International Journal of Food Properties

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Myofibrillar protein denaturation in frozen and

stored Clanis bilineata larvae as affected by
pullulan

Saikun Pan, Longfa Jiang, Xu Meng & Shengjun Wu

To cite this article: Saikun Pan, Longfa Jiang, Xu Meng & Shengjun Wu (2017) Myofibrillar protein
denaturation in frozen and stored Clanis�bilineata larvae as affected by pullulan, International
Journal of Food Properties, 20:sup3, S2342-S2348, DOI: 10.1080/10942912.2017.1362432

To link to this article: https://doi.org/10.1080/10942912.2017.1362432

© 2017 Taylor & Francis Group, LLC

Published online: 03 Jan 2018.

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INTERNATIONAL JOURNAL OF FOOD PROPERTIES
2017, VOL. 20, NO. S3, S2342–S2348
https://doi.org/10.1080/10942912.2018.1362432

Myofibrillar protein denaturation in frozen and stored Clanis

bilineata larvae as affected by pullulan
Saikun Pana,b,c,d, Longfa Jianga,b,c,d, Xu Menga,b,c,d, and Shengjun Wua,b,c,d
a
College of Marine Life and Fisheries, Huaihai Institute of Technology, Lianyungang, China; bMarine Resources
Development Institute of Jiangsu, Huaihai Institute of Technology, Lianyungang, China; cCo-Innovation Center of
Jiangsu Marine Bio-industry Technology, Lianyungang, China; dJiangsu Key Laboratory of Marine Biotechnology,
Huaihai Institute of Technology, Lianyungang, China

ABSTRACT ARTICLE HISTORY

Placing Clanis bilineata larvae in cold storage results in denaturation of Received 23 March 2017
their myofibrillar (MF) proteins, which alters their texture. In this study, the Accepted 28 July 2017
effect of pullulan on denaturation of MF proteins in C. bilineata larvae
KEYWORDS
frozen at −18°C for 6 months was investigated. Different amounts of Pullulan; Unfrozen water
pullulan (2.5–7.5 g/100 g wet weight) were added to MF proteins. content; Sulfhydryl content;
Alterations in MF protein properties, such as unfrozen water content, ATPase activity; Solubility
sulfhydryl content, calcium-adenylpyrophosphatase activity, and solubi-
lity, during frozen storage were evaluated. Pullulan enhanced these prop-
erties, suggesting that it is a promising cryoprotectant for C. bilineata
larvae meat during frozen storage.

Introduction
Clanis bilineata belongs to the subfamily Ambulicinae (Sphingidae, Lepidoptera). C. bilineata larvae
are edible and nutritious. Dried C. bilineata has the following characteristics: protein content, 65.5%
(w/w); essential amino acid ratio, 52.84% (w/w); fat content, 23.68% (w/w); ratio of unsaturated fatty
acids, 64.17% (w/w); and linolenic acid, which is a functional fatty acid, 36.53% (w/w) of total fatty
acids.[1] China is the largest consumer of C. bilineata larvae, with annual consumption of approxi-
mately 6000 t.
C. bilineata larva meat is usually placed in frozen storage because of seasonality. However, the
protein in C. bilineata larvae is prone to denaturation during frozen storage, resulting in deteriora-
tion of quality over time. Deteriorated meat has lower commercial and consumption values. Several
studies have investigated the influence of various agents, such as sodium chloride, sodium nitrite,
phosphate, amino acids, organic acids, protein hydrolysates, and sugars, on protein denaturation to
explore ways to inhibit the denaturation process.[2–12]
Pullulan is a polysaccharide composed mainly of maltotriose repeating units interconnected by α-
(1→6) linkages (Fig. 1). Pullulan possesses high water solubility and structural flexibility[13] and has
been widely applied in the food, cosmetic, and medical industries.[14] However, data regarding the
effects of pullulan addition on protein denaturation at freezing conditions are limited. In this study,
the protective effects of pullulan against protein denaturation in C. bilineata larva meat subjected to
freezing conditions were investigated. Alterations in unfrozen water, Ca-adenylpyrophosphatase
(ATPase) activity, sulfhydryl content, and solubility of proteins were examined.

CONTACT Shengjun Wu wusjhhit@126.com College of Marine Life and Fisheries, Huaihai Institute of Technology, 59
Cangwu Road, Haizhou, Lianyungang 222005, China.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ljfp.
© 2017 Taylor & Francis Group, LLC
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S2343

Figure 1. Molecular structure of pullulan.

Materials and methods

Materials
Fifth instar larvae of C. bilineata were purchased from a local agricultural market in Yishan City,
Lianyungang, China. All reagents used were of analytical grade.

Preparation of myofibrillar (MF) protein

MF proteins of C. bilineata larvae were prepared according to the method described by Hossain
et al.[4] Pullulan (2.5–7.5 g/100 g wet weight) was added to MF protein samples. Sodium hydroxide
(0.01 M) was added to the mixture to adjust pH to 7.0. The resulting solution was mixed at ~3°C for
20 min. Approximately 5 g of the mixture was then sealed in a test tube (inner diameter, 12 mm;
length, 75 mm) and stored at –18°C for 6 months. Control samples consisting of C. bilineata larvae
MF proteins without pullulan were stored similarly. The approximate components of MF proteins of
C. bilineata larvae were 85.76% water, 13.57% crude protein, 0.03% crude lipid, and 0.37% crude ash.

Measurement of unfrozen water

The unfrozen water content in frozen C. bilineata larvae MF was determined through methods
described previously by Yamashita et al.[11] The heat of fusion of 5–25 mg of distilled water was
determined by raising the temperature from −40°C to 25°C at a rate of 1°C/min until a linear
relationship was observed between the amount of water and the heat of fusion (79.9 cal/g H2O).
Frozen MF protein (20 mg) was placed in a tightly sealed aluminium cell and weighed. The heat of
fusion of water in the frozen MF was also measured by an identical method. The amount of unfrozen
water is considered bound water and partially bound water.

Determination of sulfhydryl content

Sulfhydryl content was assayed according to the method of Slyer et al.[15] MF protein (1 g) was
mixed with 50 mL of cold distilled water and then homogenized. The protein in the homogenate
was diluted to 2 mg/mL with 0.1 M phosphate buffer (pH 7.4). Protein content in the homo-
genate was assayed by the biuret method. Approximately 0.5 mL of the homogenate was
transferred to a tube and dissolved in urea buffer (1:1). After incubation with 0.5 mL of 5,5ˊ-
dithiobis (2-nitrobenzoic acid) at room temperature for 15 min, the absorbance of the solution
was measured at 412 nm. Sample blanks with 0.5 mL of phosphate buffer without 5,5ˊ-dithiobis
S2344 S. PAN ET AL.

(2-nitrobenzoic acid) and reagent blanks with only water were prepared. Sulfhydryl content was
calculated using a molar extinction coefficient of 11,400 M−1 cm−1 for 5,5ˊ-dithiobis at this
wavelength. The results, expressed in ŋmol, indicated the total free sulfhydryl groups per 1 mg
of protein.

Ca-ATPase activity determination

MF protein samples were subjected to different frozen storage periods. The samples were treated
according to the procedures described by Yamashita et al.[11] The Ca-ATPase activity of the MF
proteins was measured according to the methods of Katoh et al.[16] The samples (0.2–0.4 mg) were
incubated at 25°C with 100 mM KCl, 5 mM CaCl2, 25 mM Tris–maleate (pH 7.0), and 1 mM
adenosine triphosphate. The reaction was terminated by adding 30% (V/V) trichloroacetic acid to a
final concentration of 5% (v/v). The inorganic phosphate liberated in the supernatant was deter-
mined according to the method of Fiske and Subbarow.[17] Measurement results were expressed as
the ratio of the specific activity before freezing (relative %). The ratio was expressed as micromoles of
inorganic phosphate released per 1 mg of protein per minute.

Determination of solubilisation of MF protein

After being subjected to various frozen storage conditions, the samples were centrifuged at 9000 × g
at 4°C for 60 min. The protein concentration in the supernatant was then measured to obtain the
concentration of salt-soluble protein. This value was used to calculate the percentage of the initial
MF proteins remaining after frozen storage.

Analytical methods
The pH of the solution was recorded using a digital pH meter (Model: PHS-3C, CD Instruments,
China). Protein concentrations were determined using the biuret method, and bovine serum
albumin was used as a standard.[18]

Statistical analysis
All data are presented as mean ± standard deviation. Statistical analysis was performed using
Statgraphics Centurion XV version 15.1.02. Multifactor ANOVA with posterior multiple range test
was used to determine significant differences among the groups.

Results
Changes in unfrozen water contents in the C. bilineata larvae MF samples are shown in Fig. 2. The
unfrozen water content in the control samples sharply decreased initially to approximately 0.32%
over the first 2 months of frozen storage and then levelled off. In the pullulan-containing group,
although the unfrozen water contents in the C. bilineata larvae MF samples decreased slowly, they
were consistently higher than those of the control group during the entire frozen storage period. The
sulfhydryl contents in all MF protein samples decreased during the entire frozen storage period
(Fig. 3), but decreased sharply only in the initial 2 months of frozen storage in the control group.
The sulfhydryl contents in the experimental groups were consistently higher than those of the
control group during the entire frozen storage period despite gradual decrease in these contents in
the experimental groups.
The Ca-ATPase activity of the control group decreased sharply in the first 2 months and
decreased gradually thereafter. The Ca-ATPase activity of the pullulan-containing groups was higher
than those of the control group during the entire frozen storage period (Fig. 4).
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S2345

0.9

Unfrozen water (g H2O/100 g dry matter)

0.8
0.7

0.6 Control
0.5 2.5% pullulan
0.4 5% pullulan

0.3 7.5% pullulan

0.2
0.1

0
0 1 2 3 4 5 6
T ime (month)

Figure 2. Effect of pullulan on unfrozen water content in the Clanis bilineata larvae myofibrillar during frozen storage. Bars
represent the standard deviation. Data are shown as mean ± SD (n = 3).

1.4

1.2
Sulfhydryl content (ηmol/g)

1
Control
0.8 2.5% pullulan

0.6 5% pullulan
7.5% pullulan
0.4

0.2

0
0 1 2 3 4 5 6
Time (month)

Figure 3. Effect of pullulan on the Clanis bilineata larvae myofibrillar sulfhydryl content during frozen storage. Bars represent the
standard deviation. Data are shown as mean ± SD (n = 3).

120

100
Relative Ca-ATPase (%)

80 Control
2.5% pullulan
60
5% pullulan

40 7.5% pullulan

20

0
0 1 2 3 4 5 6
T ime (month)

Figure 4. Effect of pullulan on the Ca-ATPase activity of the Clanis Bilineata larvae myofibrillar during frozen storage. Bars
represent the standard deviation. Data are shown as mean ± SD (n = 3).
S2346 S. PAN ET AL.

120

100

80
Mf solubility (%)
Control
2.5% pullulan
60
5% pullulan
7.5% pullulan
40

20

0
0 1 2 3 4 5 6
T ime (month)

Figure 5. Effect of pullulan on the Clanis bilineata larvae myofibrillar solubility during frozen storage. Bars represent the standard
deviation. Data are shown as mean ± SD (n = 3).

The solubility of the MF protein in all samples decreased throughout the frozen storage period
(Fig. 5). The solubility of the control group decreased sharply during the initial 2 months of frozen
storage and then levelled off. By contrast, the solubilities of the pullulan-containing groups decreased
gradually during the entire frozen storage, and the values were higher than those of the control
group (Fig. 5; p < 0.05).

Discussion
Proteins are three-dimensional structures formed and stabilized by several factors, such as hydro-
phobic interactions, hydrogen bonds, and hydration of polar residues. Thus, disrupting these factors
alters the protein structures and can lead to loss of physiological activities, such as biocatalytic
activity. Protein denaturation during freezing can be induced by various physical and chemical
factors, such as changes in pH, high osmotic pressure, dehydration, and interactions with hydrolase.-
[4]
Sugars can stabilize proteins by stabilizing the structure of the water surrounding the proteins.
This stabilizing effect depends on the concentration of sugar in the liquid.[5]
Unfrozen water content is a critical index for the stability of meat-based food. High unfrozen
water content indicates adequate binding water, leading to the preservation of protein structures
during frozen storage.[4] In the present study, the pullulan-containing group showed higher
unfrozen water contents than the control group. This characteristic indicates that pullulan can
constrain the water in the samples and consequently inhibit the denaturation of the C. bilineata
larva MF proteins.
Formation of ice crystals alters the three-dimensional structures of MF proteins, which exposes
the sulfhydryl groups at the intramolecular level. Oxidation of sulfhydryl groups leads to formation
of disulfide bonds, which consequently decreases the sulfhydryl content of samples in frozen storage.
We found that addition of pullulan inhibited the oxidation of sulfhydryl groups, preventing decrease
in sulfhydryl content. These effects stabilized the C. bilineata larva MF proteins during frozen
storage.
We investigated the denaturation of frozen C. bilineata larva MF protein in the presence of
pullulan by measuring Ca-ATPase activity. Addition of pullulan increased Ca-ATPase activity of the
MF protein, but no increase was observed in the control group. These findings indicated that
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES S2347

inactivation of Ca-ATPase activity in the control group of the C. bilineata larvae MF protein
followed two first-order equations.
Addition of pullulan also increased solubility, unfrozen water content, sulfhydryl content, and Ca-
ATPase activity of MF proteins during frozen storage in a dose-dependent manner. The 5% and
7.5% pullulan groups showed higher efficiencies than the 2.5% pullulan group. Notably, no sig-
nificant difference was observed between the 5% and 7.5% pullulan groups, indicating that 5%
pullulan was the optimum amount for use during frozen storage.

Conclusions
The effects of pullulan on denaturation of C. bilineata larva MF proteins during frozen storage were
investigated. Addition of pullulan effectively inhibited denaturation of frozen MF proteins in a dose-
dependent manner. Thus, pullulan is a potential food cryoprotectant agent.

Funding
This research was supported by the Natural Science Fund Project of Huaihai Institute of Technology (Z2016002).

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