International Journal of Biological Macromolecules 115 (2018) 1174–1182

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Physicochemical, molecular, emulsifying and rheological

characterizations of sage (Salvia splendens) seed gum
Soo Young Seo, Yu-Ra Kang, Yun-Kyung Lee, Jin Hye Lee, Yoon Hyuk Chang ⁎
Department of Food and Nutrition, Kyung Hee University, Seoul 02447, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: The objective was to investigate the physicochemical, molecular, rheological, and emulsifying properties of water
Received 9 February 2018 soluble-sage seed gum (WSG). WSG mainly comprised galacturonic acid and xylose. FTIR and NMR analyses con-
Received in revised form 27 March 2018 firmed the presence of pectic polysaccharides in WSG. Additionally, the molecular weight of WSG was higher
Accepted 30 April 2018
than that of pectin standard. Compared to pectin standard solutions, WSG solutions exhibited higher shear thin-
Available online 03 May 2018
ning behavior and higher values of apparent viscosity (ηa,100) and consistency index (K) in steady shear measure-
Keywords:
ments. According to the results of frequency sweep test, the dynamic moduli (G′ and G″) for WSG solutions were
Sage (Salvia splendens) seed gum increased with increasing frequency and concentration. The changes in dynamic moduli of WSG solutions as a
Chemical structure function of aging time at 4 °C indicated that WSG could form a more rigid network than pectin standard. Accord-
Rheological properties ing to the results of temperature sweep test, the dynamic moduli of WSG solutions were higher than those of pec-
Emulsifying properties tin standard solutions. Emulsion capacity and stability analyses indicated that WSG (58 and 56%, respectively)
had better emulsifying properties than pectin standard (46 and 37%). In conclusion, compared to pectin standard,
superior rheological and emulsifying properties observed in WSG might be related to higher molecular weight
and protein content, respectively.
© 2018 Published by Elsevier B.V.

1. Introduction
Salvia splendens is the plants of belongs to Labiatae family, a large
number of annual and perennial plant species and is one of several gen-
era commonly referred to as sage [1]. In addition to Salvia splendens, dif-
ferent Salvia species, such as Salvia macrosiphon, Salvia officinalis, Salvia
sclarea, Salvia miltiorrhiza and Salvia grandiflora, have been used for food
and pharmaceutical purposes [2–4]. Sage seeds are small rounded seed,
which quickly swells in water to give mucilaginous gum [4]. The sage
seed gum has viscous suspension properties which are comparable
with commercial food hydrocolloids, and therefore it is regarded as a
new source of hydrocolloid [5–7]. Gum is hydrophilic high molecular
weight biopolymers which have the ability to greatly hydrate in contact
with water, thus producing colloidal systems of different structures [8].
Rheological and emulsifying characterizations of gum depend on con-
centration, temperature, dispersion, dissolution, electrolyte, mechanical
treatment and structure that influence food process conditions [9].
Gums, such as pectin, guar gum, locust bean gum and tara gum, have
been widely used as a thickener, emulsifier, stabilizer and gel strength-
ener in the food industry due to their ability to form viscous solutions at
relatively low concentrations [10,11].
⁎ Corresponding author.
E-mail address: yhchang@khu.ac.kr (Y.H. Chang).
Dynamic rheological properties can be used along with steady shear
rheological properties to provide insight on the structure of the gum so-
lutions [12]. Dynamic shear rheological tests have been used to charac-
terize or classify the viscoelastic properties of macromolecular
dispersions [13]. In a dynamic test, the storage modulus (G′), loss mod-
ulus (G″) and complex viscosity (η*) have been determined using a si-
nusoidal strain cycle. The dynamic test is a non-destructive technique
enabling measurements to be made without structural breakdown to
the sample and it allows researchers to relate dynamic rheological pa-
rameters with the molecular structure of the sample. Thus, dynamic
rheology is one of the most extensively used methods to assess the vis-
coelastic behavior of gum solutions or gels [14].
At present, the food industry has placed a significant amount of at-
tention on gums for their roles as stabilizers in oil in water (O/W) emul-
sions. Most of gums can form stable O/W emulsions because they are
adsorbed at the oil-water interfaces [15]. The adsorption properties
are seemed to be attributed to the hydrophobic groups or proteinaceous
moieties related to gums [16]. Therefore, it is important to extract gums
from natural sources and investigate relation between their structure
and emulsifying capacities.
Until now, a few researchers have focused on the structural and
physicochemical properties of sage gums extracted from Salvia
macrosiphon seed [4]. It has been reported that the gum extracted
from Salvia macrosiphon seed was a galactomannan (mannose to galac-
tose ratio of 1.78–1.93:1) containing 28.2–32.2% uronic acids and could

https://doi.org/10.1016/j.ijbiomac.2018.04.173
0141-8130/© 2018 Published by Elsevier B.V.
 S.Y. Seo et al. / International Journal of Biological Macromolecules 115 (2018) 1174–1182
be used as stabilizer, thickener, and emulsifier in food products because
of its characteristic physicochemical and rheological properties [4,17].
In general, the physicochemical, molecular, emulsifying, and rheological
properties of gums can be remarkably affected by the species, growth
conditions, and origin of Salvia. However, no detailed studies on sage
seed gums extracted from Salvia splendens seed have been found in pre-
vious literatures. Therefore, the present study was carried out to inves-
tigate the chemical composition, molecular weight distribution, partial
structure, and rheological and emulsion properties of water soluble-
sage seed gum (WSG) to explore its potential application as a thickening
agent and emulsion stabilizer in the food industry.
2. Materials and methods
2.1. Materials
The sage seed (Salvia splendens) was obtained from Danong Ltd.
(Namyangju, Korea). The sage seed was grinded into fine powder
using a laboratory blender (7011S, Waring Blender, Torrington, CT,
USA), sieved and stored at −20 °C until further use. Commercial pectin
standard was provided from CP Kelco [citrus pectin, high methoxyl (DE
N 50%), Lille Skensved, Denmark]. Monosaccharide standards of
rhamnose, arabinose, glucose, xylose, galactose and mannose were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other
chemicals and solvents used were of analytical grade unless otherwise
specified.
2.2. Extraction of water soluble-sage seed gum
The extraction procedure of WSG followed the method of Razavi
et al. [4] with slight modifications. First, the dried powder of sage seed
was mixed with acetone, hexane and 95% ethanol to remove lipids.
The residue was placed in a beaker and distilled water was added
in 1:20 (w/v). The temperature during extraction was maintained at
70 °C for 24 h using a shaking water bath (BS-11, Jeio tech Co., Ltd., Dae-
jeon, Korea) and the extract was centrifuged (VS-5000N, Vision scien-
tific Co., Ltd., Daejeon, Korea) at 3500 rpm for 10 min. The collected
supernatants were mixed with 95% ethanol and kept at 4 °C for over-
night. The ethanol-insoluble precipitate was collected by centrifugation
at 3500 rpm for 10 min and dried in a dry oven (Isotemp 500 Series,
Fisher Scientific Inc., Pittsburgh, PA, USA) at 60 °C for 24 h.
2.3. Chemical composition and monosaccharide composition analysis
The chemical composition of WSG including moisture content, ash
content, protein content and fat content was determined according to
A.O.A.C. methods [18] by 925.09B, 923.03, 979.09 and 920.39C, respec-
tively. The content of total sugar was determined by the phenol-
sulfuric acid method [19]. The uronic acid content was measured by
m-hydroxyphenyl colorimetric method [20] with D-galacturonic acid
(Sigma Chemical Co., St. Louis, MO, USA) as the standard.
The neutral monosaccharide composition of WSG was determined
by the procedure of Cui and Chang [21] with slight modifications.
WSG (10 mg) was hydrolyzed with 2 M TFA (2 mL) at 121 °C for 1 h.
After TFA was removed by a rotary vacuum evaporator (N-N1, Eyela,
Japan), distilled water (1 mL) was added to dissolve the residue. The
sample was passed through a 0.45 μm filter and injected to a high-
performance anion exchange chromatography with pulsed ampero-
metric detection (HPAEC-PAD) (Dionex, Sunnyvale, CA, USA). Separa-
tion of each neutral sugar was performed on a CarboPac PA1 column
(250 mm × 4 mm, Dionex, Sunnyvale, CA, USA). The temperature was
kept at 30 °C and the injection volume was 15 μL. The solvents were
A: 18 mM NaOH and B: 200 mM NaOH. The solvent flow rate was
1 mL/min. The rhamnose, arabinose, glucose, xylose, galactose and
mannose were used as reference standards.
1175
2.4. Fourier transform-infrared spectroscopy
Fourier transform infrared (FTIR) spectrum of WSG was determined
using a Fourier transform infrared spectrophotometer (Spectrum GX,
Perkin Elmer, Massachusetts, USA). The sample was mixed with 100 mg
of KBr under the anhydrous condition, and then pressed into a KBr pellet.
The pellet was scanned in the spectra range of 400–4000 cm−1.
2.5. 1H and 13C NMR spectroscopy
WSG (20 mg) was dissolved in D2O (99.9 atom %) and kept at room
temperature. 1H and 13C NMR spectra were performed by 300 NMR
spectrometer (JNM-AL300, JEOL, Tokyo, Japan), with a frequency of
75 MHz at 25 °C. The chemical shifts (δ) were reported in ppm units
using tetramethylsilane (TMS) as an internal standard; coupling
constants (J) were given in Hertz and referred to apparent peak
multiplicities.
2.6. Molecular weight distribution
The molecular weight distribution of WSG was analyzed using a
high-performance lipid chromatography system (Agilent 1100, Palo
Alto, CA, USA) equipped with a refractive index detector (RID). WSG
(10 mg) was dissolved in distilled water (10 mL) and passed through
a gel filtration chromatographic column of PL aquagel-OH MIXED-H
column (300 × 7.5 mm, Agilent, Amherst, MA, USA). The column was
operated at 30 °C and eluted by triple distilled water at a flow rate of
1 mL/min with an injection volume of 20 μL. The molecular weight
was estimated by the standard curve which was calibrated with dextran
standards (Mw 12,000, 50,000, 150,000, and 410,000 Da, Sigma Chem-
ical Co., St. Louis, MO, USA).
2.7. Rheological properties
2.7.1. Sample preparation
WSG was moderately stirred for 30 min at room temperature in a
magnetic stirrer for hydration and then heated at 70 °C for 1 h in a
water bath with mild agitation provided by a magnetic stirrer. During
heating, a screw-cap Erlenmeyer flask was used to prevent water evap-
oration. At the end of the heating period, the sample mixture was cooled
at room temperature and placed on a rheometer plate for measurement
of its rheological properties.
2.7.2. Steady shear rheological analysis
The steady shear rheological properties of pectin standard and WSG
solutions (2, 4, 6 and 8%, w/w) at 25 °C on a strain controlled Phyusica
MCR 102 rheometer (Anton Paar, Graz, Austria) using a plate/plate
geometry (50 mm diameter, 0.5 mm gap). The steady shear (shear
stress and shear rate) data were obtained over a shear rate rage of
0.1–1000 s−1. After loading, the gum solution was held for 5 min at
the initial test temperature before testing to allow stress relaxation
and temperature equilibration. In order to describe the variation in the
rheological properties of gum solutions under steady shear, the data
were fitted to the well-known power law (Eq. (1)) model, which is
used extensively to describe the flow properties of non-Newtonian liq-
uids in theoretical analysis as well as in practical engineering applica-
tions [22]:
σ ¼ Kγ_
n
ð1Þ
where σ is the shear stress (Pa), γ_ is the shear rate (s−1), K is the consis-
tency index (Pa·sn), n is the flow behavior index (dimensionless). The
apparent viscosity (ηa,100) at 100 s−1 was calculated using the magni-
tudes of K and n.
1176 S.Y. Seo et al. / International Journal of Biological Macromolecules 115 (2018) 1174–1182
2.8. Dynamic shear rheological properties
2.8.1. Frequency sweep
Frequency sweep measurements were performed to investigate
the dynamic rheological properties [storage modulus (G′), loss modulus
(G″), complex viscosity (η*) and tan δ (G″/G′)] of pectin standard and
WSG solutions (2, 4, 6, 8, 10, and 12%, w/w) on a strain controlled
MCR 102 rheometer (Anton Paar, Graz, Austria). Prior to the frequency
sweep tests, a strain sweep test at a constant frequency of 6.3 rad/s
was carried out to determine the linear viscoelastic region. The fre-
quency sweep tests were performed at a strain value of 0.02 (2%)
(within the linear viscoelastic region). Frequency sweep tests of WSG
were performed using a plate/plate geometry (50 mm diameter,
0.5 mm gap) at 25 °C and frequencies (ω) from 0.63 to 63 rad/s.
2.8.2. Time sweep
For time sweep measurements in the aging process, pectin standard
and WSG solutions (8 and 10%, w/w) were loaded onto the rheometer
plate at 4 °C. The exposed sample edge was covered with a thin layer
of light paraffin oil to prevent evaporation during measurements. The
G′ and G″ values were monitored during aging at 4 °C for 1 h at
6.28 rad/s and a strain value of 0.01 (1%) (within the linear viscoelastic
region).
2.8.3. Temperature sweep
The temperature sweep measurements were performed at strain
value of 0.01 (1%) (within the linear viscoelastic region), while the fre-
quency was fixed at 6.28 rad/s. To investigate the effect of temperature
on the rheological properties of pectin standard and WSG solutions (8
and 10%, w/w), a program was set up. WSG was transferred to the rhe-
ometer plate at 4 °C. The exposed sample edge was covered with a thin
layer of light paraffin oil to prevent evaporation during measurements.
Briefly, the programs included: (1) a heating step with a linear temper-
ature increased from 4 to 95 °C, using heating rate of 5 °C/min, (2) a
cooling step with a linear temperature decreased from 95 to 4 °C,
using cooling rate of 5 °C/min.
2.9. Emulsion capacity and stability
The emulsion capacity and stability were determined based on the
method described by Chang et al. [23].
2.10. Statistical analysis
All statistical analyses were performed using SAS version 9.3 (SAS
Institute Inc., Cary, NC, USA). Analysis of variance (ANOVA) was per-
formed using the general linear models (GLM) procedure to determine
significant differences among the samples. Means were compared by
using Fisher's least significant difference (LSD) procedure. Significance
was defined at the 5% level.
3. Results and discussion
3.1. Chemical composition
The extraction yield of WSG was 7.93% (data are not shown). The
chemical composition of WSG is shown in Table 1. WSG contained
8.73% moisture, 14.16% crude ash, 16.62% crude protein and 62.79%
total sugar (Table 1). WSG had 31.75% of total uronic acid contents,
reflecting a poly-electrolyte nature and the relative amount of acidic
polysaccharides in the gum. Razavi et al. [4] also reported that sage
seed gum extracted from Salvia macrosiphon contained 28.2–32.2% of
uronic acid.
Neutral sugar analysis by high performance anion exchange chro-
matography (HPAEC) revealed the presence of xylose as the main neu-
tral sugar component in WSG (Table 1). Minor amounts of fructose,
Table 1
Chemical composition and monosaccharide composition of water soluble-
sage seed gum (WSG).
Chemical composition WSG
Moisture content (%) 8.73 ± 0.54
Ash content (%) 14.16 ± 0.27
Protein content (%) 16.62 ± 0.19
Fat content (%) ND
Total sugar (%) 62.79 ± 0.38
Monosaccharide relative (%)
Fucose 0.13 ± 0.00
Rhamnose 0.80 ± 0.10
Arabinose 2.44 ± 0.04
Galactose 2.18 ± 0.23
Glucose 2.48 ± 0.41
Xylose 22.89 ± 0.75
Total uronic acid (%) 31.75 ± 0.45
All measurements are on a dry weight basis except for moisture.
Data presented are means of three replicates ± the standard deviations.
ND: not detected.
rhamnose, arabinose, galactose and glucose were also found in WSG.
The presence of various neutral sugars in WSG can be attributed to a
more complex polysaccharide structure. This finding was contradictory
with the previous results from Razavi et al. [4], who reported that the
mannose and galactose (mostly galactomannans) were the main
component of monosaccharides in the sage seed gum extracted from
Salvia macrosiphon. Therefore, it was found that different Salvia species
had the different monosaccharide composition.
3.2. Fourier transform-infrared spectroscopy (FTIR)
The FTIR spectrum in Fig. 1(A) shows the characteristic absorption
peaks of WSG. The peaks observed in the regions between 1000 and
1150 cm−1 were corresponded to the skeletal C\\O and C\\C vibration
bands of glycosidic bonds and pyranoid ring, respectively [24–26]. Wu
et al. [27] reported that pectic saccharides extracted from boat-fruited
sterculia seeds showed the bands at 1146, 1070, and 1035 cm−1,
which were characteristic peaks of pectic polysaccharides.
The peaks in the regions between 1200 and 1800 cm−1 reflected the
stretching modes of carboxyl groups [24]. The peak at about 1740 cm−1
was attributed to C_O stretching vibration of the methyl esterified car-
boxylic acid, while the peak at about 1415 cm−1 were attributed to
C_O stretching vibration of nonesterified carboxyl groups [28,29].
The strong and broad absorption peak at 3432.03 cm−1 was assigned
to the O\\H stretch vibration of hydroxyl groups [30] and the signal at
2922.10 cm−1 was attributed to C\\H stretching (including CH, CH2,
and CH3) vibration of the methyl groups [24].
3.3. 1H and 13C NMR analysis
The signals of WSG in 1H NMR spectra (Fig. 1(B)) were assigned
based on the chemical shifts compared with the previous literatures.
The signal at 4.73 ppm can be assigned to the strong chemical shift of
solvent D2O. In the anomeric protons, the signals at 5.08 and 4.84 ppm
were assigned to H-1 of nonesterified and methyl esterified 1,4-linked
α-D-GalpA, respectively [31]. The signal at 3.76 ppm of WSG was
attributed to H-2 of methyl groups binding to carboxyl groups of 1,4-
linked α-D-GalpA, corresponding to the previous results reported by
Košťálová et al. [32]. The low-intensity signal at around 2.20 ppm was
derived from O-acetyl groups of GalpA [33]. The methyl rhamnose sig-
nal at 1.05 ppm of WSG indicated the O-2,4-linked α-L-Rhap [34]. The
two distinguished peaks at 4.42 and 4.19 ppm of WSG apparently
indicated the unesterified and esterified β-D-Xylp, respectively [35].
The 13C NMR spectrum of WSG and its derivatives is presented
in Fig. 1(C). In intense anomeric regions, the signals at 100.2 and
99.0 ppm were attributed to C-1 of methyl esterified and non-
esterified 1,4-linked α-D-GalpA, respectively [36]. The signals at 71.01,
 S.Y. Seo et al. / International Journal of Biological Macromolecules 115 (2018) 1174–1182 1177

Fig. 1. FTIR and 1D NMR spectra of water soluble-sage seed gum (WSG); (A) FTIR, (B) 1H NMR, and (C) 13C NMR.

71.8, 73.17, and 76.95 ppm of WSG were attributed to the C-2, C-3, C-4,
and C-5 of α-D-GalpA, respectively [29]. Signals at 176.2 and 174.7 ppm
were attributed to C-6 of the carboxyl groups of 1,4-linked α-D-GalpA
and methyl estered carboxyl group of α-D-GalpA, respectively [37].
3.4. Molecular weight distribution
The results on molecular weights of commercial pectin standard
and WSG showed that the different molecular weights were ob-
tained by various parameters, such as weight average molecular
weight (Mw), z-average molecular weight (Mz), polydispersity
(Mw/Mn), and number average molecular weight (Mn) (Table 2).
The molecular weight (Mw) (3.214 × 10 11) of WSG was higher
than that (1.268 × 1011 ) of pectin standard. The number-average
molecular weight (Mn) and z-average molecular weight (Mz) of
WSG were also higher than those of pectin standard. Comparison re-
vealed that the polydispersity (Mw/Mn) of WSG was lower than that
of pectin standard, indicating a broad range of molecular weight dis-
tribution [38]. Therefore, WSG showed a macromolecular compound
in comparison to pectin standard.
1178 S.Y. Seo et al. / International Journal of Biological Macromolecules 115 (2018) 1174–1182

Table 2
Molecular characterization, emulsion capacity and emulsion stability of pectin standard and water soluble-sage seed gum (WSG).

Sample Molecular characterization Emulsion capacity (%) Emulsion stability (%)

Mw × 1011 Mn × 1010 Mz × 1012 Mw/Mn × 10

Pectin standard 1.268 0.380 1.020 3.340 46.13 ± 0.09b 36.93 ± 1.06b
WSG 3.214 1.847 1.669 1.740 57.77 ± 1.61a 55.80 ± 0.63a

Mw, weight average molecular weight; Mn, number average molecular weight; Mz, z-average molecular weight; Mw/Mn, polydispersity.
Data presented are means of three replicates ± the standard deviations.
Means (n = 3) within the same column with different superscript letters differ significantly (p b 0.05).

3.5. Steady shear rheological analysis
The analysis of flow behavior of pectin standard and WSG solu-
tions by power law model (Eq. (1)) is shown in Table 3. In general,
power law model presented coefficients of determination (R2 ) N
0.97 for all samples. The flow behavior indexes (n), which indicate
the extent of shear thinning behavior as it deviates away from 1,
ranged from 0.44 to 0.85, reflecting pseudoplastic characteristics of
all samples. The observed shear thinning behavior of pectin standard
and WSG solutions can be explained by disruption of intermolecular
entanglements between polymer chains during shearing, as de-
scribed by Morris [39]. In the same concentration range, the n values
of WSG solutions were significantly lower than those of pectin
standard solutions, explaining a more pronounced shear thinning
behavior of WSG solutions. Morris et al. [40] reported that polysac-
charides with high molecular weight presented a more pronounced
shear thinning behavior. Therefore, the result of the higher shear
thinning behavior of WSG solutions may be explained by the higher
molecular weight of WSG compared to pectin standard, as indicated
in Table 2.
A concentration (2–8%) had a distinct effect on the n values of pectin
standard and WSG solutions (Table 3). For instance, an increase in the
concentration of pectin standard and WSG solutions from 2 to 8% caused
a significant reduction of n values from 0.85 to 0.60 and 0.67 to 0.44, re-
spectively, showing the higher shear thinning behavior of all sample so-
lutions at the higher concentration. Anvari et al. [41] noted that the
higher shear thinning behavior at the higher concentration can be ex-
plained by the increased amounts of high molecular weight molecules
in the liquid phase.
In the same concentration range, the apparent viscosity (ηa,100) and
consistency index (K) values of WSG solutions were significantly higher
than those of pectin standard solutions (Table 3). According to Wood
[42], an increase in the molecular weight of polysaccharides caused
the increase in their viscosity. Therefore, it was speculated that higher
ηa,100 and K values of WSG solutions might have resulted from higher
molecular weight of WSG compared to that of pectin standard as
shown in Table 2.
The ηa,100 and K values for pectin standard and WSG solutions were
significantly affected by their concentration (Table 3). For instance, ele-
vating concentration of all sample solutions from 2 to 8% resulted in a
substantial increase in the values of ηa,100 and K. The increase in the
values of ηa,100 and K for all sample solutions at the higher concentration
could be attributed to the greater number of junction zones which were
formed in polysaccharide solutions. These junction zones in polysaccha-
ride solutions are known to cause flow interruption [43]. Accordingly, it
is plausible in the present study that the molecules could be drawing
closer to one another and junction zones could be more readily formed
with increasing the concentration, which could cause an increase in the
ηa,100 and K values of all sample solutions at higher concentration.
3.6. Frequency sweep measurements
Fig. 2 shows changes in storage modulus (G′), loss modulus (G″),
and complex viscosity (η*) as a function of frequency (ω) for pectin
standard and WSG solutions at different concentration (2, 4, 6, 8, 10,
and 12%, w/w) at 25 °C. As seen in Fig. 2, pectin standard and WSG so-
lutions at the identical concentration showed the same trends, but the
magnitudes of G′ and G″, and η* of WSG solutions were much higher
than those of pectin standard solutions in the frequency range from
6.3 to 63 rad/s. The magnitudes of G′ and G″ increased with an increase
in frequency, showing a frequency dependency for all concentration of
sample solutions. Furthermore, for all concentration of sample solu-
tions, the η* values decreased linearly with an increase in frequency, in-
dicating a strong shear thinning behavior of them. The increase in the
magnitudes of G′, G″, and η* at the higher concentration of sample solu-
tions was also confirmed. The foregoing results could be explained by
the greater network development in higher concentration of sample so-
lutions. The network development is known to be formed by the strong
interaction between closed-packed molecules in solution [44].
In the experimental range of frequency indicated in Fig. 2, the visco-
elastic behavior of all sample solutions was obviously dependent on
sample concentration. At low sample concentration (b6%), G″ was supe-
rior to G′, showing that sample solutions tended to present mainly vis-
cous characteristics instead of a clear tendency to form a gel. Whereas,

Table 3
Apparent viscosity (ηa,100), consistency index (K), flow behavior index (n), and coefficients of determination (R2) of pectin standard and water soluble-sage seed gum (WSG) solution with
different concentrations at 25 °C.

Concentration (%) Apparent viscosity Consistency index Flow behavior index R2

n
ηa,100 (Pa·s) K (Pa·s ) n (–)

Pectin standard
2 0.08 ± 0.00d 0.16 ± 0.00d 0.85 ± 0.01a 1.00 ± 0.00
4 0.64 ± 0.00c 1.41 ± 0.01c 0.83 ± 0.00b 1.00 ± 0.00
6 2.30 ± 0.03b 7.97 ± 0.07b 0.73 ± 0.00c 0.99 ± 0.00
8 5.62 ± 0.07a 35.75 ± 0.37a 0.60 ± 0.00d 0.98 ± 0.00

WSG
2 0.22 ± 0.01d 1.00 ± 0.06d 0.67 ± 0.00a 1.00 ± 0.00
4 1.28 ± 0.03c 8.38 ± 0.20c 0.59 ± 0.00b 1.00 ± 0.00
6 4.32 ± 0.02b 40.33 ± 0.39b 0.51 ± 0.00c 0.99 ± 0.00
8 9.95 ± 0.35a 129.57 ± 4.84a 0.44 ± 0.01d 0.97 ± 0.00

Data presented are means of three replicates ± the standard deviations.

Means (n = 3) within the same column with different superscript letters differ significantly (p b 0.05).
 S.Y. Seo et al. / International Journal of Biological Macromolecules 115 (2018) 1174–1182 1179

Fig. 2. Plots of log G′ (▲), G″ (□), and η* (+) versus log ω for pectin standard and water soluble-sage seed gum (WSG) at different concentration (2, 4, 6, 8, 10, and 12%) at 25 °C.

sample solutions with concentration of 8 and 10% initially exhibit vis-
cous behavior and, above the cross-over point, G′ values dominated
over G″ values, representing elastic behavior. These rheological patterns
of sample solutions (8 and 10%) were typical of viscoelastic fluids which
were consistent with those of other gums as describe in previous studies
[22]. Finally, in sample solution with concentration of 12%, G′ exceeded
G″ over most of the frequency range investigated, suggesting a clear ten-
dency to form weak-gel network.
The G′, G″, and η* values of pectin standard and WSG solutions at
6.3 rad/s are shown in Table 4. The G′ and G″ values of WSG solutions

Table 4
Storage modulus (G′), loss modulus (G″), complex viscosity (η*), and tan δ at 6.3 rad/s of pectin standard and water soluble-sage seed gum (WSG) at 25 °C.

Sample Concentration (%) G′ (Pa) G″ (Pa) η* (Pa s) tan δ

Pectin standard 2 0.20 ± 0.05e 0.65 ± 0.04e 0.11 ± 0.01e 3.43 ± 0.75a
4 2.43 ± 0.46e 6.93 ± 0.30e 1.17 ± 0.07e 2.92 ± 0.47a
6 14.32 ± 0.51d 31.83 ± 0.85d 5.54 ± 0.16d 2.22 ± 0.02b
8 60.53 ± 1.76c 97.75 ± 2.63c 18.25 ± 0.50c 1.62 ± 0.00bc
10 194.85 ± 15.24b 225.95 ± 13.21b 47.36 ± 3.16b 1.16 ± 0.02cd
12 412.38 ± 3.98a 389.09 ± 4.27a 89.99 ± 0.91a 0.94 ± 0.00d
WSG 2 3.55 ± 0.49e 5.81 ± 0.58e 1.08 ± 0.12d 1.64 ± 0.07a
4 17.01 ± 0.16e 27.52 ± 0.37e 5.14 ± 0.06d 1.62 ± 0.01a
6 90.18 ± 1.94d 120.06 ± 1.33d 23.83 ± 0.35d 1.33 ± 0.01b
8 337.53 ± 8.92c 361.24 ± 3.46c 78.48 ± 1.35c 1.07 ± 0.02c
10 952.93 ± 4.44b 854.78 ± 5.95b 203.20 ± 1.05b 0.90 ± 0.00d
12 2096.01 ± 84.56a 1621.66 ± 48.09a 408.03 ± 32.76a 0.78 ± 0.01e

Data presented are means of three replicates ± the standard deviations.

Means (n = 3) within the same column with different superscript letters differ significantly (p b 0.05).
1180 S.Y. Seo et al. / International Journal of Biological Macromolecules 115 (2018) 1174–1182
at all the concentration were significantly higher than those of pectin
standard solutions, indicating that WSG solutions exhibited the greater
viscoelastic properties compared to pectin standard solutions. The η*
values for WSG solutions were significantly higher than those for pectin
standard solutions at the same concentration. This finding was consis-
tent with our previous results on apparent viscosity where WSG solu-
tions exhibited the significantly higher apparent viscosity than pectin
standard solutions (Table 3).
The tan δ values of WSG solutions with concentration of 2, 4, 6, and
8% were in the range of 1.07–1.64, which demonstrated that the sample
solutions exhibited mainly viscous properties instead of elastic proper-
ties. On the other hand, the tan δ values of WSG solutions with concen-
tration of 10 and 12% were 0.90 and 0.78, respectively, indicating the
sample solutions were more elastic than viscous (Table 4). For pectin
standard solutions, only sample solution with concentration of 12%
exhibited elastic properties instead of viscous properties at 6.3 rad/s
(tan δ = 0.94). In view of foregoing results, superior viscoelastic proper-
ties were confirmed in WSG solutions compared to pectin standard
solutions, reflecting the greater network development in WSG
solutions. The differences in macromolecular conformation between
pectin standard and WSG might be a reason for the foregoing results
because the conformation of macromolecule could contribute to the
inter- and/or intra-molecular interactions essential for strengthening
polymer network [43]. Moreover, the viscoelastic properties of pectin
standard and WSG solutions were strongly influenced by the sample
concentration.
3.7. Time sweep measurements
Changes in G′ and G″ as a function of aging time for 1 h at 4 °C were
measured for pectin standard and WSG solutions at different concentra-
tion (8 and 10%), as illustrated in Fig. 3. For both pectin standard and
WSG solutions, the G′ and G″ values were not remarkably changed dur-
ing aging for 1 h. This phenomenon can support that WSG solutions as
well as pectin standard solutions could be very stable during storage
at refrigeration temperature (4 °C). For WSG solution with concentra-
tions of 8%, G″ values were higher than G′ during all the aging time,
Fig. 3. Time evolution of storage (G′; open symbols) and loss (G″; filled symbols) moduli
for pectin standard (○) and water soluble-sage seed gum (WSG, □) solutions at different
concentration (8 and 10%, w/w) at 4 °C for 1 h.
indicating that viscous properties predominated over the elastic compo-
nent G′. Whereas, WSG solution with concentration of 10% had elastic
properties rather than viscous properties because G′ was higher than
G″ during all the aging time, representing a progressive reinforcement
of the polymer networks. Similar trends were also observed in pectin
standard solutions. However, G′ and G″ values of WSG solutions were
largely higher than those of pectin standard solutions throughout the
entire time window. These results were probably due to the formation
of a more rigid polymeric network in WSG solutions compared to pectin
standard solutions [45]. Moreover, under the experimented conditions,
the magnitudes of G′ and G″ of WSG solutions were increased with an
increase in sample concentration, suggesting enhanced formation of
macromolecular network at higher concentration.
3.8. Temperature sweep measurements
Changes in magnitudes of G′ and G″ during heating from 4 to 95 °C
for pectin standard and WSG solutions with different concentration (8
and 10%) are shown in Fig. 4. During the initial heating, G′ and G″ values
decreased quickly as temperature increased, reaching a minimum value
at 95 °C. The decrease in G′ can be related to the increase in fluidity with
increasing temperature. Also, this decrease may be attributed to the en-
ergy dissipation movement of the molecules and a decrease in intermo-
lecular interactions, which in turn decrease the energy needed for the
flow, thus decreasing the interference of the hydrodynamic domains.
The reduction in G′ values with increasing temperature is well-known
and usually explained by an increase in thermal activity of molecules,
resulting in an increase in molecule free volume and a simultaneous
decrease in intermolecular and/or intramolecular hydrogen bonding
interactions [46,47].
The G′ and G″ values of all samples steadily increased with decreas-
ing temperature from 95 to 4 °C (Fig. 4). In all samples, the G′ values dra-
matically increased when temperature was decreased up to a critical
value. Such increase in G′ values can be attributed to the formation of
a three-dimensional network structure due to the strong interactions
in the samples. Hesarinejad et al. [14] reported that the increase in G′
values was associated with the structural changes, such as formation
of hydrogen bonds, occurred during cooling of sample solutions.
The gelation temperature for a biopolymer is defined as the temper-
ature where the G′ value equals the G″ value (the sol-gel transition)
[48]. For WSG solutions with concentration of 8 and 10%, during heating
and cooling, G′ values were higher than G″ values in the whole range of
investigated temperatures. There was no cross-over point for G′ and G″,
which means WSG tended to exist in aggregated helical structures to
form three-dimensional networks. In other words, WSG solutions
with concentration of 8 and 10% showed a typical gel-like behavior.
On the other hand, for pectin standard solution with concentration
of 8%, G″ values were higher than G′ values during heating and cooling.
It means that 8% pectin standard solution tended to exist in a random
coil state, subsequently presenting the predominance of the viscous
character. In the case of pectin standard solution with concentration of
10%, the sample solution showed gel-like behavior in the beginning of
the heating step (G′ N G″). The G′ and G″ values decreased with an in-
crease in temperature and G′ values decreased much faster than G″
values. Then, a cross-over point between G′ and G″ values were ob-
served at around 20 °C (melting point). After the melting point, the vis-
cous behavior became predominant until 95 °C (G″ N G′), which means
that the dissociation of the aggregated helices happened, consequently
resulting in helix to coil transition (gel-sol transition). During cooling
from 95 to 4 °C for 10% pectin standard, the sol-gel transition occurred
at around 20 °C (gelation point) and the elastic behavior became pre-
dominant after gelation point. It seems that pectin standard solution
with concentration of 10% might undergo a structural transformation
from random coil to helical structure. Moreover, at lower temperature
than gelation point, the side by side association of the helical structure
 S.Y. Seo et al. / International Journal of Biological Macromolecules 115 (2018) 1174–1182
Fig. 4. Temperature dependence of storage (G′; △) and loss (G″; ○) moduli for pectin standard (A) and water soluble-sage seed gum (WSG; B) solutions at different concentration (8 and
10%, w/w) during initial heating from 4 to 95 °C and subsequent cooling from 95 to 4 °C at a rate of 5 °C/min.
might occur to form three-dimensional network, as seen in the higher
G′ values than G″ values.
A thermal hysteresis was found for all concentration of pectin stan-
dard and WSG solutions during the testing temperature range. More-
over, thermal hysteresis area decreased when the concentration of
sample solutions increased from 8 to 10%. The decreasing hysteresis
area might be due to a denser and greater network formation in higher
concentration of sample solutions.
Finally, during heating and cooling procedures, the dynamic moduli
(G′ and G″) of WSG solutions were remarkably higher than those of pec-
tin standard solutions. According to Yaich et al. [49], the variation in dy-
namic moduli was associated with the molecular weight. In other
words, when polysaccharides were degraded, a decrease in the dynamic
moduli could be observed due to the reduced interactions between
small ions and charged chains in solutions. Furthermore, Yaich et al.
[49] described that the higher dynamic moduli might be caused by the
greater network formation in sample solution due to the existence of
expanded and high molecules which can lead to polymer-polymer asso-
ciations. Accordingly, in the present study, higher dynamic moduli of
WSG compared to those of pectin standard could be associated with
greater molecular weight of WSG (Table 2). Also, the dynamic moduli
of pectin standard and WSG solutions increased with an increase in
concentration of sample solutions. It can be explained that the sample
solution with higher concentration has a denser network with more
interconnected chains. The denser network can retain the greater
amount of aqueous phase, resulting in formation of stronger gel [49].
3.9. Emulsion capacity and emulsion stability
The emulsion capacity and stability of pectin standard and WSG are
shown in Table 2. The emulsion capacity and stability of WSG were
1181
57.77 and 55.80%, respectively, and were significantly higher than
those of pectin standard. In general, it has been widely known that
many commercial polysaccharides, such as gums and pectins, include
a small amount of protein, either as a contaminant or as an intrinsic
part of their structure. These proteins can provide good emulsifying
ability to the polysaccharides, since those are usually strongly hydro-
phobic. That is, the hydrophobic proteins in polysaccharides can adsorb
at oil-water interfaces to form stabilized layers around oil droplets [21].
Due to their ability to act as the strong anchor at an oil-water interfacial
film, gums have often been used as ingredients to stabilize oil-in-water
food emulsions [50]. Therefore, when compared to pectin standard, the
higher emulsion capacity and stability of WSG can be due to the higher
protein content (16.62%, Table 1) of WSG than that of pectin standard
(1.47%, data are not shown).
4. Conclusion
The water soluble gum (WSG) from sage (Salvia splendens) seed was
isolated and its structural, physicochemical, and rheological properties
were studied. Molecular weight distribution analysis indicated that
WSG had slightly higher molecular weight than pectin standard. FTIR,
1
H NMR, and 13C NMR spectra confirmed successful extraction of WSG
from sage seed gum. According to the results of steady shear rheological
analysis, compared to commercial pectin standard, WSG solutions
showed higher shear thinning behaviors, apparent viscosity (ηa,100),
and consistency index (K). Furthermore, the dynamic shear rheological
analyses indicated that WSG tended to form the more viscoelastic solu-
tions compared to commercial pectin standard. Besides, we confirmed
that WSG had better emulsifying capacities than pectin standard. There-
fore, it is suggested in the present study that WSG could be used in food
industry as a potential surfactant, thickening, stabilizing, and gelling
1182 S.Y. Seo et al. / International Journal of Biological Macromolecules 115 (2018) 1174–1182

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