Food Chemistry 382 (2022) 132329

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Effect of κ-carrageenan on the gelation properties of oyster protein

Suisui Jiang a, b, 1, Yuyang Ma a, 1, Yahui Wang a, c, Rui Wang a, d, Mingyong Zeng a, *
a
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China
b
Institute of Nutrition and Health, School of Public Health, Qingdao University, Qingdao 266021, China
c
Dongxihu District Bureau of Commerce, Wuhan 430040, China
d
MHOME Industrial Co., Ltd, Guangzhou 510530, China

A R T I C L E I N F O A B S T R A C T

Keywords: Proteins and polysaccharides commonly coexist in the food system, forming complexes and coacervates to make
Oyster protein tailor-made food. In this study, the effects of κ-carrageenan on the rheological behavior, network structure,
κ-carrageenan textures, and molecular force of oyster protein (OP treated with high-pressure homogenization were investigated.
Gel properties
Rheological results showed that κ-carrageenan improved the storage modulus of OP, and the higher concen­
Rheological properties
Molecular force
tration of κ-carrageenan accelerated the gelation of OP. The second derivative of infrared spectroscopy revealed
that κ-carrageenan contributed to the formation of β-sheet in OP. Molecular force and texture analysis showed
that κ-carrageenan might promote the increase of hydrophobic bonds and disulfide bonds, which was helpful to
enhance gel strength. The microstructure showed that the OP gel with 1.5% κ-carrageenan had a compact
network structure with abundant minor mesh and sheet edge. This study reveals the gelation mechanism of OP/
κ-carrageenan and provides the theoretical basis for developing innovative oyster products.

1. Introduction
Oyster is abundant with essential amino acids and minerals (Jiang,
Liu, Xu, Zeng, & Zhao, 2019), widely consumed throughout the world.
By now, the development of oysters, primarily consumed as fresh food,
is limited due to preservation and transportation (Felici, Vittori, Meli­
grana, & Roncarati, 2020). Canned and dried oysters are low in tech­
nology, and they are the main commercial productions (Neri, Jung,
Jang, Kang, & Choi, 2021) with relatively low value. As the continuous
expansion of oysters in aquatic breeding, it is always the hot point of the
oyster industry to develop new products. Our earlier research found that
oyster protein (OP) prepared by alkali-extraction and acid-precipitation
has potential properties in gelation (Wang, Jiang, Zhao, & Zeng, 2020).
In daily life, surimi, sausages, and tofu are prepared by gelation. Surimi
manufactures with pleasant textures, cohesion, and elasticity are pop­
ular marine products by consumers. However, the water holding ca­
pacity (WHC) of surimi prepared with only oyster protein was weak.
WHC plays a crucial role in gel textures, further affecting the chewiness
and mouthfeel.
Proteins and polysaccharides usually coexist in the food system,
forming complexes and coacervates to make tailor-made food. Kappa
(κ)-carrageenan, extracted from red seaweed, is one of the essential in­
gredients in many foods processing, which plays a prominent role in
stabilizing and regulating gelling characteristics (Stenner, Matubayasi,
& Shimizu, 2016). It is reported that κ-carrageenan can improve the
rheological properties, WHC, textures, and microstructure of gel, which
further modify the quality of final products. The κ-carrageenan
improved the elastic property and the degree of syneresis of locust bean
gum (Glicksman, 1979). Moreover, κ-carrageenan increased the vis­
cosity of pea protein gel by affecting rheological properties (Musampa,
Alves, & Maia, 2007). The gelation process and viscoelasticity of gelatin
were enhanced by the synergistic effect of κ-carrageenan (Derkach,
Ilyin, Maklakova, Kulichikhin, & Malkin, 2015). Similarly, κ-carra­
geenan contributed to the viscoelasticity of egg yolk protein gel (Agui­
lar, Cordobés, Raymundo, & Guerrero, 2017). Moreover, κ-carrageenan
facilitated the increase of elastic moduli of scallop male gonads hydro­
lysate, which formed a compact network (Yan, Shang, Zhao, Han, Jin,
Wang, et al., 2019). The textures and WHC of krill protein were
improved by κ-carrageenan (Zheng, Beamer, Matak, & Jaczynski, 2019).
These results demonstrated that κ-carrageenan could interact with
protein and further affect the gel properties. However, extensive ag­
gregation of double-helix in the κ-carrageenan forms a stiff network that

* Corresponding author.
E-mail address: mingyz@ouc.edu.cn (M. Zeng).
1
Suisui Jiang and Yuyang Ma contributed equally to this work and are co-first authors.

https://doi.org/10.1016/j.foodchem.2022.132329
Received 3 September 2021; Received in revised form 30 January 2022; Accepted 31 January 2022
Available online 3 February 2022
0308-8146/© 2022 Elsevier Ltd. All rights reserved.
S. Jiang et al.
is strong and brittle. In addition, our team found that the gel strength of
OP was low and tender. Thus, adding κ-carrageenan to optimize the OP
gel properties and improving the physicochemical properties of final
food products is practical.
In the present study, an excellent gel compounded with oyster pro­
tein and κ-carrageenan was prepared for the first time. Moreover, the
influence of κ-carrageenan on the rheological behavior, textures, WHC,
macrostructure, and molecular forces were studied to elucidate the
formation mechanism of OP/κ-carrageenan gel. This study reveals the
gel mechanism of OP/κ-carrageenan, and provides a new reference for
developing oyster protein nutrition gel products.
2. Materials and methods
2.1. Materials
Oysters (Crassostrea gigas) were obtained from Qidong Road aquatic
market (Qingdao, Shandong Province, China). KBr, κ-carrageenan, and
8-anilino-1-naphthalene sulphonic acid (ANS) were purchased from
Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China), and
5,5′ -dithiobis-(2-nitrobenzoic acid) was obtained from Sigma-Aldrich
Co., Ltd. (Shanghai, China). Oyster protein (OP) was prepared using
the method according to our previous report (Wang, Jiang, Zhao, &
Zeng, 2020).
2.2. The OP/κ-carrageenan gel preparation
Oyster protein (OP) was dissolved in deionized water at a final
concentration of 100 mg/mL and then stirred for 30 min at room tem­
perature. Subsequently, different proportions (0, 0.5, 1, 1.5, and 2.0%,
m/v) of κ-carrageenan was added to the OP solution and stirred for 30
min. The mixture was adjusted to pH 8.0 using 0.1 M NaOH and then
subjected to heat treatment at 80 ◦ C for 30 min. Finally, OP/κ-carra­
geenan gel was obtained and stored at 4 ◦ C for subsequent analysis. All
analyses were conducted within 72 h.
2.3. Rheological measurements
A rotary rheometer (MCR-101, Anton Paar Co. Ltd., Graz, Austria)
with a parallel plate (diameter = 50 mm, gap = 1 mm) was used to
monitor the dynamic rheological properties of OP and OP/κ-carra­
geenan gel.
Place 2–3 mL of the mixture prepared by 2.2 section on a plate and
equilibrated for 5 min, followed by strain sweep measurement. A fixed
frequency of 1 Hz and a temperature of 25 ◦ C were set, and then storage
modulus (G’) of gel at strains ranging from 0.1 to 100% was measured.
A frequency of 1 Hz and strain value of 1% were selected for the
dynamic temperature sweep of gels. OP and OP/κ-carrageenan gel was
equilibrated for 1 min at 5 ◦ C and then rose from 5 ◦ C to 100 ◦ C at a rate
of 2 ◦ C/min. Subsequently, samples were balanced at 100 ◦ C for 1 min
and then cooled from 100 ◦ C to 5 ◦ C at a rate of 2 ◦ C/min. The storage
modulus (G’) was recorded during the whole process.
A constant strain value (1 Hz) and temperature (25 ◦ C) were selected
for frequency sweep measurement. The G′ and loss modulus (G′′ ) of
frequency sweep were recorded within a frequency range varying from 1
to 20 Hz.
2.4. Water holding capacity (WHC)
WHC of gels was detected according to the previous method (Cai,
Feng, Cao, Tian, Wang, Liu, et al., 2018), with slight modifications. The
gels were wrapped using a filter and then placed in a centrifuge tube.
Subsequently, these gels were centrifuged at 10000×g for 15 min. The
WHC of gels was calculated as the following equation:
2
Food Chemistry 382 (2022) 132329
W1
WHC = (1)
W2
where W1 is the initial weight of gels, W2 is the weight after
centrifugation.
2.5. Low-field NMR (LF-NMR)
Water distribution of OP and OP/κ-carrageenan gels was determined
using an LF-NMR analyzer (Model MR25, Niumag Electronic Technol­
ogy Ltd., Shanghai, CN). The transverse relaxation time (T2) and in­
tensity were determined. Test parameters were set as follows: resonance
frequency at 22.6 Hz, the temperature at 32 ◦ C, and τ value at 200 μs.
The NIUMAG software was performed to fit the T2 relaxation curve to a
multiple-exponential curve using a synchronous iterative reconstruction
technique (SIRT) algorithm. Each sample was measured at least 6 times
and acquired 32 scan repetitions.
2.6. Texture profile analysis (TPA)
The gels were cut into 2 cm cube and placed on the plate of the
texture analyzer (Texture Technologies Corp., Surrey, UK). Then, sam­
ples were compressed to 50% original height using cylindrical prob (P/
36R) to obtain cohesiveness, springiness, gumminess, and chewiness.
During the test, the test speed was 1.0 mm/s, and the post-test speed of
1.0 mm/s.
2.7. Gel force analysis
OP or OP/κ-carrageenan gel was cut into a 2 cm cube and placed on
the plate of the texture analyzer (Texture Technologies Corp., Surrey,
UK). With a fixed trigger force of 5 g, the cylindrical probe (diameter =
5 mm) was used to puncture gel for 1 cm at a rate of 1 mm/s to measure
the gel strength.
2.8. Fourier-transform infrared spectroscopy (FTIR)
The potassium bromide (KBr) disc technique was performed for
sample preparation. The sample and KBr in proportion (1:100, w/w)
were ground into a fine powder and then compressed to disc. The
spectrum results were obtained at wavenumbers of 4000–400 cm− 1 in
64 scans with a resolution of 4 cm− 1. Fourier self-deconvolution was
applied to analyze the secondary structure of samples.
2.9. Molecular forces analysis
Molecular forces of gel were measured according to the previous
reports (Ni, Wang, He, Wang, Pan, Li, et al., 2014), with some modifi­
cation. The gel samples (2.0 g) were separately added into of gel to 20
mL of SA (0.05 M sodium chloride), SB (0.6 M sodium chloride), SC (1.5
M urea + 0.6 M sodium chloride), SD (8.0 M urea + 0.6 M sodium
chloride), and SE (8.0 M urea + 0.6 M sodium chloride + 0.6 β-Mer­
captoethanol) solution. Then the mixture was homogenized at 10000
rpm for 20 s and stirred at 4 ◦ C for 1 h. After centrifugation, the protein
content of the supernatant was tested.
2.10. Surface hydrophobicity (H0) and free sulfhydryl groups content
The gel samples (5.0 g) were added to 10 mL of Tris-HCl (pH 7.0, 20
mM) and homogenized for 1 min at 10000 rpm. Then, the mixture was
centrifuged at 10,000 g for 15 min. Protein concentration of the super­
natant was diluted to 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL, which were used
to measure the H0 according to ANS prob. Moreover, 0.2 mg/mL of gel
supernatant was used to measure the sulfhydryl groups content (Ni,
et al., 2014).
S. Jiang et al.
2.11. Scanning electron microscopy (SEM)
The samples were cut into 2–3 mm squares, soaked in glutaraldehyde
solution (2.5%, V/V) for 1 h, and then rinsed three times using PBS (pH
7.3). Subsequently, the gels were dehydrated in 30, 50, 70, 90, and
100% ethanol solutions, respectively. The specimens were soaked in
hexamethyldisilazane for 15 min and freeze-dried for 72 h. Finally,
samples coated with gold were observed using SEM (JSM-5800 LV, JEOL
Ltd., Tokyo, Japan) at an accelerating voltage of 20 kV.
2.12. Data analysis
All tests were carried out in triplicate, and data were presented as
mean ± SD (standard deviation). ANOVA and Duncan’s multiple range
tests were used to analyze statistical analysis. A level of p < 0.05 was
considered a significant difference.
3. Results
3.1. Rheological behavior of OP/κ-carrageenan
The rheological behavior of OP and OP/κ-carrageenan was shown in
Fig. S1-2 and Fig. 1. The storage moduli (G′ ) of all gels maintained
steady at low strain values (Fig. S1). When the strain values were over
10%, the G′ gradually decreased and then achieved critical strain (γ0).
However, κ-carrageenan had little effect on the γ0, indicating no sig­
nificant difference in fracture strength between different groups under
Fig. 1. Effect of κ-carrageenan on the rheological characteristics of oyster protein (OP). Temperature dependence of storage modulus (G’) of OP with κ-carrageenan
(a); Frequency dependence of G’ (b), loss modulus (G’’) (c), and loss factor (tanδ) (d). OP: oyster protein, without the addition of κ-carrageenan, κ-carrageenan: pure
κ-carrageenan (2.0 wt%), OP/κC0.5: OP + 0.5% κ-carrageenan, OP/κC1.0: OP + 1.0% κ-carrageenan, OP/κC1.5: OP + 1.5% κ-carrageenan, OP/κC2.0: OP + 2.0%
κ-carrageenan.
3
Food Chemistry 382 (2022) 132329
the same stress (Lin, Zhang, Li, Zheng, Rea, & Miao, 2019). According to
the above results, 1% strain was selected for further rheological
experiments.
Heating is one of the crucial steps for OP gelation formation. Fig. 1a
showed the storage moduli (G′ ) variation of the mixture of OP/
κ-carrageenan as a function of temperature, implying the gelling pro­
files. The change of G′ value of OP has two stages in the heating process.
At 5–37 ◦ C, with the increase of temperature, the value of G′ decreases
gradually. However, when the temperature was higher than 37 ◦ C, the
value of G′ increased significantly. This change differs from pure
κ-carrageenan (2.0 wt%). G’ value of κ-carrageenan decreased rapidly
during the heating process, which was attributed to the changes of
molecular chain mobility (Fig. S2). Moreover, the G′ trend of the mix of
OP/κ-carrageenan groups was similar to the OP group during the
heating ramp. The κ-carrageenan might promote the hydrogen bond or
ion interaction of OP, leading to the increase of G′ , suggesting that the
gel strength was boosted remarkably by κ-carrageenan. Most gelation of
animal protein is divided into three stages: weak gel, gel weakening, and
gel-strengthening period. During the weak gel period, denaturation of
myosin after heating followed by the formation of weaker chemical
bonds between proteins, resulting in the G′ of protein slightly increased
and forming the soft gel structure. The next stage is the decrease of the G′
value as the light chain subunits of the myosin gradually dissociate,
increasing the fluidity of the solution (Zhang, Xue, Li, Wang, & Xue,
2015). The third phase is to form a solid three-dimensional structure by
cross-linking between myosin. As shown in Fig. 1a, there was no weak
gel period during forming the OP gel, which was ascribed to the low
S. Jiang et al.
Fig. 2. Effect of κ-carrageenan on the distribution of T2 relaxation time (a) and water types (b). OP: oyster protein, without the addition of κ-carrageenan, OP/κC0.5:
OP + 0.5% κ-carrageenan, OP/κC1.0: OP + 1.0% κ-carrageenan, OP/κC1.5: OP + 1.5% κ-carrageenan, OP/κC2.0: OP + 2.0% κ-carrageenan.
content of tropomyosin. The weak crosslinking forces of oyster myosin
at low temperatures were not sufficient to form a soft gel. The G′ of all
groups was gradually increased with decreasing the temperature, indi­
cating that the gel was formed and strengthened during the cooling
stage.
To further explore the effect of κ-carrageenan on OP rheological
properties, the G′ , G′′ , and loss factor (tan δ) of all gels were tested. For
the OP group, the G′ gradually increased versus frequency (Fig. 1b). The
G′ increased as the κ-carrageenan concentration increased, which was
similar to the rheological strain scan results. In addition, the G′ value of
the pure κ-carrageenan group (2.0 wt%) was significantly lower than
that of the OP/κ-C0.5 group. This indicated that OP and κ-carrageenan
had gel synergy. However, excessive κ-carrageenan reduced the G′ of
OP/κ-carrageenan gel. The κ-carrageenan was a brittle gel, and excess
κ-carrageenan level caused the oyster protein gel to become more rigid
or brittle, but negatively affected the elasticity of the gel. In addition,
similar results have been observed in previous reports (Sinthusamran,
Benjakul, Swedlund, & Hemar, 2017).
Moreover, G′′ also showed a similar variation trend (Fig. 1c). For all
samples, the value of G′ was higher than that of G′′ , indicating that all
gels exhibited elastic properties. Furthermore, the tan δ value slightly
increased with the increase of frequency (Fig. 1d). The addition of
κ-carrageenan caused the reduction of the tan δ and enhancement of
overall network denser. This was because κ-carrageenan, as a liner
anionic hydrocolloid, interacted with protein and limited the free water
or network chain mobility at the same time.
Index-law equation was used to visually exhibit viscoelastic char­
acteristics between different samples (Table S1). The constant n’ is
closely related to the gel frequency dependence, and the closer this value
is to 0, the more covalent bonds are involved in forming the gel network
(Wang, Chang, Xue, Li, Wang, & Xue, 2016). The higher value of K’
dictates the higher hardness of samples. After κ-carrageenan was added,
the n’ increased rapidly and the K’ increased, implying that κ-carra­
geenan had a contribution to the formation of covalent bonds between
κ-carrageenan and OP to enhance the hardness of gel. However, the
excessive κ-carrageenan (2.0%) could not improve the quality of OP gel
due to the excessive aggregation rather than the interaction between
κ-carrageenan helices and OP.
3.2. Water holding capacity (WHC) of gel
Water holding capacity (WHC) is one of the important indicators for
evaluating the structural quality of protein gel network. In general,
lower WHC values exhibited poor overall gel quality and low texture
stability (Zheng, Beamer, Matak, & Jaczynski, 2019). The effect of
κ-carrageenan on the WHC of OP gel was shown in Fig. S3. After the
4
Food Chemistry 382 (2022) 132329
addition of κ-carrageenan, the value of WHC gradually increased. The
water holding capacity in food mainly involves weak electrostatic force
and hydrogen bond (Ma, Chen, Zheng, Zhou, Cai, & Han, 2013). Water
holding capacity of recombinant muscle food generally increases after
adding κ-carrageenan ascribed to the formation of hydrogen bonds be­
tween κ-carrageenan and water (Zheng, Beamer, Matak, & Jaczynski,
2019). Moreover, κ-carrageenan is a hydrophilic colloid that in­
corporates a portion of water into the gel network to increase the water-
holding capacity of the gel. Similarly, a previous study revealed that
κ-carrageenan contributed to the increase of WHC in surimi (Chen,
Deng, Wang, Mi, Yi, Li, et al., 2020). However, the addition of 2%
κ-carrageenan did not change the WHC of the gel, which might be the
mixed gel becoming water-saturated.
The water distribution and its mobility of gel were investigated using
low-field NMR. Generally, the alteration of relaxation times reflects the
properties of the gel. Relaxation time at 0.1 ~ 10 ms (T21) is assigned to
the tightly bound water combined with macromolecules. The T22 (10 ~
100 ms) and T23 (100 ~ 1000 ms) peaks represented immobilized water
and free water. As shown in Fig. 2a, the peak of T22 and T23 shifted to the
left, indicating the alteration of water in the gel. Moreover, the pro­
portion of T21 was not remarkably different between different groups
(Fig. 2b). The proportion of T22 was elevated significantly, and T23
decreased after the addition of κ-carrageenan (0.5–1.5%). It is specu­
lated that κ-carrageenan might have interacted with water by hydrogen
bond, and some water shifted from free water to immobilized water.
Compared to the OP/κ-carrageenan1.5 group, the proportion of T22 in
the OP/κ-carrageenan2.0 group was reduced. This was consistent with
rheological results. The higher value of WHC corresponded to stress
resistance. This was because the flexible gel with higher water content
was difficult to destroy than brittle gel. Therefore, it is suggested that
κ-carrageenan with optimal concentration had a good influence on the
WHC of the gel.
3.3. Effect of κ-carrageenan on textural properties of OP gel
The effect of κ-carrageenan on textural properties was measured
using texture profile analysis (TPA). As seen in Fig. 3a, the weak OP gel
was formed by OP treated with high-pressure homogenization due to the
low content of myofibrillar in OP. The gel force increased remarkably
after adding κ-carrageenan (p < 0.05). Polysaccharides participated in
the gel formation process, resulting in the product’s breaking force
(Zhang, Xue, Li, Wang, & Xue, 2015). Additionally, κ-carrageenan is
filled in the protein structure and interacted with myosin, promoting gel
strength (Zhou, Chen, Chen, Li, Ma, & Lu, 2014). Moreover, κ-carra­
geenan is structurally a sulfated galactan and contains sulfate group
(Bernal, Smajda, Smith, & Stanley, 1987). The negatively charged
S. Jiang et al. Food Chemistry 382 (2022) 132329

Fig. 3. Effect of κ-carrageenan on the gel strength (a), FTIR curves (b), hydrophobic force (c), hydrogen bond (d), disulfide bond (e), and ionic bond (f) of oyster
protein gel. OP: oyster protein, without the addition of κ-carrageenan, OP/κC0.5: OP + 0.5% κ-carrageenan, OP/κC1.0: OP + 1.0% κ-carrageenan, OP/κC1.5: OP +
1.5% κ-carrageenan, OP/κC2.0: OP + 2.0% κ-carrageenan. Different letters indicate the data are significantly different at p < 0.05.

domain of the sulfate group activated embedded disulfide bond groups
and hydrophobic interaction of unfolded protein (Tang, Tan, Chen,
Zhang, Li, & Chen, 2021). Subsequently, aggregation behavior occurs in
protein molecules due to the interaction between the exposed functional
groups, and finally a gel network structure is formed (Catsimpoolas &
Meyer, 1970). With the increase of κ-carrageenan level, the interaction
between functional groups increased, and the gel strength became
strong.
Compared with the OP group, the hardness of OP/κ-carrageenan
increased, implying that OP/κ-carrageenan needed more force to
compress in the mouth (Table 1). The cohesiveness, springiness, gum­
miness, and chewiness also increased with the increase of κ-carra­
geenan. The above results demonstrated that κ-carrageenan had the
potential to improve the tastes of OP gel. These changes of tastes and
properties of the gel were usually related to the alteration of tertiary,
secondary, and chemical bonds, which were further detected in the
study.
3.4. Surface hydrophobicity (H0) and sulfhydryl groups
Non-covalent interactions and breaking are the main factors driving
the tertiary changes of the protein. H0 reflected the exposure content of
buried amino groups and protein conformation changes. As shown in
Fig. S4a, the H0 was decreased with the increase of κ-carrageenan, which
implied that the hydrophobic interaction of gel was elevated. The pro­
tein structure unfolded after heating, and some hydrophobic residues
were exposed to the surface to form new binding sites and generated
hydrophobic interactions. The appropriate exposure rate of hydrophobic
groups played a crucial role in gel formation.
The reactive sulfhydryl groups of the OP and OP/κ-carrageenan gel
were depicted in Fig. S4b. The addition of κ-carrageenan had no sig­
nificant effect on the total sulfhydryl group contents of the gel, which
was about 30 μmol/g protein. However, the content of the free sulfhy­
dryl group decreased from 12.68 ± 0.65 μmol/g protein to 8.84 ± 0.64
μmol/g protein. After heating, the sulfhydryl groups were exposed and
then easily oxidized to form S–S. The results of sulfhydryl groups
implied that some of the free sulfhydryl groups in the gel were oxidized
to form disulfide bonds during heating. Chen et al. (2018) reported that
the sulfhydryl groups in myosin were exposed by heating and formed
new disulfide bonds between the molecules. The gel with high κ-carra­
geenan content exhibited the lower range of free sulfhydryl group,
indicating that κ-carrageenan might promote the formation of the di­
sulfide bond in gel and improve the structure of the gel. This was
consistent with previous reports that κ-carrageenan activated sulfhydryl

Table 1
Textural properties of oyster protein gels with κ-carrageenan at different levels.
Hardness (N) Cohesiveness Springiness (mm) Tackiness Chewiness (mJ)

OP 0.12 ± 0.01e 0.33 ± 0.05c 1.27 ± 0.04e 0.05 ± 0.01e 0.06 ± 0.01e
OP100/κ-C0.5 0.48 ± 0.03d 0.34 ± 0.02c 1.13 ± 0.11f 0.28 ± 0.03d 0.38 ± 0.03d
OP100/κ-C1.0 0.83 ± 0.04c 0.63 ± 0.02b 1.63 ± 0.14c 0.51 ± 0.04c 0.58 ± 0.05c
OP100/κ-C1.5 1.77 ± 0.30b 0.71 ± 0.02a 3.67 ± 0.37a 0.58 ± 0.05b 1.03 ± 0.15b
OP100/κ-C2.0 2.00 ± 0.09a 0.64 ± 0.03b 3.23 ± 0.47b 0.68 ± 0.04a 2.20 ± 0.38a

Note: a-f: mean value in the same column with different letters was significantly different (p < 0.05) by a Tukey test (n = 3). OP: oyster protein, without the addition of
κ-carrageenan, OP/κC0.5: OP + 0.5% κ-carrageenan, OP/κC1.0: OP + 1.0% κ-carrageenan, OP/κC1.5: OP + 1.5% κ-carrageenan, OP/κC2.0: OP + 2.0% κ-carrageenan.

5
S. Jiang et al.
functional groups and induced the bridging of new intermolecular di­
sulfide bonds, and directly promoted the formation of protein gel net­
works (Catsimpoolas & Meyer, 1970).
3.5. Effect of κ-carrageenan on conformation characterization of OP gel
The secondary structure changes in gel with or without κ-carra­
geenan were analyzed using FTIR. As shown in Fig. 3b, there were five
characteristic absorption bands in OP. The PK1 (3100–3500 cm− 1) ab­
sorption band is attributed to –OH stretching (Barth, 2007). The ab­
sorption bands near PK2 (2800–3000 cm− 1) exhibited –CH vibration
(Arrondo & Goñi, 1999). PK3 (1600–1700 cm− 1) was the amide I band,
reflecting the tensile vibration of the C–
– O double bond (Chen, Xu, Han,
Zhou, Chen, & Li, 2016). PK4 (1500–1600 cm− 1) was expressed as an
amide II band, which is related to the bending vibration of the N–H
bond and the tensile vibration of C–N bond (Krimm & Bandekar, 1986).
The absorption band near PK5 (800–1200 cm− 1) reflected the stretching
of the C–C and C–O (Souza & Garcia-Rojas, 2017). Furthermore, after
κ-carrageenan was added, the infrared spectra of all the samples showed
similar skeleton, and the functional groups did not increase or decrease,
indicating that κ-carrageenan had no effect on the type of functional
groups. However, the positions of typical absorption peaks have
changed to different degrees. As shown in Table S2, after the addition of
κ-carrageenan, the PK1 changed slightly from 3289.00 cm− 1 to 3292.32
cm− 1, demonstrating that κ-carrageenan elevated the intensity of
hydrogen bonds. PK2 did not shift and concentrated near the 2925 cm− 1
absorption band. Furthermore, the PK3 absorption band shifted to the
right from 1657.00 cm− 1 to 1648.33 cm− 1. PK4 absorption band was not
changed with the addition of different κ-carrageenan concentrations.
The PK3 and PK4 absorption bands represent the β-sheet and α-helix
content in the secondary structure (Kobayashi, Mayer, & Park, 2017).
The above results suggested κ-carrageenan contributed to the formation
of β-sheet in OP. β-sheet is a more ordered and cross-linked network
structure, which binds κ-carrageenan to form a denser gel (Sánchez-
González, Carmona, Moreno, Borderías, Sánchez-Alonso, Rodríguez-
Casado, et al., 2008). Similarly, Cando et al. (2016) found that β-sheet
content in cod surimi was significantly increased after adding some
additives. Finally, the change in PK5 shifted most notably from 1080.66
to 1042.10 cm− 1 compared with other absorption bands. PK5 repre­
sented C–C and C–O stretching of the colloidal polysaccharide.
Therefore, it was speculated that the transfer of PK5 might be due to
vibration caused by the addition of κ-carrageenan.
3.6. Effect of κ-carrageenan on molecular forces of OP gel
The interactive force of OP and OP/κ-carrageenan gel was shown in
Fig. 3c-f. The proportions of hydrophobic force and disulfide bonds in
OP and OP/κ-carrageenan gels were much higher than that of ionic and
hydrogen bonds, suggesting that they were the main chemical forces to
maintain the gel network structure. The hydrophobic force was elevated
2-fold when the gel contained 1.5% κ-carrageenan in the system.
Additionally, disulfide bonds in OP/κ-carrageenan gel exhibited
remarkable improvement. The intensity of disulfide bonds in OP/κ-C-1.5
had quadrupled compared with the OP group. These results illustrated
that the hydrophobic bond and disulfide bonds had a crucial role in
maintaining OP/κ-carrageenan gel.
After heating, protein denaturation increased the exposure of func­
tional groups such as hydrophobic force and sulfhydryl groups in the
side chain of protein amino acid. At this time, hydrophobic interaction
occurs in the system, which makes myosin molecules aggregate, and
finally promotes the gel network structure (Yongsawatdigul & Sinsu­
wan, 2007). Furthermore, the active sulfhydryl approaches each other
and undergo oxidation or –SH/S–S exchange reaction to form disulfide
bonds (Careche, Alvarez, & Tejada, 1995). Disulfide bonds are impor­
tant covalent bonds that stabilize the protein conformation and help to
maintain the three-dimensional structure of the protein (Riebroy,
6
Food Chemistry 382 (2022) 132329
Benjakul, Visessanguan, Erikson, & Rustad, 2009). The addition of
κ-carrageenan activated the embedded disulfide bonds and induced a
new intermolecular disulfide bridge, thus promoting the formation of
the protein gel network (Catsimpoolas & Meyer, 1970). In the present
study, the addition of κ-carrageenan significantly increased the hydro­
phobic interaction force and disulfide bond of the composite gel. This
was consistent with the results of many reports (Lin, Zhang, Li, Zheng,
Rea, & Miao, 2019; Xu, Xia, Yang, & Nie, 2010; Zheng, Beamer, Matak,
& Jaczynski, 2019). Furthermore, the proportion of ionic bonds and
hydrogen bonds was low in the OP/κ-carrageenan gel system, indicating
that ionic bonds and hydrogen bonds were not the main chemical forces
to maintain the stable conformation of the gel. It was speculated that the
ionic and hydrogen bonds participated in the entanglement of myosin
tails, but not in the aggregation of the heads (Benjakul, Visessanguan, &
Pecharat, 2004).
3.7. Effect of κ-carrageenan on the microstructure of OP gel
A scanning electron micrograph (SEM) was used to observe the
microstructure and surface morphological of the gel. As seen in Fig. 4,
the OP gel exhibited a network with relatively smooth and irregular
cavities. The κ-carrageenan exhibited a ridge-like structure, with thin
wall layers and more wrinkles, which was attributed to the syneresis of
κ-carrageenan to form the wrinkled wall layers. Moreover, after adding
κ-carrageenan, the OP/κ-carrageenan gels showed a network structure
with abundant minor mesh and sheet edge. The κ-carrageenan aggre­
gated and formed some ridge-like structure as supporting frames to in­
crease the strength of a gel. Canola protein and κ-carrageenan formed
compact gel containing sheet structure by synergistic interaction
(Uruakpa & Arntfield, 2006). There were more thick sheet objects and
larger pores in the gel when the 2% κ-carrageenan was added. This was
because the saturation of OP surface was reached by bridging floccula­
tion and excessive κ-carrageenan aggregated in the gel system
(Schorsch, Jones, & Norton, 2000). Arltoft et al. (2007) reported similar
results that high levels of κ-carrageenan led to larger aggregates with
flocculated microstructure.
3.8. Proposed gelation mechanism of OP/κ-carrageenan gel
The proposed gelation mechanism of OP/κ-carrageenan gel was
depicted in Fig. 5. OP and κ-carrageenan in the system unfolded after
heating, exposing the internal groups of protein and helices of κ-carra­
geenan. During the cooling period, κ-carrageenan was filled into the
oyster protein and gathered to form a skeletal support. Meanwhile,
κ-carrageenan promotes hydrophobic interactions and the formation of
disulfide bonds, resulting in a more ordered and crosslinked OP network
gel structure. However, flocculation would occur when the OP surface
was saturated by κ-carrageenan.
4. Conclusion
In the current study, κ-carrageenan had a substantial impact on the
gelation behavior of OP. Based on the results, κ-carrageenan remarkably
reduced the tan δ of OP and boosted the gel formation process. The
proportion of β-sheet in OP gel was increased by κ-carrageenan, forming
a more ordered and cross-linked network structure. Compared with OP
gel, OP/κ-carrageenan gel had strong gel strength, which mainly de­
pends on disulfide and hydrophobic force. Incorporating κ-carrageenan
restricted the water motion and strengthened the WHC of OP gel.
Flexible gel with higher water content was difficult to destroy, while
texture profile analysis showed OP/κ-carrageenan gel exhibited high
cohesiveness, springiness, tackiness, and chewiness. Salt is an essential
component in the food system; the influence of KCl or NaCl on the
aquatic-derived complex gel needs to be further investigated.
S. Jiang et al. Food Chemistry 382 (2022) 132329

Fig. 4. Representative microstructure of OP gel with κ-carrageenan concentratio. OP: oyster protein, without the addition of κ-carrageenan, κ-carrageenan: pure
κ-carrageenan (2.0 wt%), OP/κC0.5: OP + 0.5% κ-carrageenan, OP/κC1.0: OP + 1.0% κ-carrageenan, OP/κC1.5: OP + 1.5% κ-carrageenan, OP/κC2.0: OP + 2.0%
κ-carrageenan.

Fig. 5. Suggested formation mechanism of OP/κ-carrageenan gel.

CRediT authorship contribution statement Acknowledgements

Suisui Jiang: Data curation, Formal analysis, Writing – original This work was supported by the National Key R&D Program of China
draft, Writing – review & editing. Yuyang Ma: Investigation, Writing – (2018YFD0901005)
original draft, Methodology, Writing – review & editing. Yahui Wang:
Data curation, Software, Methodology. Rui Wang: Investigation, Su­ Appendix A. Supplementary data
pervision, Visualization. Mingyong Zeng: Supervision, Funding
acquisition. Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2022.132329.

Declaration of Competing Interest References

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