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Food Hydrocolloids: Sha Zhang, Fu-Hung Hsieh, Bongkosh Vardhanabhuti
Food Hydrocolloids: Sha Zhang, Fu-Hung Hsieh, Bongkosh Vardhanabhuti
Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd
a r t i c l e i n f o a b s t r a c t
Article history: Heated whey protein and pectin soluble complexes were formed at near neutral pH (6.2e7.0) by heating
Received 11 April 2013 the mixtures (5% w/w protein and 0.05e0.5% w/w pectin) at 85 C for 30 min, and acid-induced gels were
Accepted 29 July 2013 formed by addition of glucono-delta-lactone. Gel properties were characterized by large and small
deformation and microstructural analysis as well as water holding capacity and turbidity measurements.
Keywords: Gel properties were compared to those made from whey protein polymers and pectin where proteins
Whey protein
were heated alone to form the polymers and mixed with pectin before acid-induced gelation. Both small
Polysaccharide
and large deformation results showed that gels made by heated soluble complexes at pH 7.0 had
Cold gelation
Protein polysaccharide interaction
significantly improved gel strength than the traditional polymer and pectin gels (polymer/pectin gels).
Microstructure No difference in gel strength was observed between complex gels and polymer/pectin gels when solu-
Phase separation tions were heated at pH 6.5 or pH 6.2. However, complex gels showed significantly improved water
holding capacity at all pH values, especially at higher pectin concentrations. The enhanced electrostatic
interactions between whey proteins and pectin during heating led to finer gel microstructure formation
with less porosity and smoother gel network compared to polymer/pectin gels which had limited
interaction between the biopolymers. The interaction between protein and pectin increased with
lowering the initial pH of the soluble complexes. As a result, a change from a coarse strand, phase
separated to a homogenous microstructure upon lowering the initial pH from 7.0 to 6.2 was observed for
complex gels.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Rosenberg, & Van de Velde, 2009). Little has been done on acid-
induced gelation of heated proteinepolysaccharide soluble com-
Acid-induced gelation of whey protein can be achieved by plex (i.e., formation of heated soluble protein and polysaccharide
heating protein to form aggregates, followed by reducing the complex followed by acid-induced gelation).
electrostatic repulsion between the aggregates by decreasing the Proteinepolysaccharide complex mostly originated from elec-
pH to or near the isoelectric point (pI) of the protein (Alting, Hamer, trostatic attraction between two oppositely charged biopolymers
de Kruif, & Visschers, 2003). Polysaccharides are used to alter the (Turgeon, Schmitt, & Sanchez, 2007). The most important factors
properties of acid-induced gelation of proteins. Addition of poly- that affect electrostatic-driven complex formation are pH, ionic
saccharides during cold gelation process results in the formation of strength, biopolymer ratio and total biopolymer concentration
gels with a wide range of different microstructures, depending on (Schmitt & Turgeon, 2011). Through its role in ionization of protein
the properties of protein and polysaccharides (such as size, and polysaccharides, pH is the most significant factor that de-
conformation, and charge density), biopolymer ratio, and the na- termines protein/polysaccharide interaction. The optimum com-
ture and strength of protein/polysaccharide interactions. In studies plex formation has been found when protein and polysaccharide
that investigated the effect of polysaccharides on gelation proper- were mixed at pH near pI of the protein (Chourpa, Ducel, Richard,
ties of whey protein, polysaccharides are typically added after Dubois, & Boury, 2006; Girard, Turgeon, & Gauthier, 2003;
protein aggregate formation (Cavallieri & Cunha, 2009; de Jong & Jiménez-Alvarado, Beristain, Medina-Torres, Román-Guerrero, &
van de Velde, 2007; Van den Berg, Rosenberg, Van Boekel, Vernon-Carter, 2009; Weinbreck, Nieuwenhuijse, Robijn, & de
Kruif, 2003). However, even at neutral pH, where both biopolymers
carry a net negative charge and the interaction is minimal, soluble
* Corresponding author. Tel.: þ1 573 882 1374; fax: þ1 573 884 7964. complex can be formed during heat treatment. Unfolding of protein
E-mail address: [email protected] (B. Vardhanabhuti). exposes the positively charged local domain on protein, allowing
0268-005X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodhyd.2013.07.029
S. Zhang et al. / Food Hydrocolloids 36 (2014) 76e84 77
the interaction between positively charged domain and anionic 2.2. Sample preparation
polysaccharides (Girard, Turgeon, & Gauthier, 2002; Klemmer,
Waldner, Stone, Low, & Nickerson, 2012; Vardhanabhuti, Yucel, WPI stock solution (10% w/w) was prepared by slowly dissolving
Coupland, & Foegeding, 2009). The resulting heated soluble com- the protein in Millipore water (18.2 MU) with continuous stirring
plex differs in size and shape compared to heated protein aggre- for at least 2 h at room temperature (25 C). Pectin stock solution
gates. It has been reported that the molecular weight of whey (4% w/w) was prepared by hydrating pectin at 85 C for 2 h under
protein aggregates formed in the presence of k-carrageenan were continuous stirring. The stock solutions were then kept in the
lower than the aggregates formed in whey protein solution heated refrigerator (4 C) overnight for complete hydration. On the next
in the absence of k-carrageenan (De la Fuente, Hemar, & Singh, day, these stock solutions were warmed to room temperature prior
2004). Beaulieu, Corredig, Turgeon, Wicker, and Doublier (2005) to mixing.
found that low methylester (LM) pectin has a protective effect on
whey protein during heating, resulting in complex with smaller 2.2.1. Heated soluble complex
molecular weight than heated whey protein aggregates. During Stock solutions of WPI and pectin were mixed at appropriate
complex formation at near neutral pH, significantly different extent amount and their pH was adjusted to 7.0, 6.5, and 6.2. Deionized
of interactions between protein and polysaccharides could be ob- (DI) water was added such that the final protein concentration was
tained by heating the biopolymers together at slightly different pH. 6% (w/w) and pectin concentrations ranged from 0.06 to 0.6% (w/
For example, dextran sulfate altered heat aggregation of b-lacto- w). The mixtures were gently stirred at room temperature for 2 h
globulin (b-LG) at pH 5.8 and 6.0 but not at pH 6.2 (Vardhanabhuti before being heated in a temperature-controlled water bath at
et al., 2009). Pectin has been found to increase heat stability of 85 C for 30 min and cooled using running tap water. DI water was
whey protein at pH 6.0 and 6.2, but the effect is less evident at pH added to all the samples to produce the final protein concentration
6.4 (Zhang, Zhang, Lin, & Vardhanabhuti, 2012). of 5% (w/w), and pectin concentration ranged from 0.05% to 0.5%
As mentioned above, polysaccharides are typically added after (w/w). Samples were kept at 4 C for 18 h before GDL addition.
protein aggregation in acid-induced gelation process. The in-
teractions between protein aggregates and polysaccharides mainly 2.2.2. Polymer with addition of pectin (polymer/pectin)
depend on the charge density of polysaccharides. It has been re- Whey protein polymer was prepared by heating the WPI solu-
ported that the interactions between proteins and polysaccharides tion with an initial concentration of 6% (w/w) at pH 7.0, 6.5, and 6.2
increase with increasing charge density of polysaccharides, and no at 85 C for 30 min. After polymer solutions were cooled using
interaction is observed for neutral polysaccharides (De Jong, Klok, & running tap water, stock pectin solution was added at appropriate
van de Velde, 2009). The limited interaction between protein and amount and the pH was adjusted to the required values. DI water
polysaccharides is one of the most important reasons that cause was added such that the final protein concentration was at 5% (w/
phase separation between the two incompatible components w) and pectin concentration ranged from 0.05 to 0.5% (w/w).
during gelation. Since interactions between proteins and poly- Samples were gently stirred at room temperature for 2 h and then
saccharides could be enhanced during heating, the formation of kept in the refrigerator for 18 h for complete interaction between
heated soluble complex can potentially alter gel microstructure of protein and polysaccharide before GDL addition.
acid-induced gels. The objective of this study was to investigate
acid-induced gelation of heated soluble complex formed by heating 2.2.3. Acid-induced gelation
whey protein and pectin together at pH 7.0, 6.5, and 6.2. Pectin, an GDL was added to the heated solutions at different GDL/WPI
anionic carboxylated polysaccharide, was selected as it is ratio to reach a final pH of 4.7 0.1 after 24 h of incubation. The
frequently used in whey protein/polysaccharide mixed gel and low mixtures were gently stirred for 30 s and then 1.5 mL of the sample
pH dairy gels such as yogurt. The reasons that we chose these pH was taken out to a centrifuge tube for water holding capacity
values are 1) heated protein aggregates are typically formed at near measurement. Samples were covered with parafilm and left over-
neutral pH in acid induced gelation applications, and 2) change of night at 4 C. After 24 h, gels were removed from the refrigerator
pH in this range can yield significant difference in degree of elec- and left to warm up to room temperature (22 2 C) for 2 h before
trostatic interactions according to our previous work (Zhang et al., analysis.
2012). Hence, this pH range would allow the study of whether
enhancement in electrostatic interactions between biopolymers
2.3. Zeta potential and particle size measurements
could lead to different gel properties. Gel formation, physical
properties, and microstructures of gels formed from heated soluble
The average particle diameter and electrical charge (zeta po-
WPI and pectin complex were studied and compared to those
tential) of the heated soluble complex and polymer/pectin were
formed from heated WPI (heated alone) with added pectin.
measured by the Zetasizer Nano ZS (Malvern Instruments Ltd.,
Worcestershire, United Kingdom) equipped with 633 nm laser and
2. Materials and methods
173 detection optics. Samples were diluted with DI water to a final
protein concentration of 0.3% w/w for particle size measurement.
2.1. Materials
The particle size was reported as the Z-average mean diameter. Zeta
potential was measured without any dilution.
Whey protein isolate (WPI, BiPro) was the gift from Davisco
Foods International (Le Sueur, MN). As stated by the manufacturer,
the powdered WPI was constituted of 97.9 wt% protein, 2.1 wt% ash, 2.4. Turbidity measurement
and 0.3 wt% fat (dry weight basis) and 4.7 wt% moisture (wet
weight basis). Pectin LM-18 was kindly donated by CP Kelco Inc. Turbidity measurements were made on Ultra Microplate Reader
(Lille Skensved, Denmark). It is derived from citrus peels, and has a (BioTek Instruments Inc., Winooski, USA) at a wavelength of 630 nm
degree of methyl esterification of 40%. Glucono-d-lactone (GDL) and 25 C. Samples (200 mL) were distributed in a 96-well micro-
and Rhodamine B were purchased from Sigma Chemical Co. Ltd (St. plate and covered with a thin layer of mineral oil to prevent water
Louis, MO). Other chemicals used in this study were of analytical evaporation. The turbidity of gel was monitored in situ at 5-min
grade. intervals for 24 h. The pH was recorded simultaneously at
78 S. Zhang et al. / Food Hydrocolloids 36 (2014) 76e84
60
Soluble complex and polymer/pectin mixture (36 mL) were
used to form 30-mm-thick gels at 4 C. Gelation was induced by the 40
addition of GDL as previously described in Section 2.2. Large
deformation tests were performed using a texture analyser (TA- 20
HDi, Texture Technologies Corp, Scarsdale, NY) with a 5-kg load cell
and 13-mm-diameter cylindrical plunger, according to the method 0
of Picone and da Cunha (2010) with modifications. Before analysis, 0 0.1 0.2 0.3 0.4 0.5
samples were equilibrated at ambient temperature for 2 h. The Pectin concentration (%)
penetration distance was fixed to 10% of the original gel thickness (b)
with a deformation rate of 10 mm/s. The force required to maintain 100
60
2.6. Small deformation properties
40
For small deformation measurements, a Kinexus rheometer
(Malvern Instruments Ltd., Worcestershire, United Kingdom) with 20
plate and plate geometry (50 mm diameter upper plate) was used
0
for all experiments. Before the rheological measurements, GDL was
0 0.1 0.2 0.3 0.4 0.5
added to the sample and gently stirred for 30 s. An aliquot of 1.2 mL
was immediately transferred to the rheometer lower plate, and a Pectin concentration (%)
humidity cover and solvent trap were used to prevent water (c)
evaporation. The development of elastic modulus (G0 ) was moni- 150
Z-average diameter (nm)
tored in the linear region (1% strain) at a constant frequency of 1 Hz
120
at 4 C for a period of 24 h. A strain sweep test was performed to
check that measurements have been done within the linearity 90
limits of the viscoelastic behavior.
60
2.7. Water holding capacity
30
Water holding capacity (WHC) was measured according to other 0
references (Gu, Mao, Xiao, & Yang, 2011; Picone & da Cunha, 2010). 0 0.1 0.2 0.3 0.4 0.5
Gel samples (1.5 g) formed in the centrifuge tubes were used to Pectin concentration (%)
measure water holding capacity. Loss of water was determined
from the gel weight before and after centrifugation at 10 000 g for Fig. 1. Particle size of heated WPIepectin complexes (-) and polymer/pectin (:)
10 min. The WHC value was calculated by weight difference: solutions with increasing pectin concentration at initial pH of 7.0 (a), 6.5 (b), and 6.2
(c).
.
WHCð%Þ ¼ mwater
gel mwater
loss mwater
gel 100
2.9. Statistical analysis
where mwater
gel
is the amount of water in gel before centrifugation (g)
and mwater
loss
is the amount of water lost during centrifugation. All Statistical analysis of results was performed using SPSS software
measurements were done in triplicate. (SPSS Inc., Ver.19, Chicago, IL). A one-way analysis of variance
(ANOVA) was employed to determine the statistical difference.
Significant differences (p < 0.05) between means were identified
2.8. Confocal laser scanning microscopy (CLSM)
using Duncan’s multiple range test.
For CLSM imaging, samples were stained with Rhodamine B
solution (20 mL of a 0.2 wt% solution/mL of sample) prior to acidi- 3. Results and discussion
fication. After GDL addition, approximately 70 mL of the dyed pro-
tein solution was placed into a welled slide, covered with a 0.17 mm 3.1. Particle size and zeta potential of heated solutions
coverslip. Samples were left to gel at 4 C and were observed under
the microscope at room temperature. The microstructure of gel was In order to confirm the formation of heated soluble complex at
recorded by a Zeiss LSM 510 META confocal laser scanning micro- near neutral pH, the particle size of complex and polymer/pectin
scope (Cal Zeiss, Jena, Germany) with 100 and 63 water im- was measured. The Z-average mean diameters of native WPI and
mersion objectives. An Ar/Kr visible light laser was used with an pectin were 5 and 700 nm, respectively, which were in agreement
excitation wavelength of 543 nm. Digital image files were acquired with what previously reported (Huang, Wang, Yen, Jiang, & Wu,
in 1024 1024 pixels. Z-stacks of xy-scans were recorded between 2011; Kazmierski & Corredig, 2003). After the biopolymers were
3 and 60 mm penetrations, with an interval of 3 mm. The reported heated together, the heated soluble aggregates showed a single
images in this paper were recorded at a penetration depth of 18 mm. peak with the size between native WPI and pectin, while the peaks
S. Zhang et al. / Food Hydrocolloids 36 (2014) 76e84 79
Table 1
Zeta-potential of heated soluble complex and polymer/pectin solutions at different
pH.
All of the values are the means standard deviations of at least three measure-
ments. Different letters at the same pH values indicate significant differences
(P < 0.05).
concentrations.
150
Table 2
Gelation time and final storage modulus (G0 ) after addition of GDL for 24 h of
a 100 e ee ee ee e e
concentrations.
Gel formed by WPI polymer alone showed the lowest final G0 80
after 24 h of acidification. The final G0 increased with increasing
pectin concentration from 0% to 0.25%, but decreased at 0.5% 60
pectin concentration. Acid gels from heated soluble complex had a
higher final G0 than polymer/pectin gels at all three pectin con- 40
centrations. This was consistent with the results obtained from
large strain deformation. The trend of the final G0 for gel samples 20
formed at pH 6.5 and pH 6.2 were also consistent with the results
of large deformation properties (data not shown). In this study,
0
small and large deformation properties of gels were determined
0 0.05 0.1 0.15 0.25 0.375 0.5
by rheological measurements and gel penetration measurements,
respectively. The initial force recorded in penetration measure- Pectin concentration (%)
ment indicates the force needed to penetrate and break the gel.
Fig. 4. Water holding capacity of gels formed by WPIepectin complex (dark gray) and
Gel hardness is determined by both the number of the effective polymer/pectin (light gray) at different pectin concentrations and different initial pH:
strands in the gel and the modulus of the protein strands (Van (a) pH 7.0; (b) pH 6.5; (c) pH 6.2. Bars with different letters are significantly different,
Vliet, 1998). The elastic modulus of the gel measured in rheolog- p < 0.05.
ical measurement, on the other hand, is governed by the number
of the elastic effective junctions between strands (Alting et al.,
2004). The two techniques yield similar trend in this study. 3.4. Water holding capacity
Compared to polymer/pectin, complex formed at pH 7.0 signifi-
cantly improved the gelation properties. Our previous work Water holding capacity (WHC) of acid-induced gels is shown in
(Zhang et al., 2012) found that a-helical and b-structure of protein Fig. 4. WHC of pure polymer gels at pH 7.0, 6.5, and 6.2 were 92.8%,
increased during the presence of pectin during heating, indicating 94.3%, and 97.0%, respectively. For polymer/pectin gels made at pH
enhanced hydrogen bonding between biopolymers. These sec- 7.0, addition of pectin at lower than 0.15% did not have significant
ondary structural changes could be responsible to the improved effect on WHC, while higher pectin concentrations led to a signif-
gel strength of complex gel. Badii and Howell (2006) used Raman icant decrease in WHC. However, pectin did not affect the WHC of
spectroscopy to investigate the secondary structure of different complex gel until the concentration reached 0.5%. For example, the
kinds of gelation, and found that tilapia and horse mackerel WHC of gels made by complex and polymer/pectin at pH 7 with
gelatin have higher helical content and hydrogen bonding which 0.25% pectin concentration were 92.3% and 82.1%, respectively.
could contribute to the their higher gel strength compared to Similar trend was observed for pH 6.5 and pH 6.2. No difference in
other gelatin. WHC was shown between complex and polymer/pectin gel at
82 S. Zhang et al. / Food Hydrocolloids 36 (2014) 76e84
Fig. 5. CLSM images of WPIepectin complex gel vs. polymer/pectin gels at different pectin concentrations and different initial pH (7.0, 6.5, and 6.2).
pectin concentration lower than 0.25%; however, WHC of complex The WHC of polymer/pectin system decreased with increasing
gel was significantly enhanced compared to that of polymer/pectin pectin concentration because higher pectin content enhanced the
at higher pectin concentrations. Overall, gel from solutions heated interaction between protein and pectin such that less hydrophilic
at lower pH values showed higher WHC for both complex and sites remained available to bind water. During complex formation,
polymer/pectin gel. Improvement of WHC of complex gels was the unfolding and aggregation of protein differs from that in the
observed at high pectin concentrations across all pH values. absence of pectin. In our previous study using Raman spectroscopy
Whey proteins are extremely hydrophilic molecules since they to investigate the structural changes of whey protein after heating
are mainly formed by polar amino acids (Picone & da Cunha, 2010). together with pectin, it has been found that tyrosine residue
S. Zhang et al. / Food Hydrocolloids 36 (2014) 76e84 83
became more exposed in the presence of pectin than protein alone, smaller G0 of polymer/pectin gels compared to complex gels. At
which could increase hydrogen bonding between biopolymers. As a initial pH of 7.0, the period in which the systems were kept at
result, in comparison with polymer/pectin, formation of complex pH > pI is longer than other two pH values. In such a condition, the
could results in more hydrophilic sites exposed on the surface of thermodynamic incompatibility was favored among the polymer
protein, further favored the water binding during gelation. and pectin during acidification, resulting in more discontinuous
structure. However, the enhanced interaction between protein and
3.5. CLSM pectin in complex samples retard the micro phase separation,
leading to more homogenous gel structure with improved gel
CLSM images of gels formed from samples with different initial strength. On the contrast, no improvement of gel strength was
pH were shown in Fig. 5. No difference in microstructure was observed at pH 6.5 and 6.2. The period for the system to reach pI
observed between gels made from pure whey protein with was much shorter for systems at pH 6.5 and 6.2, hence there is not
different initial pH values. All microstructure were homogenous. enough time for the phase separation before gelation occurs. Even
For gels made from solutions heated at the initial pH of 7.0, no if the electrostatic interaction between biopolymers was enhanced
phase separation was observed at pectin concentration of 0.1%, and in complex at these pH values, it does not contribute to the final gel
gel microstructure was homogenous and smooth. With increasing strength.
pectin concentration, gels became coarser, and micro phase sepa- The difference in WHC between complex gels and polymer/
ration was observed at 0.5% pectin concentration. At pectin con- pectin gels could also be explained by their microstructures.
centrations of 0.25 and 0.5%, complex gel clearly showed smoother Addition of pectin at low concentration to whey protein aggregates
microstructure and less phase separation than polymer/pectin gels. had no effect on WHC of WPIepectin mixed systems, while high
This further supports the rheological data, which showed improved pectin concentration had severe adverse effect on WHC. At high
gel strength by complex formation at pH 7.0. In the case of pH 6.5, pectin concentrations, the effect of thermodynamic incompatibility
all gels were homogenously smooth at 0.1% and 0.25% pectin. dominates, which promotes the micro-phase separated structure
Although the coarseness of the gel increased with higher pectin with a coarse network which can be seen from its microstructure,
concentration, the gel microstructure was less phase-separated leading to a lowered WHC. Microstructures of complex gels
than that at pH 7.0, and complex gel had less porosity than poly- exhibited homogenous networks with considerably low porosity
mer/pectin gel at pectin concentration of 0.5%. With regard to pH even at high pectin concentrations, resulting in higher WHC than
6.2, the microstructures of gels with 0.1 and 0.25% pectin showed polymer/pectin gels at all pH values.
homogenous protein network. Similarly, phase separation occurred The microstructure of protein/polysaccharide mixed gels mainly
at 0.5% pectin concentration for polymer/pectin gel, while complex depends on the nature of polysaccharides and biopolymer ratio. It
gel was still homogenous. has been reported that when the biopolymer concentrations in-
For gels formed by WPI/pectin complex at pH 6.5, the micro- crease such that they exceed a certain critical value, the large size
structure was less coarse than that at pH 7.0. This is probably a and rigidity of the macromolecules may lead to thermodynamic
result of a shorter time available for the phase separation. During incompatibility and finally phase separation (Picone & da Cunha,
acidification the molecular interactions associated with protein and 2010). In this study, phase separation was not observed until
pectin molecular rearrangements occurred simultaneously with pectin concentration reached 0.5%. The porosity of polymer/pectin
the pH lowering. For samples with initial pH of 7.0, there was gels at such pectin concentration is attributed to an obstruction of
enough time for protein and pectin to interact and consequently proteineprotein interaction by pectin. However, complex gels at
the network structure was more organized. In the case of pH 6.5, this pectin concentration showed much less porosity and smoother
the time it took to decrease the pH to the pI of the protein was much network, and no incompatibility seemed to occur. In addition, the
shorter than that of gels formed from solutions at pH 7.0, hence, gel repulsion between protein and pectin is positively related to the pH
microstructure was formed before sufficient molecular rearrange- of the solution. The enhanced association between biopolymers at
ment, resulting in less phase separation. Comparable results were low pH values explains the change from a coarse strand, phase
obtained by De Jong et al. (2009), who found that at higher incu- separated to a homogenous microstructure upon lowering the
bation temperature the microstructure of the final gel was less initial pH from 7.0 to 6.2.
coarse because higher acidification rate resulted in a shorter time There are several benefits regarding to the application of soluble
available for phase separation. Similar pattern was observed for pH proteinepectin complex. Firstly, the enhanced gel strength and
6.2 at 0.1% and 0.25% pectin concentration. However, polymer/ water holding capacity of complex gel has its potential to be applied
pectin gel showed severe phase separation at highest pectin con- in yogurt or novel high protein low pH gel products. Pectin has been
centration probably due to the large particle formation before used in high protein yogurt as stabilizer, but usually is added after
acidification. At pH 6.2, WPI carries less net negative charges than protein is heated. By heating whey protein and pectin together to
higher pH, resulting in less repulsion between protein and protein form the soluble complex, it is highly possible that it could improve
and protein and pectin which further led to large cross-linked the texture of the yogurt, as well as reduce the syneresis by
particles with low solubility. improving the water holding capacity of the protein. Secondly,
The difference in the acidification rate among samples with during the mixing of polymer and pectin, we observed that it took
different initial pH are also responsible for the results obtained in much longer time for pectin to dissolve in the polymer than in the
the rheological properties, where complex at pH 7.0 showed native protein solution as for complex preparation.
significantly improvement in gel strength compared to polymer/ In conclusion, this study demonstrated that gelation properties
pectin, with the highest gel strength observed at pectin concen- of whey protein/pectin mixed systems were improved by heated
tration of 0.25%. As shown in their microstructure, although no soluble complex formation, which has not been shown previously.
phase separation was observed under such pectin concentrations, Heating whey protein and pectin together to form the soluble
polymer/pectin gels showed coarser network. CLSM images of complex promoted the interactions between the two biopolymers,
polymer/pectin gels also exhibited lower degree of inter- which resulted in better gel microstructure having less porosity and
connectivity than complex gels, which could lead to decreased smoother network. The extent of the interaction between protein
number of the effective strands and thus weaker gel network. and pectin was greatly influenced by the initial pH of the complex.
Correspondingly, lower degree of interconnectivity also resulted in The mechanical properties of gels were improved by forming
84 S. Zhang et al. / Food Hydrocolloids 36 (2014) 76e84
complex at pH 7.0, while no difference was observed between Girard, M., Turgeon, S. L., & Gauthier, S. F. (2002). Interbiopolymer complexing
between b-lactoglobulin and low-and high-methylated pectin measured by
complex gel and polymer/pectin gel at initial pH of 6.5 and 6.2. At
potentiometric titration and ultrafiltration. Food Hydrocolloids, 16(6), 585e591.
all the three pH values studies, complex gels showed improved Girard, M., Turgeon, S. L., & Gauthier, S. F. (2003). Thermodynamic parameters of b-
water holding capacity than polymer/pectin gels, especially at high lactoglobulin-pectin complexes assessed by isothermal titration calorimetry.
pectin concentrations (>0.15%). Journal of Agricultural and Food chemistry, 51(15), 4450e4455.
Gu, J., Mao, X., Xiao, Y., & Yang, S. (2011). Effect of gelation factors on the formation
and characteristics of protease-induced whey protein gel. International Journal
Acknowledgments of Dairy Technology, 64(4), 473e479.
Hoffmann, M. A., & van Mil, P. J. (1997). Heat-induced aggregation of b-lactoglob-
ulin: role of the free thiol group and disulfide bonds. Journal of Agricultural and
Support from Dairy Research Institute is greatly acknowledged. Food Chemistry, 45(8), 2942e2948.
The authors thank Davisco Foods International, Inc. and CP Kelco for Huang, A. C., Wang, Y. T., Yen, H. H., Jiang, C. M., & Wu, M. C. (2011). Transacylation
donating whey protein isolate and pectin, respectively. properties of pectin methyl esterase from Aspergillus niger. African Journal of
Food Science, 5(13), 710e716.
Jiménez-Alvarado, R., Beristain, C., Medina-Torres, L., Román-Guerrero, A., & Ver-
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