International Journal of Biological Macromolecules 128 (2019) 629–637

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International Journal of Biological Macromolecules

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

Physicochemical and rheological characterization of pectin-rich fraction

from blueberry (Vaccinium ashei) wine pomace
Liyuan Feng a,d,1, Yun Zhou a,c,1, Tolulope Joshua Ashaolu a, Fayin Ye a, Guohua Zhao a,b,⁎
a
College of Food Science, Southwest University, Chongqing 400715, PR China
b
Chongqing Engineering Research Center of Regional Foods, Chongqing 400715, PR China
c
Guangxi Key Laboratory of Agricultural Resources Chemistry and Biotechnology, Yulin 537000, PR China
d
School of Resources and Environment Sciences, Baoshan University, Baoshan 678000, PR China

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

Article history: Blueberry wine pomace is rich in pectin, which has been extensively used as a functional dietary fiber and a rhe-
Received 8 October 2018 ological modifier in the food industry. This paper reports a method to extract the pectin-rich fraction (PF) with a
Received in revised form 22 January 2019 mediate degree of esterification of 51.66% from blueberry wine pomace and provides insight into its relationship
Accepted 28 January 2019
between the structure and rheological properties. The impacts of related extrinsic factors, such as sucrose, ions
Available online 29 January 2019
and pH, were also studied in view of food applications. The viscosity of PF aqueous dispersion gradually increased
Keywords:
with its concentration. The addition of sucrose, CaCl2 or NaCl to the solution resulted in increased viscosity. How-
Pectin ever, the elevations in temperature and pH led to decrease in solution viscosity. The viscoelastic property of PF
Blueberry dispersion displayed strong temperature dependence but weak frequency dependence. This was largely due to
Rehological properties PF concentration, sucrose, CaCl2 and solution pH. The present study revealed the unique characteristic of
medium-methoxylated pectin fraction and the obtained results are helpful in value-added utilization of blue-
berry wine pomace.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction
Blueberry (Vaccinium spp.), a very popular berry fruit, is widely con-
sumed as fresh fruit and in processed products such as fruity yogurt, jam
and beverages. The health significance of blueberry is attributed to the
presence of a diverse range of phytochemicals, such as anthocyanins,
flavonols and chlorogenic acids and pectin-rich dietary fibers. On the
average, the pectin content in fresh blueberry fruits is up to 4 g/kg [1].
Blueberry is very suitable for wine processing. During wine process-
ing, the fruits are pressed to release the sweet juice used for fermenta-
tion along with their skins and seeds. Similar to grapes, anthocyanins
of blueberries are present mainly in the skins, while tannins and pheno-
lic acids are abundantly predominant in the seeds [2]. As fermentation
proceeds, these compounds are transferred into wine contributing to
the color, mouthfeel, and antioxidant properties of wine. The skin of
blueberries are abundant in dietary fibers and other bioactive com-
pounds [3]. However, the residues of wine processing, the blueberry
pomace, which accounts for approximately 60% of the total waste, is a
low-value by-product and usually discarded.
⁎ Corresponding author at: College of Food Science, Southwest University, 2 Tiansheng
Road, Chongqing 400715, PR China.
E-mail address: zhaogh@swu.edu.cn (G. Zhao).
1
These authors contributed equally to this work.
Innovative ways are sought to utilize berry pomace in food process-
ing to minimize the amount of wastes, thereby generating a new food
source. Press residues of red/green currant and sea buckthorn berry
could be used as stabilizers and food supplements [4]. Swamp cranberry
pomace extracts could assist in the antimicrobial protection of minced
pork meat [5] and inhibit unfavorable storage changes of lipids and
muscle pigments as food additives for meat products [6]. Incorporation
of dried black currant pomace in wheat dough is a possible way to in-
crease the water absorption and development time of dough [7]. A pre-
liminary study on the blueberry wine pomace showed that the
phenolics extracts obtained by supercritical CO2 fluid extraction exhib-
ited a higher antioxidant activity than that recorded by the conventional
methanol extraction [8]. A pressurized liquid extraction method was
used to recover phenolic compounds, antioxidants and anthocyanins
from blueberry residues [9].
The blueberry wine pomace is rich in dietary fibers, including
5.12 g/100 g soluble dietary fiber and 77.74 g/100 g insoluble dietary
fiber (Supplementary material 1). The chemical composition of dietary
fiber was also investigated, indicating that there was 3.33 wt% pectin,
8.38 wt% cellulose and 20.91 wt% hemicellulose. The content of free
phenol and bound phenol of blueberry wine pomace was 150.83 mg
and 252.73 mg of garlic acid equivalents per 100 g extract, respectively,
on a dry-weight basis. It can be observed that blueberry wine pomace is

https://doi.org/10.1016/j.ijbiomac.2019.01.166
0141-8130/© 2019 Elsevier B.V. All rights reserved.
630 L. Feng et al. / International Journal of Biological Macromolecules 128 (2019) 629–637
rich in phenol compounds, especially the bound ones. Regenerating di-
etary fibers from blueberry wine pomace could improve the commercial
values of wine wastes and the utilization rate of related functional com-
ponents of blueberry.
Pectins, a heterogeneous polysaccharide found in the primary cell
wall and the middle lamella, has been extensively used as a rheology
modifier in the food industry. Depending on the degree of esterification
(DE), pectin can be divided into high methoxyl pectin (HMP) with DE
N 50% (N7% methoxyl), and low methoxyl pectin (LMP) with DE b 50%
(3%–6% methoxyl) [10,11]. DE value of pectin could determine its rheo-
logical properties and gel characteristics of pectin-incorporated food
matrix, along with other intrinsic molecular parameters including mo-
lecular weight, monosaccharide composition, and extrinsic conditions
like temperature and pH [12]. The concentration of pectin and co-
solutes could also influence the viscoelasticity of pectin solution and
gels by influencing intermolecular interactions such as hydrogen bond-
ing, electrostatic and hydrophobic interactions.
Commercial pectin are mostly obtained from citrus peel and apple
pomace. Pectins extracted from various plant species show different
physicochemical properties. Gels formed by pectins extracted from
berry press residues by microwave-assisted extraction were weaker in
gel strength than that of commercial citrus pectin, but stronger than
that of pectin extracted from apple [13]. In this paper, pectin-rich con-
centrate from blueberry wine pomace was obtained by acid-soluble al-
cohol precipitation, and its physicochemical characteristics were
analyzed. The influences of some extrinsic factors on the thickness and
gel-forming properties of pectin were explored in detail.
2. Materials and methods
2.1. Materials
Fresh blueberry wine pomace (BWP) was provided by Tiantaishan
Blueberry Wine Co., Ltd. (Chongqing, China). The pomace was immedi-
ately dried at 50 °C in a thin layer in the oven for 48 h. The moisture con-
tent of the dried pomace was determined as 4.30 g/100 g (wet basis).
The contents of ash, crude protein, fat, total fiber, and pectin were
0.47 g/100 g, 4.46 g/100 g, 5.54 g/100 g, 82.86 g/100 g and
3.33 g/100 g (dry basis), respectively. Analytical grade chemicals were
purchased from Chengdu Kelong Chemical Reagent Co., Ltd. (Chengdu,
China). Galacturonic acid standard (97%) for galacturonic acid content
determination was purchased from Sigma-Aldrich Co., Ltd. (USA). The
standard substances of molecular weight (5, 50, 150, 670 and
110 kDa) and monosaccharides (GalA, Man, Ara, Glu, Rha, Fuc, GluA
and Gal) were abtained from Sigma-Aldrich Co., Ltd. (USA) and
YIFANG S & T Co., Ltd. (Tianjin, China) for monosaccharide composition
and molecular weight characterization, respectively. All the HPLC-grade
reagents were purchased from Tjshield Co., Ltd. (Hebei, China).
2.2. Extraction of pectin-rich fraction
The pectin-rich fraction was extracted according to Fig. 1, which is a
modified protocol of Pasandide et al. [14]. Approximately 100 g blue-
berry wine pomace was dried at 5 °C for 24 h until the attainment of a
constant weight. The dried blueberry wine pomace was grounded and
milled through a 0.15 mm sieve. Hydrochloric acid solution was added
to the blueberry wine pomace powder (pH = 1, 1:15 g/mL) and stirred
for 2 h at 85 °C. The resulting slurries were centrifuged at 4000 rpm for
20 min to remove sediments. The supernatants were filtered and pre-
cipitated with absolute ethanol (1:1, v/v) for 12 h at ambient tempera-
ture. The ethanol extraction was repeated twice. The pectin
concentrates were filtered and washed successively with 80%, 95% and
100% ethanol (1:2, v/v). The pectin-rich fraction was dried by vacuum
lyophilization for further use. The extraction yield was estimated to be
1.62 wt% on a dry basis and the pectin concentration in the fraction
was colorimetrically determined as 65.88 (wt%) by using a meta-
phenyldiphenol assay.
2.3. Physicochemical properties
2.3.1. Galacturonic acid (GalA) content
The content of Gal-A was determined by the colorimetrically meta-
phenyldiphenol assay at 525 nm, using GalA as the standard and
NaOH solution as the blank group [15].
2.3.2. Monosaccharide composition
Monosaccharide composition analysis was performed as described
by Zhang et al. [16]. Briefly, 2 mg pectin was hydrolyzed with 2 mL of
2 M TFA at 110 °C for 3 h. The monosaccharides were derivatized by
the 1-phenyl-3-methyl-5-pyrazolone (PMP) method. The derivatives
were analyzed by HPLC on a Thermo BDS-C18 column (250 × 4.6 mm
i.d, 5 μm) with a UV detector (Shimadzu, Tokyo, Japan) at 30 °C. 20 μL
of PMP derivative were injected, eluted with continuous gradient elu-
tion mobile phase (A phase: 15% (v/v) acetonitrile + 0.05 mol/L
KH2PO4-NaOH buffer, pH 7.1; B phase: 40% (v/v) acetonitrile + 0.05
mol/L KH2PO4-NaOH buffer, pH 7.1) at a flow rate of 0.7 mL/min and
monitored by UV absorbance at 250 nm. Elution time stage is 0 min–
10 min-40 min-50 min-57 min, corresponding to the B concentration
gradient change being 0%—10%—30%—0%—0%. The correction factor is
defined by the equation of f(Lactose) = (Alac × Cmon)/(Amon × Clac),
where f (Lactose) is the correction factor. Alac and Amon are the peak
areas for lactose and a standard monosaccharide, respectively. Clac and
Cmon are the values of concentration (mmol/L) of lactose and a standard
monosaccharide, respectively.
The linear standard curve equations are expressed by the concentra-
tion (mmol/L) of standard monosaccharide as x, while the ratio value of
peak area for corresponding monosaccharide of tested samples and lac-
tose as y.
2.3.3. Molecular weight
The molecular weight was measured by high-performance size-
exclusion chromatography (HPSEC) on a TSK G4000 PWXL-SEC column
(7.8 × 300 mm i.d.) [17]. Pectin-rich fraction (0.5 mg/mL) was dissolved
in deionized water and filtered through a 0.45 μm cellulose acetate
membrane. 20 μL of sample was injected, eluted with 0.02% NaN3 (w/
v) at a flow rate of 0.6 mL/min and monitored using a refractive index
RID-10A detector (Shimadzu, Tokyo, Japan). Dextran of molecular
weight of 5, 50, 150, 670, 110 kDa were used for building the standard
curve. The linear standard curve was expressed by the equation of y
= −0.4497x +10.11, R2 = 0.9986, where x represents retention time,
y as log Mw.
2.4. Rheological properties
All rheological measurements were carried out on a DHR rheometer
(TA Instruments, USA). The parallel plate geometry (60 mm diameter)
was used for both steady shear and oscillatory tests. The prepared sam-
ples were equilibrated at 25 °C for 1 h before being loaded to the rheom-
eter plate with a spoon. Prior to each measurement, sample was left for
10 min to recover the structural breakdown upon loading. The exposed
sample edge was covered with a thin layer of silicon oil in order to pre-
vent moisture evaporation.
2.4.1. Flow behavior
In steady shear measurements, pectin dispersions with varying con-
centrations were prepared by suspending PF in distilled water with con-
tinuous magnetic stirring for 2 h at room temperature. The effects of PF
concentration (0–2.5%, w/w), CaCl2 (0–81.0 mM), NaCl (0–0.7%, w/w),
sucrose (5–20%, w/w), temperature (25–100 °C) and pH (2.9–10.0)
were investigated. The steady shear flow analyses were performed at
25 °C via a ramp of shear rate from 18 s−1 to 1000 s−1in 10 min. The
 L. Feng et al. / International Journal of Biological Macromolecules 128 (2019) 629–637 631

Fig. 1. Schematic flow chart of the extraction of pectin-rich fraction (PF) from blueberry wine pomace (BWP).

results were fitted with Power law models: τ = K × γ_ n, where τ is the
shear stress (Pa), γ_ is shear rate (s−1), K is the consistency index
(Pa·sn) and n is the dimensionless flow behavior index.
2.4.2. Viscoelastic behavior
In visoelastic properties of PF gels were investigated by temperature
sweep and frequency tests. The effects of PF concentrations (0–2.5%, w/
w), CaCl2 (0–81.0 mM), sucrose (10–50%, w/w) and pH (3.0–7.0) were
investigated. In preparing samples for oscillatory tests, PF and sucrose
were first dissolved in 0.1 M citric acid sodium buffer with a specific
pH. Then the dispersions were heated until boiling and thoroughly
mixed with a portion of CaCl2 solution to result in a pre-settled concen-
tration. The resultant dispersion was then cooled to room temperature
and rested for 24 h at 4 °C prior to measurements. Temperature
sweep was performed from temperature decreasing from 85 °C to 25
°C at the rate of 5 °C/min at 1 Hz and 0.5% strain. For frequency sweep,
it was operated from 0.1 to 20 Hz at 5 °C. All oscillatory measurements
632 L. Feng et al. / International Journal of Biological Macromolecules 128 (2019) 629–637

were carried out at a strain % of 0.5% within the linear viscosity region. methoxyl pectin, since its DE was very close to 50%. It is traditionally
The traces of G′ and G″ as a function of temperature or frequency were recognized that soluble solid can facilitate the gelation of high methoxyl
obtained. pectin by reducing water activity and thus increasing the size and num-
ber of cross-linking zones. For a specific pectin solution, a decrease in pH
2.5. Statistical analysis not only enhances the hydrogen bonding between water molecules and
the carboxyl groups of pectin molecules but also weakens the electro-
The level of statistically significant difference was set at p b 0.05 by static repulsion between pectin molecules. Regarding the gelation of
Duncan's procedure using the SPSS statistics 16. All experiments were low methoxyl pectin, it usually requires the presence of metal cations,
done in triplicate and results were expressed as mean ± standard especially divalent calcium ions. In this case, one calcium ion can elec-
deviation. trostatically interact with two negatively charged carboxyl groups of
pectin molecules and this ultimately triggers the formation of a three-
3. Results and discussion dimensional mesh structure called “egg box” [23]. Therefore, the factors
influencing both high methoxyl pectin and low methoxyl pectin should
3.1. Characterization of pectin-rich fraction (PF) be taken into account in evaluating the functionality of PF.
The monosaccharide composition of PF was analyzed by a PMP-pre-
The chemical characteristics of PF are summarized in Table 1. In column derivatization HPLC method. The results revealed that PF con-
order to evaluate the purity of PF, hydroxybiphenyl method was used tains seven monosaccharides, namely GalA, Man, Ara, Glu, Rha, Fuc
to determine its galacturonic acid (GalA) content. The results showed and GluA in a decreasing molar ratio order (Supplementary material
that GalA content (65.88 wt%) in PF extracted from blueberry wine 3). GalA was the most abundant among these monosaccharides, ac-
pomace was slightly higher than the minimum value (≥65 wt%) re- counting for 44.14 mol% while Man is the richest neutral monosaccha-
quired by Food Chemicals Codex (FCC) for commercial pectin products. ride, although it is relatively low in gold kiwifruit pectins [22]. It is
However, the present GalA content was higher than that of the pectin worth noting that galactose (Gal) could not be found in the present PF
from whole blueberry power (51.6 wt%) [18]. samples, although it was present in other pectin-rich pomaces of vari-
Generally, high-performance size-exclusion chromatography was ous fruits sources [22]. This shows that the monosacchride profile of a
used to characterize the molecular weight distribution of various poly- pectin matter certainly depends on its plant origin [24].
saccharides. For the molecular weight distribution pattern of the pres- Rha/GalA value was usually applied to identify the structure of pec-
ent PF, a dominant big peak as well as several small peaks were tin polymers. The present PF was concluded with a Rha/GalA value of
observed in the resultant eluting curve (Supplementary material 2). 0.03 (b0.05), indicating it belongs to HG and RG-II groups [25]. On the
The weight-average molecular weigh (Mw), number-average molecular other hand, the value of (Gal + Ara)/Rha always reflect the extension
weigh (Mn) and polydispersity index (PDI, Mw/Mn) of PF were 641 kDa, of the neutral side chains in a pectin matter [26]. The present PF showed
460 kDa and 1.39, respectively (Table 1). These data were comparable to a relatively low (Gal + Ara)/Rha value (10.45) suggesting the shortness
those reported for the pectin extracted from whole blueberry powder of its side chains of RG-I.
[18]. It was also observed that Mw of PF was higher than that of commer-
cial pectins such as citrus pectin (100–200 kDa) and sugar beet pectin
(400 kDa) [19–21], but lower than that of the pectin from gold kiwifruit 3.2. Flow behaviors of PF solutions
pomace (670 kDa) [22].
As determined by FCC titration method, the degree of esterification Almost all polymer solutions present shear thinning behavior, i.e.
(DE) of PF was 51.66%, which was much higher than that (26%–36%) apparent viscosity decreases as shear rate increases. This behavior is
of the pectins extracted from blueberry by different methods [18]. This usually interpreted as a consequence of insufficient polymer chain en-
indicated the pectin from blueberry wine pomace could be classified tanglements. However, in most cases of pectin-containing samples,
as high methoxyl pectin. However, it could be expected that the present co-solutes and environmental factors such as sugar concentration and
pectin fraction might show some specific properties or functions of low temperature delivered substantial impacts on the rheological properties
of their solutions. As shown in Fig. 2, PF solutions under different condi-
Table 1
tions clearly exhibited a shear thinning behavior, signifying that the
Chemical characterization of pectin-rich fraction from blueberry wine samples were pseudoplastic. However, the situations were more or
pomace. less different among the cases with different co-solutes and environ-
mental parameters. For the cases on PF concentration, sucrose, NaCl,
Character Value
temperature and pH, the shear thinning behavior was mainly observed
Galacturonic acid (wt%)a 65.88 ± 0.06
in low shear rate scope (b30 s−1). However, continuous and significant
Mw (×105 Da) 6.41 ± 0.18
Mn (×105 Da) 4.60 ± 0.21 shear thinning behaviors were observed for CaCl2 cases across the
Mw/Mn (PDI) 1.39 ± 0.11 whole shear rate scope.
DE (%)b 51.66 ± 0.05 The steady rheological characteristics of PF solutions were described
Monosaccharides (mol %) by fitting the shear rate and shear stress to power law equation and the
Galacturonic acid (GalA) 44.14 ± 2.63 results are shown in Table 2. When PF concentration is under consider-
Mannose (Man) 33.59 ± 3.72 ation, the results clearly revealed that the higher PF concentration was
Arabinose (Ara) 16.11 ± 1.73 inevitably accompanied with a higher value in consistency coefficient,
Glucose (Glu) 3.22 ± 0.26
K. The K value increased from 0.872 to 4.777 as PF concentration varied
Rhamnose (Rha) 1.54 ± 0.11
Fructose (Fuc) 0.84 ± 1.12 from 0.1 wt% to 2.5 wt% (Fig. 2a). At the same time, the n value de-
Glucuronic acid (GluA) 0.58 ± 0.12 creases from 1.008 to 0.967. This attested that PF solutions become
Rha/GalA 0.03 ± 0.00 more non-Newtonian as pF concentration increased and the present
(Gal + Ara)/Rha 10.45 ± 0.37
PF system shows the typical behavior of polymer-rich systems. With
HGc 42.60 ± 2.52
RG-Id 19.18 ± 1.95 the aid of statistical analysis, it was found that the K value of the tested
a
systems is linearly correlated to PF concentration (R = 0.9863). This fact
Based on dry matter.
b
DE refers to the degree of esterification.
suggests that the present PF solutions behave as diluted solutions. In
c
HG = GalA-Rha. other words, even at a concentration of 2.5 wt%, the present PF could
d
In this case, RG-I = 2Rha + Gal + Ara. not form a highly viscous liquid via molecular entanglements.
 L. Feng et al. / International Journal of Biological Macromolecules 128 (2019) 629–637 633

Fig. 2. The steady shear flow curves of the dispersion of pectin-rich fraction (PF) from blueberry wine pomace as affected by substrate concentration (a, natural pH), sucrose (b, PF 1.5%,
pH 2.85), CaCl2 (c, PF 1.5%, pH 2.85), NaCl (d, PF 1.5%, pH 2.85), temperature (e, PF 1.5%, pH 2.85) and pH (f, PF 1.5%, pH 2.85).

Regarding the impacts of sucrose, the experiments were carried out
at a PF concentration of 1.5 wt%. The obtained data showed that a signif-
icant increasing tendency in K value (2.85 → 3.79) but a weak increasing
tendency in n value (0.976 → 0.999) were concluded as the sucrose in
the solutions increased from 0 wt% to 20 wt%. In details, either in low
(b30 s−1) or high (N30 s−1) shear rate scope, the resultant amplitudes
of shear thinning gradually weakened as the sucrose concentration in-
creased. At the end of shear, increase in the sucrose concentration of
the solution led to increased final viscosity. A previous study revealed
that the presence of sucrose could accelerate preferential hydration

Table 2
Rheological parameters of solutions of PF based on Power Law equation.

Systema τ = K × γ_ n

K (mPa·sn) n R2

PF 0.1% 0.87 ± 0.12 1.008 ± 0.027 0.9986

PF 0.5% 1.23 ± 0.17 0.994 ± 0.013 0.9995
PF 1.0% 1.80 ± 0.06 0.979 ± 0.024 0.9976
PF 1.5% 2.85 ± 0.11 0.976 ± 0.005 0.9997
PF 2.0% 3.41 ± 0.40 0.978 ± 0.008 0.9999
PF 2.5% 4.78 ± 0.28 0.967 ± 0.008 0.9999
PF 1.5%-Sucrose 5% 2.92 ± 0.07 0.983 ± 0.005 0.9998
PF 1.5%-Sucrose 10% 3.18 ± 0.08 0.988 ± 0.005 0.9999
PF 1.5%-Sucrose 15% 3.52 ± 0.02 0.990 ± 0.001 0.9999
PF 1.5%-Sucrose 20% 3.79 ± 0.06 0.999 ± 0.006 0.9999
PF 1.5%-CaCl2 13.5 mM 141.6 ± 10.5 0.524 ± 0.011 0.9964
PF 1.5%- CaCl2 27.0 mM 86.3 ± 2.40 0.598 ± 0.004 0.9981
PF 1.5%- CaCl2 40.5 mM 61.2 ± 5.03 0.641 ± 0.010 0.9990
PF 1.5%- CaCl2 54.0 mM 45.9 ± 4.71 0.673 ± 0.013 0.9984
PF 1.5%- CaCl2 67.5 mM 41.6 ± 4.23 0.680 ± 0.012 0.9987
PF 1.5%- CaCl2 81.0 mM 46.4 ± 7.85 0.671 ± 0.023 0.9991
PF 1.5%-NaCl 0.1% 2.79 ± 0.06 0.982 ± 0.005 0.9997
PF 1.5%-NaCl 0.3% 3.58 ± 0.11 0.960 ± 0.005 0.9999
PF 1.5%-NaCl 0.5% 4.69 ± 0.20 0.928 ± 0.011 0.9999
PF 1.5%-NaCl 0.7% 5.89 ± 0.11 0.900 ± 0.002 0.9999
PF 1.5%-40 °C 3.03 ± 0.25 0.971 ± 0.008 0.9997
PF 1.5%-60 °C 2.60 ± 0.06 0.983 ± 0.006 0.9999
PF 1.5%-80 °C 2.59 ± 0.09 0.989 ± 0.000 0.9998
PF 1.5%-100 °C 2.42 ± 0.08 0.994 ± 0.004 0.9997
PF 1.5%-pH 2.85 2.85 ± 0.11 0.976 ± 0.005 0.9997
PF 1.5%-pH 4.07 2.88 ± 0.06 0.975 ± 0.005 0.9997
PF 1.5%-pH 6.01 2.58 ± 0.10 0.968 ± 0.011 0.9996
PF 1.5%-pH 8.10 2.30 ± 0.02 0.980 ± 0.001 0.9997
PF 1.5%-pH 10.04 2.40 ± 0.00 0.983 ± 0.000 0.9998
a
The concentrations of PF and sucrose were in wt%.
634 L. Feng et al. / International Journal of Biological Macromolecules 128 (2019) 629–637
[27], by which more water molecules bind to sucrose via hydrogen
bonds [28]. This promoted the crosslinking formation among polymer
chains and gave rise to increased viscosity of high methoxyl pectin solu-
tions [29]. Simultaneously, the hydrophobic interactions among pectin
chains were promoted with sucrose incorporation and thus again pro-
mote crosslinking within polymer chains [30].
Calcium ion (Ca2+) can generate a bridge between the two carboxyl
groups of pectin molecules and thus trigger the formation of a gel net-
work structure with a semi-stable configuration between pectin chains
and Ca2+, known as “egg box” model [31]. As illustrated in Fig. 2c, the
addition of CaCl2 at any concentration (13.5–81.0 mM) brought about
a substantial increase in the viscosity of pectin solution. However, the
resultant amplitude in viscosity does not linearly depend on the concen-
tration of CaCl2 applied. Specifically, the CaCl2 addition at 13.5 mM pro-
duced the largest amplitude in viscosity changes. With the further
increase in CaCl2 concentration from 13.5 mM to 54.0 mM, the resultant
viscosity changes decreased gradually. No significant changes in viscos-
ity were observed upon further increase of CaCl2 concentration from
54.0 mM to 81.0 mM. The significant viscosity increase at 13.5 mM
should be ascribed to the crosslinking of pectin molecules via calcium
bridge. However, as previously pointed out that excessive Ca2+ will
bring about the agglomeration of pectin molecules, thus weakening
the viscosity [11,32]. These results convey that the present PF indeed
showed an ability to chelate divalent cation. This was assumed as a con-
sequence of a certain number of consecutive free carboxyl groups, as
available frequently in low methoxyl pectin. For high methoxyl pectin,
it also can form calcium bridges upon the demethoxylation achieved
by using pectin methyl esterases [33].
It has been proven that pectin, an anionic biopolymer, tends to form
aggregates upon salts addition, being responsible for the increase in vis-
cosity of the pectin involved system [34]. The natural pH of 1.5 wt% PF
solution was determined as 2.85 in the present study. Under these cir-
cumstances, the chains of PF polymers would be negatively charged at
a low density [35]. Upon the addition of NaCl over 0.3 wt%, the resultant
increase in ion strength of the system significantly screened the electro-
static repulsion among pectin molecules. This brought the adjacent
polymer chains closer to each other and, consequently, strengthened
the polymer-based three-dimensional network within the system.
This was ultimately confirmed by a significantly elevated viscosity of
the system at the end of shear (Fig. 2d). Therefore, the addition of
salts like NaCl to a pectin solution manifested an obvious viscosity en-
hancement via the partial shielding of electrostatic repulsive forces as
well as the significant enhancement of hydrogen bonding and
rearranging of polymer chains [10].
To investigate the effects of heating treatment on the rheological
properties of PF solution, an 1.5 wt% PF solution was preprepared and
incubated at different temperatures of 25 °C, 40 °C, 60 °C, 80 °C and
100 °C, respectively, for 20 min before viscosity measurement. The re-
sults were presented in Fig. 2e. Normally, the heating treatment cer-
tainly affected the polymer conformation and the intermolecular
interactions, such as hydrogen bonds, hydrophobic association, electro-
static repulsion and Van der Waals forces [36]. This finally resulted in
more or less changes in the viscosity of pectin solution. Clearly, the
heat treatment at 40 °C caused a slight increase in viscosity, but the
treatments at 60 °C, 80 °C and 100 °C brought minor declines in viscos-
ity. For the viscosity increase induced by the heat treatment at 40 °C, it
was assumed as a consequence of the enhanced pectin solubilization. In
other words, PF did not dissolve completely without heat treatment and
the heat treatment increased the actual pectin concentration in the
tested system [37]. However, for the viscosity decrease induced by the
heat treatments over 60 °C, they could be ascribed to the heat-induced
damages on the intermolecular interactions of pectin molecules, thus
weakening the three-dimensional network within the pectin solution.
Generally, pH is crucial to the charge distribution of the chains of
pectin molecules. The pKa value of the carboxyl groups of pectin mole-
cules is about 3.5 and approximately 50% of them were protonated at
pH 3.0 [35]. The effects of media pH on the viscosity of PF solution were
demonstrated in Fig. 2 f. Obviously, there were no significant difference
between pH 2.85 and pH 4.07 across the whole shear rate range. How-
ever, lower viscosities were observed for the pectin solutions at pH 6, 8
and 10. The present result is in agreement with the results reported for
sunflower head pectin, which presented the highest viscosity at pH 3
[36,37]. At pH higher than 4.5, the galacturonic acid molecules, forming
the pectin chains, were fully charged leading to a high electrostatic repul-
sion and consequent viscosity reduction [36,38,39]. On the contrary, at pH
lower than 3.5, the\\COOH groups are available for hydrogen bonding
thus increasing the viscosity value [40].
3.3. Viscoelastic behavior of PF solutions
3.3.1. Temperature sweep
The evolution of viscoelastic moduli against temperature was ob-
tained through a temperature sweep test from 85 °C to 25 °C, at 5 °C/
min and a constant frequency of 0.5 Hz. This was done to investigate
the phase transition occurring in the systems. The temperature-
induced phase transition was ultimately a consequence of the effects
of temperature on the intermolecular interactions involved in pectin
molecules. With temperature increase, the intermolecular hydrophobic
interactions occurring among the methoxyl groups of pectin molecules
were enhanced, but the hydrogen bonding among the hydrophilic car-
boxyl and hydroxyl groups of pectin molecules grew weak. Upon the
cooling, the hydrophobic interactions were loosened and even cleaved
but the hydrogen bonding was strengthened. The results showed that
both of the storage modulus (G′) and loss modulus (G″) increased as
the temperature decreased (Fig. 3). The crossover point of two moduli
(G′ and G″) indicated the occurrence of phase transition in the system,
namely from liquid-like (high temperature) to solid-like (low tempera-
ture) or vice versus.
Regarding the effects of PF concentration on its viscoelastic behavior,
the results in Fig. 3a showed that, regardless of the PF concentration,
both moduli gradually decreased upon the cooling from 85 °C to 25 °C.
In contrast to 0.5 wt% PF solution, the bigger changes in both moduli
were concluded for 1.5 wt% and 2.5 wt% PF solutions. For a specific mod-
ulus either G′ or G″, its amplitude increased as PF concentration in-
creased. This is consistent with the previous observation in flow
behavior measurements that a higher apparent viscosity could be ex-
pected by increasing the concentration of PF. At high polymer concen-
trations, the side chains of pectin molecules play a key role in further
tightening the network through entanglements and topological con-
straints as those explained in “reptation model” [41]. Moreover, the
temperature corresponding to the cross of the traces of G′ and G″ was
highly depended on PF concentration. Approximately, the cross of the
traces of two moduli occurred at 35 °C, 60 °C and 85 °C for the PF solu-
tions at 0.5 wt%, 1.5 wt% and 2.5 wt%, respectively. This suggested that a
more concentrated PF solution could form a gel at a higher temperature.
The effects of sucrose concentration on the viscoelastic behavior of
PF solution were illustrated in Fig. 3b. The crossover point of two moduli
for PF solutions containing 10%, 30% and 50% sucrose occurred at 42.6 °C,
72.1 °C, and 79.4 °C, respectively. Obviously, the lower values in both
moduli of the system containing 10% sucrose indicated its very soft tex-
ture nature. The critical concentration of sucrose for the cold-set gela-
tion of pectin is about 50–55 wt% [29,42], allowing the necessary
contact of the pectin chains for junction zones formed via hydrophobic
interactions and hydrogen bonds. In the early stage of cooling, both G′
and G″ of 50% sucrose system were sharply raised to 7.12 Pa (72.95
°C) and 2.83 Pa (76.72 °C). In the subsequent cooling, they increased
in less degrees. Generally, the cold set gels from high methoxyl pectin
are thermo-irreversible, which attested that the gel structure would
not be re-built during annealing. As observed for konjac glucomannan,
the conformational transformation and intermolecular aggregations
are responsible for its thermo-irreversible gelation [43].
 L. Feng et al. / International Journal of Biological Macromolecules 128 (2019) 629–637 635

Fig. 3. Evolution of storage modulus G′ (filled symbols) and loss modulus G″ (open symbols) of the dispersion of pectin-rich fraction (PF) from blueberry wine pomace with temperature
(from 85 °C to 25 °C at a rate of 10 °C/min) as affected by PF concentration (a, 20 mM CaCl2, 30% sucrose, pH 4.0), sucrose (b, 20 mM CaCl2, 1.5% PF, pH 4.0), pH (c, 1.5% PF, 20 mM CaCl2, 30%
sucrose) and CaCl2 (d, 1.5% PF, 30% sucrose, pH 4.0). The determinations were performed at 1 Hz and 0.5% strain.

Generally, pH exerts a great impact on the gel formation of pectin
materials. It was previously reported that the gelation pH for high
methoxyl pectin is lower than 3.5, while that for low methoxyl pectin
is in a wider range of pH 2.6–7 with or without sugar [29,42]. In our
work, the evolution of G′ and G″ during cooling from 85 °C to 25 °C
was studied for solutions with pH of 3, 4, 5 and 7. Clearly, among
these systems, the one at pH 4 presented the highest gel strength after
being cooled to room temperature (Fig. 3c). The gel strength at pH 5
was less than that at pH 3.0. even at pH 7, the PF solution cannot form
a gel at any temperature with its G″ values consistently higher than
the corresponding G′ values. The effects of pH should be related to its in-
fluence on the dissociation degree of the carboxyl groups in pectin mol-
ecules. At a lower pH such as 3.0, the carboxyl groups in pectin
molecules are highly protonized (\\COOH) and there are not enough
dissociated carboxyl groups (\\COO−) to interact with calcium ions to
induce the gel formation. On the other hand, as the pH increases to 5
or 7, the dissociation degree of the carboxyl groups in pectin molecules
will be substantially improved, which highly increased the electrostatic
repulsion among the negatively charged pectin molecules and thus im-
paired the gel network structure or even disabled the formation of poly-
mer based three-dimension structure [31].
The presence of calcium ions or other divalent metal ions could form
intermolecular ionic “egg-box” junction zones, which is responsible for
the gel formation of pectin solution [31]. Besides the electrostatic inter-
actions, van der Waals interactions and hydrogen bonds could stabilize
the chains when egg-boxes were formed between neighboring chains.
Fig. 3d showed that the optimal CaCl2 concentration for the gelation of
the present PF is around 10 mM. The gel strength with 30 mM CaCl2
was lower than that with 10 mM. The previous study declared that
the application of excessive CaCl2 in pectin system might cause sheet-
like aggregation and precipitation instead of gel formation [44]. In
these aggregates or precipitates, a large number of junction zones
with short distances formed between pectin molecules. They contained
many small voids but less water. Eventually, the gelling process is re-
tarded and the mechanical properties of the resultant product might
be undermined.
3.3.2. Frequency sweep
The rheological properties play an essential role in evaluating the ef-
ficacy of pectin in modifying food matrix. Typical mechanical spectra for
PF solutions present a solid-like nature, that is, the elasticity reflected by
G′ dominates over the viscocity reflected by G″. The evolution of storage
modulus G′ and loss modulus G″ against frequency (Hz) of different PF
gels are shown in Fig. 4.
Over the tested frequency range, all PF systems at concentrations of
0.5 wt%, 1.5 wt% and 2.5 wt% demonstrated a predominance of elastic-
ity. This applauded the gelling capacity of the present PF. Moreover,
the PF concentration had a significant effect on its viscoelastic moduli
(Fig. 4a). The higher the PF concentration, the stronger the gel nature
of the system was. The increase in the concentration of pectin certainly
resulted in a higher amount of crosslinks among polymers, which are
crucial to build up the gel network of certain extent of stiffness. These
results indicated that more stable and stronger gels can be obtained at
higher PF concentrations, which was in line with the results obtained
in temperature sweep test.
The impacts of sucrose on both moduli seemed to be much more
complicated than that of PF concentration (Fig. 4b). As for sucrose con-
centration, the system with 30 wt% sucrose presented the highest value
in G′ while the system with of 10 wt% sucrose presented the lowest
value in G″. For all systems, the G″ value increased with the frequency
and the similar trend was observed for G′ of the system with 30 wt% su-
crose. However, sharp drops in G′ were observed at approximately
7–8 Hz for the systems with 10 wt% and 50 wt% sucrose. This indicated
the fragility of network structure formed in these systems. An appropri-
ate level of sucrose could enhance the hydrophobic interactions among
pectin molecules. However, the excessive sucrose could damage the
636 L. Feng et al. / International Journal of Biological Macromolecules 128 (2019) 629–637
Fig. 4. The frequency dependences of G′ (filled symbols) and loss modulus G″ (open symbols) of the dispersion of pectin-rich fraction (PF) from blueberry wine pomace as affected by PF
concentration (a, 20 mM CaCl2, 30% sucrose, pH 4.0), sucrose (b, 20 mM CaCl2, 1.5% PF, pH 4.0), pH (c, 1.5% PF, 20 mM CaCl2, 30% sucrose) and CaCl2 (d, 1.5% PF, 30% sucrose, pH 4.0). The
determinations were performed at 0.5% strain.
already formed gel structure by extracting water from it and made it
more fragile. It seemed to be inconsistent with the previous discussion
of viscoelastic moduli on cooling process regarding the impact of su-
crose on gel-formation. On one hand, the increase in sucrose can pro-
mote the hydrophobic interactions favoring the approach of polymers.
On the other hand, the balance between hydrogen bonding and hydro-
phobic interaction is more crucial to a gel, which finally determine the
restraints of a considerable number of water molecules within the del-
icate network. Once the hydrophobic interaction is overwhelming, in-
tensive oscillation might lead to the fracture of architecture
maintained by hydrogen bonds, which in turn accelerates the collapse
of spongy microstructure.
The effects of matrix pH on the stability of pectin gels were illus-
trated in Fig. 4c. For the system at pH 7, the instability of the gels was ev-
ident even at low frequency and the corresponding crossover of G′ and
G″ took place at 0.141 Hz. A sharply drop of G′ was observed for this sys-
tem at approximately 2.82 Hz. The systems at pH 4 and pH 5 eventually
displayed a solid-state nature and no crossover of two moduli was ob-
served within the tested frequency range. However, for the system at
pH 3, a transition from gel to sol was observed at 4.7 Hz. This is possibly
due to the fact that the pectin chains were much less charded at pH 3,
thus weakening electrostatic repulsion among pectin molecules and im-
proving the sensitivity of pectin to Ca2+. While at pH N 4.5, the electro-
static attractions might be intensified due to the fully charged pectin
chains, and as a result, galacturonic residues turned to be insensitive
to Ca2+ [24,38,39]. The small-angle x-ray scattering analysis suggested
that both rod-like junction zones and point-like crosslinks between
neighboring chains were formed in calcium-induced low methoxyl pec-
tin gels at pH 6. However, more rod-like junction zones were replaced
by the point-like crosslinks at higher CaCl2 concentration [45].
It is interesting to note that the 10 mM CaCl2 gels experienced a gel
to sol transition at 11.2 Hz (Fig. 4d), even though 10 mM CaCl2 was op-
timal for gel formation and gel strength enhancement. Besides, both G′
and G″ moduli increased with the increase in shear frequency, and their
absolute values were arranged in a CaCl2 order of 10 mM N 30 mM
N 5 mM. Notably, the gelation of high methoxyl pectin could be acceler-
ated by CaCl2, which strengthened the molecular interactions of pectin
molecules and favored the approach of pectin molecules [46]. An opti-
mum distance between neighboring junction zones is important for
both the formation and mechanical stability of a pectin gel. If the dis-
tance between the junction zones is too long, as in the case of 5 mM
CaCl2 gels, a very fragile soft gel with low mechanical stability will be
formed. Gels with lots of rod-like junction zones most likely formed in
20 mM and 40 mM CaCl2 but failed to hold water during the redistribu-
tion of water taking place in gels aging. They are soft and vulnerable to
frequency sweep.
4. Conclusion
The present work attempts to clarify the possibility of applying the
pectin-rich fraction (PF) extracted from blueberry wine by-product as
a gelling agent in the food industry. Chemical characterization suggests
it is medium-methoxylated (DE 51.66%), mainly composed of HG and
RG-II with short side-chain of RG-I. In general, dispersions prepared at
higher concentrations of PF showed higher shear viscosity, but failed
to form very viscous liquid without divalent cations such as CaCl2 and
certain amount of soluble solids (often N50% sucrose). Both cold set ge-
lation and ionotropic gelation could induce gelling of PF. Pectin gels of
stable viscoelastic gel-like behaviors as a function of frequency could
be formed with 50% sucrose at 10 mM CaCl2 and pH 4. These results
were attributed to the unique characteristics of PF, which is bound in
medium-methoxylated pectin.
Acknowledgements
This work was supported by National Key Research and Develop-
ment Plan (2016YFD0400204-2), Fundamental Research Funds for the
Central Universities (SWU117046), Open Funds provided by Guangxi
 L. Feng et al. / International Journal of Biological Macromolecules 128 (2019) 629–637
Key Laboratory of Agricultural Resources Chemistry and Biotechnology
(KF06) and Development and Research Center of Sichuan Cuision
(CC18Z13).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2019.01.166.
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