope of the
G’ curve indicates low strength and a small slope indicate high
Commercial SPI complex viscosity (η) decreased from 16.4
to 2.4 Pa.S with increase in angular frequency hence observed a
shear thinning behavior but Spray dried, Oven dried and freeze
dried samples exhibited a slight increase in η value with increasing
angular frequency value (Figure 1(c)), hence exhibited a shear
thickening behavior. The rate of increase is less for oven dried
samples compared spray dried SPI. Koksel et al.  explained that
the viscosity increases as a result of water uptake by the samples
following hydrogen bond disruption due to heating. Granger et al.
 reviewed that increase in viscosity may be due to an increase
in droplet concentration and/or variation in the droplet size.
In commercial SPI, G’ and G” (G’ greater than G”) were followed
almost parallel path with angular frequency (Figure 2(a)), shows
the characteristics of a weak visco-elastic gel behavior. Low value
of loss tangent (tan δ) also indicates the elastic like property of
commercial SPI suspension. Where as in freeze dried, spray dried
and oven dried sample exhibited a cross over point between G’ and
G” with angular frequency below 100 1/s except for oven dried
SPI which is dried under 60 °C for 48 hours (Figure 2(b,c,d,e,f)).
Ross-Murphy  reported that the point at which G’ becomes just greater than G” (cross-over point), is taken as an empirical
indication of gel formation and was accompanied by a rapid fall
in tan δ. The rapid drop in Tan δ also indicates the transformation
of solution into gel. For commercial SPI and oven dried SPI (60 °C
for 48 hours) tan δ (less than unity) were almost constant (Figure
2(a,d)) during oscillation test indicated solid or gel property.
Whereas spray dried SPI (190 °C inlet temperature and high
air flow rate 0.25-0.3kg/cm2) tan δ decreased from 1.05 to 0.42
indicates transition from liquid or sol like characteristics to solid
or gel like characteristics. For rest of the samples, tan δ values
less than unity throughout the frequency sweep with a rapid drop
after cross over point. Salunkhe & Kadam  also found that the
spray-dried LPI produced a gel with more elastic properties than
Figure 3 shows the particle size distribution for various SPI
samples and Table 2 shows the corresponding D[4, 3], D[3,2] and
span values. Single distinct peak was observed for all samples. Most
of the samples had particle sizes above 100μm. Commercial SPI
(Sonic Biochem) and oven dried SPI samples had comparable size
distribution whereas there observed a higher peak and narrow
distribution for freeze dried sample (Figure 3(a)). This is due to
the fluffy nature of freeze-dried sample and this observation is on
par with the findings of Liu et al. . From Figure 3(b), significant
higher peak for spray dried samples at higher temperatures and
air flow rates were observed. The particle size of milk powder
is studied by Laval & Pak  and he reported that the particle
size is related to its appearance, reconstitution property and
flow characteristic. He observed that particle size distribution is
influenced by original milk characteristics, processing conditions
and type of equipment used in drying process.
Oven_40_24: oven drying at 40 °C for 24 h; Oven_40_36: oven drying at 40 °C for 36 h; Oven_40_48: oven drying at 40 °C for 48 h; Spray_170_
low: spray drying at 170 °C inlet air temperature and 0.15-0.2 kg/cm2 air pressure; Spray_170_medium: spray drying at 170 °C inlet air temperature
and 0.2-0.25 kg/cm2 air pressure; Spray_170_high: spray drying at 170 °C inlet air temperature and 0.25-0.3 kg/cm2 air pressure.
The De Brouckere Mean Diameter reflects the size of those
particles which constitute the bulk of the sample volume and
is sensitive to the presence of large particulates in the size
distribution. D[4, 3] value indicates the size of the coarse
particulates that make up the bulk of this sample. Oven dried
samples had comparatively lesser coarse particles. Spray dried
samples exhibited higher D[4, 3] value may be because of the use
of double fluid type spray nozzle for atomizing the sample. This
observation is in accordance with the findings by Elversson et al.
 and he reported that there is a linear relationship between
the droplet size and size of the powder particles.
The Sauter Mean Diameter D[3, 2] is most sensitive to the
presence of fine particulates in size distribution and this can be
used to monitor proportion of fine particles in samples. Similar
to D[4, 3] oven dried samples had lesser D[3, 2] value and spray
dried samples had higher value. Commercial SPI and freeze-dried
SPI possessed good number of fine particles. Carić  conducted
study on spray drying of milk powder and he observed that spray
dried powder particles are usually spherical with diameters
ranging from 10 to 250μm. He found that rapid dispersion
requires a particle size of about 150 to 200μm and powder with
large particle size has superior dispersibility. Singh  observed
that dispersibility of powder decreases as the percentage of fine
particles (<90μm) increases.
Relative span factor (RSF) is a dimensionless parameter indicative
of the uniformity of the drop size distribution. It provides
a practical means for comparing various size distributions, closer
number to zero more uniform the sample will be. Commercial
SPI had lesser uniformity compared to freeze dried, oven dried
and spray dried samples. Also, there observed a slight increase
in uniformity of particle distributions with the increase in drying
time and air flow rate in the case of oven drying and spray drying
respectively. Effect of inlet air temperature on particle size could
not identify clearly from the observations. Similar conclusion was
drawn by Seth et al. . Agrahar-Murugkar et al.  observed
that particle size of flour greatly influences the water absorption
capacity, density and spread of biscuits.
From Table 3, highest water absorption capacity was
observed for commercial SPI (8.83±0.02ml/g) followed by freeze
dried SPI (6.47±0.02ml/g). Oven dried, and spray dried samples
show comparatively low WAC values, and this may be due to
the denaturation of protein caused due to adopted processing
methods. Jovanovich et al.  concluded that this variation is
due to highly condensed and less porous particle morphology of
the spray dried protein powders. Lili et al.  and Singh et al.
 also observed that the proteins obtained from freeze-dried exhibited a much stronger capacity of absorbing water than that
from spray-dried powders (p<0.05). Highest WAC of commercial
SPI may due to the presence of additives.
Higher solubility of proteins helps to form stable dispersions
when incorporated into beverages and other food systems. WSI
of spray dried SPI samples were highly comparable to freeze
dried and oven dried samples but for commercial SPI was lesser.
Wang & Johnson  reviewed that functionalities such as gelling,
emulsifying and foaming are closely associated with solubility.
The high-water solubility of spray dried and freeze-dried protein
powders can be attributed to less thermal stress encountered
in drying process. Similar findings were reported by Pacheco
et al.,  and Yang et al. . Another possible reason for the
variation in WSI was explained by Wagner et al.,  and Pace
et al., , as the concentration of protein increases its solubility
decreases caused by increased protein-protein interaction.
Roesch & Corredig  concluded that when soy protein is at high
concentration it will be aggregated; while at lower concentrations
various types of soluble complexes may form. Corrigan et al. 
also reported that high energy amorphous form caused by spray
drying led to improve the functional properties of powder such
as the enhanced solubility and faster dissolution rate. Lower WSI
values for oven dried samples were due to the severity of heat
treatment as well as the higher bulk density of oven dried samples
compared with freeze dried and spray dried SPIs. This observation
is on par with the findings of Mirhosseini & Amid .
The rheological properties, protein solubility and particle
size distribution of isolate powder obtained through different
drying methods were compared with commercial SPI. Storage
and loss modulus of freeze dried, oven dried, and spray dried SPI
samples have shown more frequency dependence than that of the
commercial SPI. Commercial SPI showed almost constant G’ with
variation in angular frequency whereas rest of the samples shown
a gradual increase. G” of all samples increased with increase in
angular frequency irrespective of the treatment. Commercial SPI
showed a decrease in η with increase in angular frequency hence
observed a shear thinning behavior. Spray dried, Oven dried, and
freeze-dried samples exhibited a slight increase in η value with
angular frequency value, hence exhibited a shear thickening
Commercial SPI and oven dried SPI samples had comparable
size distribution whereas there observed a higher peak and
narrow distribution for freeze dried sample. Oven dried samples
had comparatively lesser coarse particles. Commercial SPI had
lesser uniformity compared to freeze dried, oven dried and spray
dried samples. Highest water absorption capacity was observed
for commercial SPI and oven and spray dried samples showed
comparatively low WAC values. WSI of spray dried SPI samples
were high compared to freeze dried and oven dried samples.
Commercial SPI also showed lesser WSI value than its other
We conclude the drying methods and conditions have significant
influence on rheological properties, solubility characteristics
and particle size distribution of soy protein isolate obtained
through ultrafiltration which in turn decides the functionality and
end use of isolates in food applications.