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Insights into cryoprotective roles of carrageenan oligosaccharides in peeled whiteleg shrimp (Litopenaeus vannamei) during frozen storage Bin Zhang, Hui-cheng Yang, He Tang, Gui-juan Hao, Yang-yang zhang, and Shang-gui Deng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05651 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017
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Journal of Agricultural and Food Chemistry
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Title: Insights into cryoprotective roles of carrageenan oligosaccharides in peeled
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whiteleg shrimp (Litopenaeus vannamei) during frozen storage
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Author names: Bin ZHANG1*, Hui-cheng YANG2, He TANG1, Gui-juan HAO1,
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Yang-yang ZHANG1, Shang-gui DENG1*
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Affiliations: 1. Key Laboratory of Health Risk Factors for Seafood of Zhejiang
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Province, College of Food Science and Pharmacy, Zhejiang Ocean University; 2.
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Zhejiang Marine Development Research Institute.
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author*: Bin
11
Corresponding
12
[email protected],
13
E-mail:
[email protected] or
ZHANG,
Tel: +(86)-0580-255-4781, E-mail:
[email protected];
Shang-gui
DENG,
14 15
Corresponding address*: No.1, Haida South Road, Lincheng Changzhi Island,
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Zhoushan, Zhejiang province, 316022 P.R.China
17 18 19 20 21 22
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ABSTRACT: The cryoprotective effects of carrageenan oligosaccharides on peeled
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whiteleg shrimp were investigated and compared with sodium pyrophosphate
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treatment during frozen storage, primarily the interaction mechanisms between
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oligosaccharides and shrimp myosin. Data revealed significant profitable effects on
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water-holding capacity and textural variables in oligosaccharide-treated shrimp
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compared to the control. Chemical analyses showed that these saccharides maintained
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a higher myofibrillar protein content and Ca2+-ATPase activity in frozen shrimp.
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Additionally, the hematoxylin and eosin staining results indicated that the saccharides
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significantly slowed the damage to muscle tissue structures. The assumption was that
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water replacement hypothesis played a leading role in cryoprotection of frozen shrimp.
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Furthermore, the homology modeling and molecular dynamics simulations confirmed
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that the saccharides substituted water molecules around shrimp myosin surface by
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forming hydrogen bonds with polar residues of amino acids, thereby stabilizing the
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structures in the absence of water, leading to an increase in protein stability during
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frozen storage.
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KEYWORDS: carrageenan oligosaccharides; sodium pyrophosphate; cryoprotective
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roles; frozen shrimp; myosin; molecular dynamics
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INTRODUCTION
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Whiteleg shrimp (Litopenaeus vannamei), also known as Pacific white shrimp, is
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one of the most important internationally traded fishery commodities as well as an
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economically representative aquatic resource. Generally, during handling of the catch
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(postmortem), quality deterioration of shrimp occurs, which is mainly caused by
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microbial contamination and autolytic enzymes. Therefore, they are generally
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processed, peeled, and then frozen as value-added products before they are exported,
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sold on the local market or further processed. Long-term preservation of the various
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shrimp products is a concern. Some undesirable changes such as protein denaturation,
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lipid oxidation, recrystallization of ice crystals, and drip loss in muscle tissue can still
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occur during the freeze/thaw process, which negatively affect the product quality and
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consumer acceptability of shrimp products 1. During frozen storage, the functional
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properties of shrimp muscle tissue are closely associated with the stability and
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integrity of muscle proteins. The denaturation and degradation of muscle proteins
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mainly contribute to the change and/or loss of those functionalities, which further
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directly affect the quality of the shrimp, including factors such as tenderness,
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water-holding capacity, juiciness, and flavor 2. Additionally, freeze-induced protein
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denaturation in muscle occurs in the order of decreasing solubility of myofibrillar
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protein, disappearance of ATP-induced contraction of muscle fiber, and a reduction of
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myosin ATPase activity 3.
65 66
Importantly, myosin in shrimp is a functional protein which is most affected by freezing,
resulting
in
denaturation/conformational
changes
and
subsequent
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cross-linking and aggregation of myofibrillar proteins, which leads to toughness in
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shrimp muscle 4. The cryoprotective effect of sugars was explained by their ability to
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increase the surface tension of water
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increased hydration of protein molecules, thus stabilizing the protein 6. Myosin in
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shrimp myofibrils retains high salt-solubility, which slowly decreases and can be
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denatured even during the first two weeks of frozen storage and/or when shrimp are
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exposed to repeated freeze-thaw cycles. Benjakul et al. 7 also found that freezing and
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frozen storage causes a marked decrease in Ca2+-ATPase activity, which in turn led to
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the denaturation of myosin. Various studies have indicated that there is a need to
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understand the mechanisms involved in myosin denaturation and improve the stability
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of myosin during freezing and frozen storage.
5
and the amount of bound water, resulting in
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Carrageenans, extracted from many species of red seaweed, are widely used in
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food and pharmaceutical industry. Carrageenan oligosaccharides that have been
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degraded by mild acidic/enzymatic hydrolysis have been recognized as playing
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significant roles in many important biological processes, including fertilization,
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inflammation, parasitic infection, oxidation, cell growth, cell-cell adhesion, and
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immune defense 8-9. Although the above advantages and applications are well known,
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the cryoprotective activities of carrageenan oligosaccharides and its mechanisms have
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not yet been studied. Therefore, the objectives of the current study were to understand
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the potential applications of carrageenan oligosaccharides by comparing the effects of
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phosphate on the water-holding capacity, texture, and chemical parameters of frozen
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shrimp, with particular focus on the cryoprotective mechanisms by evaluating the
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interactions of saccharide molecules and shrimp protein, i.e. myosin, during the
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frozen storage.
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MATERIALS AND METHODS
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Materials, processing, and sampling. Kappa-carrageenan oligosaccharides
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([C6H9O8SNa]n, n=2-4, 500-1000 Da) and sodium pyrophosphate (Na4P2O7) were
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purchased from Seebio Biotech (Shanghai) Co., Ltd. (Shanghai, China). All of the
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chemicals and reagents used in this study were of analytical grade.
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Fresh shrimp weighing 23.5-25.0 g and 12.5-14.0 cm in length from the Donghe
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Supermarket in Zhoushan (China) were packed in polystyrene boxes with slurry ice
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and transported to the laboratory within approximately 20 min. Upon arrival, the fresh
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shrimp were washed thoroughly with distilled water. The shrimp were sorted
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according to size, beheaded, and then peeled manually, but not deveined.
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Subsequently, the shrimp were submerged in prepared cryoprotective solutions at
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0-4°C for 1 h with constant stirring. The solutions were as follows: A, fresh water; B
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and C, 1.0% and 3.0% (w/v) Na4P2O7, respectively; D and E, 1.0% and 3.0% (w/v)
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carrageenan oligosaccharides, respectively. After 1 h of soaking, the shrimp were
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removed, drained for 1 min, and frozen in a freezer at -30°C for 3 h. Finally, the
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frozen samples from different batches were packed in polystyrene trays (20
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individuals each), which were placed in polyamide/polyethylene bags (20.0 × 25.0 cm,
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150 µm thickness), and stored at 23°C with 18.0 and 25.0 cc/m2/day atm for the
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transmission for O2 and CO2, respectively. All of the packaged batches were stored at
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-18°C for six weeks.
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Determination of water-holding capacity. Prior to analysis, the frozen samples
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were thawed for 3 h in a refrigerator at 4°C, drained for 1 min. Triplicate samples
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from each batch were subjected to the following analyses during frozen storage:
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thawing loss and cooking loss. The thawing loss (%) of frozen samples was measured
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by weighing the shrimp before and after the thawing process. The cooking loss (%) of
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shrimp was determined by weighing the samples before and after the cooking process
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which consisted of them being steamed for 5 min to achieve an internal temperature
119
of approximately 95°C.
120
Determination of myofibrillar protein content. Myofibrillar proteins were 10
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prepared and analyzed as described previously
122
5.0 g of each sample was minced and homogenized (10,000 rpm) in 10-volumes of
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ice-cold buffer (pH 7.0, 20 mmol/L Tris-maleate containing 0.05 mol/L KCl) using a
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blender for 60 sec at 0-4°C. The resulting homogenate was centrifuged at 10,000 × g
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for 15 min (4°C), and the supernatant was discarded. The sediment was then collected,
126
re-suspended in the same buffer, and extracted again. After two repeated cycles of
127
homogenization and centrifugation, the resulting sediment was added to 10-volumes
128
of the same ice-cold buffer. The mixture was then homogenized and centrifuged at
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6,000 × g for 15 min at 4°C. The supernatant was regarded as the myofibrillar protein
130
solution, the concentration of which was determined after proper dilution using the
131
method described by Lowry et al. 11.
132
with minor modifications. Briefly,
Determination of Ca2+-ATPase activity. According to the method of Ooizumi
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with minor modifications, the Ca2+-ATPase activity of myofibrillar
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and Xiong
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proteins was assayed in a pH 7.0, 0.50 mol/L Tris-maleate buffer containing 0.10
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mol/L CaCl2, 20 mmol/L adenosine 5’-triphosphate, and 1.0-2.0 mg/mL proteins. The
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reaction mixture was incubated for 5 min at 30°C in a water bath, and the reaction
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was terminated by the addition of 1.0 mL of chilled 15% (w/v) trichloroacetic acid.
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The reaction mixture was then centrifuged at 4,000 × g for 5 min. The amount of
139
inorganic phosphate liberated in the supernatant was assayed according to the method
140
described by Fiske and Subbarow 13. Ca2+-ATPase activity was expressed as µmol of
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inorganic phosphate (Pi) released/mg protein/min (µmolPi/mg/min).
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Texture profile analysis. Texture profile analysis (TPA) was performed using a
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texture analyzer (TMS-PRO, FTC, VA, USA). The sample was placed on the platform,
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and a P/50 cylindrical Perspex probe (50 mm diameter) simulated the chewing
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process. TPA was performed under the following conditions: constant test speed, 1.0
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mm/s; sample deformation, 30%; and hold-time between cycles, 3 sec. The texture
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analysis parameters were calculated from the force-time curves generated from each
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sample using FTC-PRO software.
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Hematoxylin and eosin (H&E) staining. The shrimp were fixed in Davidson’s
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fixative (50 mL of 37% formalin, 75 mL of alcohol, 25 mL of glacial acetic acid, and
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75 mL of tap water) for 15 h at room temperature. The tissues were then washed three
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times with 50% ethanol, dehydrated using an ascending ethanol series, and further
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embedded in paraffin blocks. Next, the obtained paraffin-embedded tissues were
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sectioned into five-µm-thick sections, and stained with H&E. The sections were then
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examined using the light microscopy (BX51, Olympus Co., Ltd., Beijing, China).
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Computational studies of molecular dynamics. To evaluate the cryprotective
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properties of carrageenan oligosaccharides in frozen shrimp, molecular dynamics
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(MD) simulations of shrimp myosin (as the typical representative) in different
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solutions were conducted by the computational studies in this experiment.
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Sequence alignment and template. Our literature review indicated that the protein
161
sequence of myosin in whiteleg shrimp has not been reported to date. Besides, the
162
amino acid sequence of the myosin heavy chain (MHC) in Marsupenaeus japonicus
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was earlier reported by Koyama et al.
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database, GenBank Acc. No. AB613205.1). However, no suitable template of whole
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myosin was found by iterative searches in the Protein Data Bank (PDB) or other
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databases. Therefore, the extracted sequence of the MHC (abbreviated as myosin
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thereafter) from M. japonicus was used as the model in this study to represent myosin,
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which was also useful in performing homology modeling and MD simulations.
14
and is available from NCBI (PubMed
169
Homology modeling. The suitable protein template (PDB ID: 1C1G) showing the
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closest homologues of myosin in shrimp was extracted from the PDB. The sequence
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identity between 1C1G (template) and myosin (target) was 42%, thereby indicating
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that 1C1G is a suitable template for modeling the structure of myosin in shrimp. The
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three-dimensional (3D) structure of myosin based on the obtained template was built
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by using Modeller software (University of California, San Francisco, CA, USA).
175 176
MD simulation. MD simulations of myosin in water and oligosaccharide systems were performed by using Amber 12 Software
15
. An Amber ff03 force field
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and
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Amber gaff force field
were utilized in optimization of myosin and saccharide
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molecule simulations, respectively. All systems were solvated by cubic boxes of
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TIP3P water, which extended at least 12 nm away from any given protein atom. The
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systems were subsequently equilibrated at 300 K (τT, 0.1 ps), during which a
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Berendsen et al. 18 barostat was used to maintain the pressure at 1 atm (1.01 × 105 Pa)
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(τp, 1.0 ps; compressive loading, 4.5 × 10-5/bar). During MD simulation, all bonds
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involving hydrogen atoms were constrained within the LINCS algorithm
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(integration-step, 2 fs; time, 100 ns; 1 frame every 1 ps). For the electrostatic
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interactions, the Particle-Mesh Ewald (PME) algorithm (truncation distance: 1.2 nm)
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was used in MD simulations.
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Data analysis. Statistical analyses were performed with the SPSS package (SPSS
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13.0 for Windows, SPSS Inc., IL, USA). Duncan’s test was used to determine
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significance in difference, and the means with different letters differed significantly at
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P < 0.05. The data are presented in means ± standard deviation (SD) of triplicate
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measurements of three replicates.
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RESULTS AND DISCUSSION
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Water-holding capacity (WHC) of frozen shrimp. WHC is defined as the
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ability of shrimp muscle to retain its own water during cutting, heating, grinding, and
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pressing as well as during transportation and frozen storage. WHC is also important
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for sensory quality and economic reasons. The effects of carrageenan oligosaccharides
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on WHC of peeled shrimp during frozen storage are presented in Table 1. Data
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revealed that during the six-week storage period, the thawing loss and cooking loss of
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the fresh water-treated samples (control) significantly increased from 6.08% to 8.85%
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and 8.50% to 12.28%, respectively, in agreement with the findings of previous studies
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19
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thawing loss and cooking loss were significantly lower over the six-week period; i.e.
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5.72% and 6.08% and 5.23% and 5.70%, respectively, significantly lower than the
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fresh water-treated values. The results indicated that the oligosaccharide
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soaking-treatment exhibited significantly better water-retaining capability, i.e. or
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cryoprotective effects. A possible explanation for the present observation is that the
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small molecules of oligosaccharides, which have a large hydration volume, offered
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good accessibility to functional groups in muscle proteins, which are expected to be
210
easily bound inside the muscle proteins and undergo H-bonding with water molecules,
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thus reducing the amount of water lost from the shrimp samples 2.
. However, for the 1.0% and 3.0% carrageenan oligosaccharide-treated shrimp, the
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Myofibrillar protein content and Ca2+-ATPase activity. The myofibrillar
213
protein content in shrimp muscle tissue decreased significantly (p < 0.05) with frozen
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storage time in all batches (Fig. 1A). The initial myofibrillar content of fresh muscle
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tissue was 129.8 mg/g. After frozen storage, the myofibrillar content of samples
216
treated with water decreased to 98.2 mg/g at week 6. However, the Na4P2O7- and
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carrageenan oligosaccharide-treated samples were in comparatively good condition,
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and their myofibrillar content ranged from 104.8-108.0 mg/g and 104.0-111.3 mg/g
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after six weeks of frozen storage, respectively. The decreased myofibrillar protein
220
content in muscle tissue, the deterioration of texture, and reduced water retention
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capacity have all been reported previously. Moreover, the degradation of myofibrillar
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and intramuscular connective tissue can occur during freezing because of the
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increased intracellular ionic strength following the migration of water into the
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extracellular spaces 20. In the current study, the oligosaccharide treatment significantly
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reduced the decrease in myofibrillar protein during storage compared with the control
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group. These results might be explained by the considerable reduction in the size of
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the ice crystals that formed in the frozen samples, thereby avoiding the irreversible
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destruction of the myofibrils. Therefore, it’s suggested that the incorporation of
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cryoprotective saccharides into muscle tissue prior to freezing could positively affect
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muscle quality during freezing and thawing of peeled shrimp.
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The Ca2+-ATPase activity in shrimp muscle tissue was also found to have
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decreased during the six-week storage period (Fig. 1B). The initial Ca2+-ATPase
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activity in shrimp muscle tissue (0 d) was 0.171 µmol Pi/mg/min. Activity then
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decreased significantly to 0.102 µmol Pi/mg/min in the control samples after six
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weeks of frozen storage, whereas activity in the carrageenan oligosaccharide-treated
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samples remained from 0.129 µmol Pi/mg/mi and 0.134 µmol Pi/mg/mi, respectively.
237
Therefore,
238
oligosaccharide-treated
239
Ca2+-ATPase activity is commonly used as a measure of actomyosin integrity as well
240
as to monitor post-mortem changes in marine species during ice shipment or frozen
241
storage. Any small microstructural change in the integrity of myofibrillar proteins can
242
decrease the activity of Ca2+-ATPase
significantly
higher
samples
Ca2+-ATPase than
in
the
activity control
was samples.
observed
in
Myofibrillar
21
. In the current study, the rapid loss in
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Ca2+-ATPase activity in the control samples was likely associated with conformational
244
changes and aggregation in the myosin globular head, which was caused by the
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generated ice crystals 21. Moreover, the rearrangement of proteins via protein-protein
246
interactions caused by decreased water-retention ability might contribute to the
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reduced Ca2+-ATPase activity. Nevertheless, the oligosaccharide-treated samples
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maintained comparatively increased Ca2+-ATPase activity, possibly by stabilizing the
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myofibrillar protein fraction, implying a decrease in product loss during storage and
250
processing.
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Texture analysis and histology staining. Fig. 2 shows the texture parameters of
252
springiness (A) and chewiness (B) values of shrimp muscle tissue after treatment with
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Na4P2O7 and carrageenan oligosaccharides during frozen storage. After six weeks of
254
storage, the shrimp treated with oligosaccharides showed springiness and chewiness
255
similar to that of shrimp treated with Na4P2O7. As a comparison, the samples treated
256
with fresh water (control) exhibited significantly lower springiness and chewiness
257
values (P < 0.05), which might be attributable to the formation of large ice crystals
258
during freezing which caused the destruction of muscle structure. Thus, it was
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possible that springiness and chewiness of the shrimp muscle were affected by the
260
presence of the saccharides and Na4P2O7. In this study, the superior stability of the
261
oligosaccharide-treated samples might be attributable to several properties of the
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saccharides glass transition state, including low free volume, restricted molecular
263
mobility, and ability to resist phase separation and crystallization during frozen
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storage.
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Regarding histology staining, hematoxylin and eosin (H&E) staining analysis of
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shrimp muscle treated with different solutions after six weeks of frozen storage is
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shown in Fig. 3. For fresh samples (Fig. 3A), the fibers were tightly connected to each
268
other, and there was little space between them. However, some of the muscle fibers in
269
the control group (Fig. 3D) were seriously disordered, forming numerous small
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fragments, and the extracellular space between the fibers was significantly larger than
271
in the fresh samples, which suggested that the mechanical strength of the muscle
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connective tissue was relatively weak and likely to be reduced further due to the
273
physical damage caused by the formation of large ice crystals. However, the muscle
274
fibers in muscle treated with oligosaccharides (Fig. 3B) were arranged in a tighter
275
manner, and the extracellular space was significantly smaller than in the control
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samples. In addition, the Na4P2O7 (Fig. 3C) treatment also preserved the physical
277
structure of the muscle significantly better than in the control samples, but the
278
cryoprotective effects were still less pronounced than in the carrageenan
279
oligosaccharide treatment groups. Previous studies reported that the freezing and
280
thawing processes could cause the shrinkage and drip loss of muscle fibers 22. It was
281
demonstrated that the ice crystals grew more rapidly in extracellular than intracellular
282
environments. This increased the solute concentration in the extracellular solution and
283
the muscle fibers began to dehydrate, which lowered the intracellular freezing point
284
and enlarged the extracellular ice crystals, leading to increased extracellular spacing
285
upon freezing and frozen storage
286
showed excellent structural preservation effects on shrimp muscle tissue, which was
23
. In this study, the oligosaccharide treatment
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likely due to the concentration of water molecules near the surface of the membrane
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and proteins, thereby protecting them from damage during freezing 24.
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Possible cryoprotective mechanisms. Currently, cryoprotectants such as
290
chitosan and its derivatives 2, konjac glucomannan, protein hydrolysate
25
291
saccharides were studied to ensure maximum functionality of frozen muscle tissue.
292
These carbohydrates stabilize proteins, prevent withdrawal of water from the proteins,
293
increase the surface tension of water, and prevent the loss of protein solubility 19. In
294
the present study, to elucidate the underlying cryoprotective mechanisms of the
295
carrageenan oligosaccharide treatment, three possible mechanisms were hypothesized
296
on the basis of previous experimental results 24:
, and other
297
і. The carrageenan oligosaccharides presumably replaced the water molecules by
298
forming large hydrogen bonds with the polar residues of the proteins in shrimp
299
muscle, thereby stabilizing their structures in the absence of water while in the frozen
300
state, thereby maintaining the protein structure and protecting these from the
301
freeze-induced damage 26-27.
302
ii. The water entrapment theory suggests it is likely that carrageenan
303
oligosaccharides moderately concentrated the water molecules near the membrane
304
and protein surface and suppressed the nucleation and growth of ice crystals, thereby
305
maintaining the protein structure upon freezing 28-29.
306
iii. The vitrification (glass formation) hypothesis states that with the addition of
307
oligosaccharides, the cellular components are brought into glassy state, which
308
prevents ice crystal growth by restricting the mobility of water molecules that may be
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caged by the saccharide glasses and thus could avoid destruction during
310
freeze-thawing 30-31.
311
However, the underlying mechanisms for the cryoprotective properties in shrimp
312
have not been elucidated. Therefore, computational studies of shrimp myosin and
313
oligosaccharide dynamics were conducted to investigate the cryoprotective roles of
314
saccharides, as well as to verify the three previously described hypotheses.
315
MD simulations. Sequence alignment and homology modeling analysis were
316
used to provide insight into the functional roles of saccharides in shrimp myosin
317
during frozen storage. Fig. 4A shows the alignment of amino acid sequences to the
318
myosin (top panel) and the template protein (1C1G, bottom panel). Previous reports
319
have indicated that when the sequence identity to the target structure was >40%, the
320
homology models were equally satisfactory
321
that when the sequence identity between the target and the template was >50%, the
322
docking performance was comparable to that of the crystal structures. In the present
323
study, the computational sequence identity between 1C1G and myosin was 42.0%,
324
thus rendering 1C1G a suitable template for modeling myosin.
32
. Similarly, Bernacki et al.
33
reported
325
Homology modeling. By using the established template structure of myosin in the
326
PDB, a myosin model (a dimer of MHC, Fig. 4B) showing both the template
327
sequence and its structural similarity to myosin was generated. In absence of crystal
328
structures, homology models are the only alternative to creating a 3D representation
329
of the target protein. The myosin molecule is a hexamer that is composed of two
330
MHC subunits (approx. 200 kDa), and four MLC subunits that were located in the
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head region (approx. 20 kDa); each MHC is associated with two MLCs. The MHC in
332
myosin has several physiologically important functions, which include ATP and actin
333
binding 14. Here, the 3D structure consisting of two MHC subunits was generated and
334
used in the subsequent MD simulations.
335
RMSD. The root-mean-squared deviation (RMSD) parameter measures the
336
overall changes in conformation from the initial or any other reference structure. A
337
plot of the RMSD for each of the simulations is shown in Fig. 5A. From the results,
338
the larger RMSD values with respect to myosin in the water system illustrated that the
339
structures in the trajectories significantly differed from that in the carrageenan
340
oligosaccharide system. Additionally, the RMSD results also indicated that the current
341
simulations included saccharide molecules, which were compared to the water-only
342
molecules in the simulation, and the overall trajectories showed similar trends.
343
However, the calculated RMSD values of the myosin structures when the saccharides
344
were incorporated showed significantly lower fluctuations than those observed in the
345
simulations that excluded the saccharides (Fig. 5A). These observations indicate that
346
the inclusion of the saccharides, which presumably affects the structure of myosin, the
347
distribution of water molecular around protein molecules
348
bonds between the hydroxyl groups of saccharides and proteins, led to a decrease in
349
RMSD fluctuations (values) and a better protection of protein stability in the MD
350
systems.
34
, and/or the hydrogen
351
RMSF. Identification of the more-flexible regions of a protein during a
352
simulation can be obtained via examination of the root-mean-square fluctuation
16
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353
(RMSF) of each residue from its time-averaged position. Fig. 5B compares the RMSF
354
of the backbone atoms of myosin in the absence or presence of the saccharide
355
molecules. Similar overall patterns of RMSF versus residue number were seen for the
356
two simulations. Nevertheless, analysis indicated greater fluctuations in the selectivity
357
filter residues for the simulations of myosin in the water system compared to the
358
saccharide system, which is in agreement with the findings on RMSD analysis. Taken
359
together, the inclusion of saccharides significantly (P < 0.05) decreased the flexibility
360
of myosin molecules. Additionally, residues 1-50 and 285-320 in the N-terminal half
361
and 250-286 and 525-568 in the C-terminal half showed higher RMSF values.
362
Furthermore, residues 99-105 (KRKLEGE) and 198-204 (LDEEVRR) also identified
363
regions of the higher mobility (RMSF values) during the simulations. Notably, these
364
residues showed the most pronounced differences in flexibility between the water and
365
saccharide systems, thereby indicated that myosin is stable in the saccharide
366
environment but is less stable in a pure water environment
367
variations of myosin flexibility also might account for the lower RMSF values
368
observed in the saccharide system.
35
. Additionally, the
369
Surface electrostatic potential. The average RMSF of backbone residues
370
indicated that the regions were undergoing large movements, the residues of which
371
were generally located either in the surface that had exposed loops/links or at N- and
372
C-termini
373
‘KRKLEGE’ and ‘LDEEVRR’ regions of myosin also showed greater mobility
374
(flexibility) during the RMSF analysis. Therefore, the electrostatic potential at the
35-36
. Furthermore, in the present study, apart from N- and C-termini, the
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375
surfaces of these two regions was further computed to explore the effects of
376
saccharides on these myosin mobility sites. The different colors in the molecular
377
surface of the two regions represent the calculated electrostatic potential (Fig. 6). The
378
amino acid residues (99-105 and 198-204) at these two protein chains, which have
379
similar electrical charges, repelled one another by virtue of their electrostatic
380
interactions. Thus, a strong electrostatic repulsion had occurred between the two
381
protein chains, thereby resulting in a greater flexibility at these two regions.
382
Nevertheless, the flexibility of these two regions could be significantly decreased by
383
the incorporation of saccharide molecules, which might account for the decrease in
384
RMSF values observed in the saccharide system 37. These observations, together with
385
RMSD and RMSF analyses, suggest that the stable properties of myosin (two chains)
386
could be significantly improved by incorporating saccharide molecules, which affect
387
the flexibility of these particular regions 38.
388
Residue cross-correlation. The examples of cross-correlation matrices between
389
the residues (at the same position) within the two chains calculated for myosin in
390
different systems are presented in Fig. 7. Positive correlations (elliptic regions) are
391
shown in the lower right triangles. The color of the diagonal portions in the maps that
392
become red indicates strong correlations between the residues of the two chains. In
393
the maps, the residues of the two chains gave rise to positive correlation triangles
394
along the diagonal. Notably, the corresponding residues within myosin in the water
395
system showed relatively weak cross-correlations (Fig. 7A). For comparison, the
396
presence of saccharides (Fig. 7B) in the solution significantly (P < 0.05) enhanced
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397
residue cross-correlations in the present analysis, thereby resulting in consistent and
398
stable correlated fluctuations in the two chains within myosin. In other words, the
399
consistency and cooperativity of internal chain motions was markedly improved with
400
the introduction of saccharides in the simulation system. In the present study, the
401
carrageenan oligosaccharides have larger hydration numbers, thus serving a larger
402
number of hydrogen bonding sites to the residues within myosin in place of water
403
molecules
404
accounting for the variations in the correlations within the myosin residues. These
405
findings were in agreement with the results of RMSD and RMSF analysis.
24
and protecting the conformational stability of myosin structures
39
, thus
406
Interaction energy between the two chains of myosin. To explore the critical roles
407
of saccharides in stabilization, the interaction energy between the two chains of
408
myosin was calculated over a trajectory range of 50-100 ns (Table 2). The results
409
indicated that the calculated van der Waals forces were significantly (P < 0.05) higher
410
than that of the electrostatic interaction forces. Significantly, the subtotal interaction
411
energy between the two chains calculated in the water system (-4,433.54 kJ/mol) was
412
stronger than its values obtained in the carrageenan oligosaccharide (-4003.65 kJ/mol)
413
system. The greater the occurrence of interaction energy between the two chains, the
414
greater the occurrence of crosslinking of myosin chains which led to the decreased
415
protein solubility and increased freeze-thaw drip. Additionally, correlation analysis of
416
RMSD values and time for simulations of each chain within myosin was also
417
performed (Fig. 8A and 8B). Importantly, the MD trajectories of each chain in the
418
saccharide system appeared to be more stable compared to its observed trajectory in
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419
the water system. The lower fluctuations of each chain illustrated that the inclusion of
420
saccharide molecules may have influenced the structure and/or stability of each chain,
421
thereby leading to a decrease in RMSD fluctuations.
422
Interaction energy between myosin and water/saccharide molecules. The
423
calculated results (Table 2) indicated that the energy of electrostatic interactions
424
largely contributed to the observed subtotal interaction energy between myosin and
425
water/saccharides in all the simulations. Electrostatic interactions play a central role in
426
a variety of functions of proteins
427
structure and stability, and appeare explicitly in the computer analysis of protein
428
conformation and dynamics
429
the present study were in good agreement with those of previous reports. For the
430
interaction between myosin and water molecules, the maximum electrostatic energy
431
in the water system was approximately -69,244.20 kJ/mol, which was significantly
432
higher than that of the carrageenan oligosaccharide (-52,424.34 kJ/mol) system. In the
433
pure water system, the bulk water molecules underwent great interactions with the
434
myosin molecules via the formation of a large number of hydrogen bonds 42, thereby
435
generating strong electrostatic interactions that primarily resulted from the effects of
436
charged amino acids. The hydrogen bonding largely contributes to the electrostatic
437
interaction energy in the water solution. In other words, the number of formed
438
hydrogen bonds is associated with the strength of the electrostatic interactions
439
However, in the saccharide system, the electrostatic interaction energy decreased
440
significantly (P < 0.05) when the saccharide molecules were added to the simulation
40
, which are an essential determinant of protein
41
. Hence, these findings of electrostatic interactions in
42
.
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441
system. Further analysis of the hydrogen bonds between myosin and water molecules
442
(Fig. 8C) confirmed a significant reduction (P < 0.05) in hydrogen bonding to myosin
443
in the saccharide system compared to that in the pure water system, which may be
444
attributable to the removal of water around the myosin surface and its replacement by
445
saccharide molecules
446
myosin surface lowered the disruption level of muscle proteins, which was mainly
447
caused by the formation of large ice crystals during frozen storage 44.
43
. The decrease in the number water molecules around the
448
Simulation analysis showed that apart from water molecules, myosin can also
449
interact with saccharide molecules. Similarly, the electrostatic interaction energy
450
played a major role in the myosin-saccharides interactions. Therefore, the more
451
favorable electrostatic interactions between myosin and saccharides may have driven
452
the saccharide molecules to attach to the myosin surface. The electrostatic interactions
453
obtained between myosin and saccharide molecules were significantly (P < 0.05)
454
lower than that of values between myosin and water molecules (Table 2). Clearly, the
455
changes in the number of hydrogen bonds formed between myosin and water
456
molecules in different systems confirmed the events in the saccharide-induced
457
weakening of electrostatic interactions
458
the interaction energy with myosin was observed during the transition from pure
459
water to the saccharide system.
43
. Accordingly, a significant stabilization in
460
Proteins are stabilized by multiple noncovalent interactions, and the disruption of
461
these bonds by either mechanical or chemical means induces protein unfolding and
462
denaturation 42. The results showed that van der Waals forces are an important factor
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463
in improving the stability of two myosin chains. Nevertheless, electrostatic
464
interactions play a major role in the interaction of myosin with water/saccharide
465
molecules. First, the improved stability of myosin in the presence of saccharide
466
molecules was mainly attributable to water replacement. The saccharide molecules
467
interacted with myosin and replaced part of the water molecules around the protein
468
surface by forming hydrogen bonds with the polar residues of charged amino acids.
469
Second, the interacted saccharides reduced the fluctuation and flexibility of each
470
myosin chain, thereby leading to an increase in stabilization. In any case, in the
471
present study, the vitrification hypothesis and entrapment theory apparently did not
472
seem to significantly contribute to the stability of myosin in the simulated systems.
473 474
ACKNOWLEDGMENTS
475
The authors would like to thank all the researchers whose data they have
476
referenced. This study is a project funded by the Natural Science Foundation of
477
Zhejiang Province (Grant No. LY15C200017); and the Public Projects of Zhejiang
478
Province
479
(www.letpub.com) for its linguistic assistance during preparation of this manuscript.
480
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481
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482
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(Grant
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and
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and denaturation of lizardfish (Saurida wanieso) myofibrillar protein during
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effective than other sugars. Arch. Biochem. Biophys. 1998, 360, 10-14.
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α-trehalose in a vacuum and in aqueous and salt solutions. J. Phys. Chem. A 2014,
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(32) Nayeem, A.; Sitkoff, D.; Krystek, S. A. Comparative study of available software
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for high-accuracy homology modeling: From sequence alignments to structural
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simulation into substrate-induced changes in protein motions of proteinase K. J.
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Biophys. J. 2000, 78, 557-570.
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alcohol dehydrogenase folding with those of sorbitol molecules on alcohol
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dehydrogenase folding using molecular dynamics simulation. J. Iran. Chem. Soc.
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2015, 12, 1973-1982.
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(39) Ces à ro, A.; De Giacomo, O.; Sussich, F. Water interplay in trehalose polymorphism. Food Chem. 2008, 106, 1318-1328. (40) Gilson, M. K.; Rashin, A.; Fine, R.; Honig, B. On the calculation of electrostatic
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interactions in proteins. J. Mol. Biol. 1985, 183, 503-516. (41) Rashin, A. A.; Honig, B. On the environment of ionizable groups in globular proteins. J. Mol. Biol. 1984, 173, 515-521. (42) Raschke, T. M. Water structure and interactions with protein surface. Curr. Opin. Struc. Biol. 2006, 16, 152-159.
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(43) Allison, S. D.; Chang, B.; Randolph, T. W.; Carpenter, J. F. Hydrogen bonding
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between sugar and protein is responsible for inhibition of dehydration-induced
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protein unfolding. Arch. Biochem. Biophys. 1999, 365, 289-298.
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(44) Xie, C.; Zhang, B.; Ma, L. K.; Sun, J. P. Cryoprotective effects of trehalose,
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alginate, and its oligosaccharide on quality of cooked-shrimp (Litopenaeus
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DOI:10.1111/jfpp.12825.
during
frozen
storage.
J.
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607 608 609 610 611 612 613 614 615 616
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FIGURE CAPTIONS
618
Figure 1. Myofibrillar protein content (A) and Ca2+-ATPase activity (B) in shrimp
619
muscle tissue treated with the control, sodium pyrophosphate, and carrageenan
620
oligosaccharides during frozen storage. Mean values of triplicate samples are shown;
621
vertical bars denote standard deviation.
622 623
Figure 2. Springiness (A) and chewiness (B) of shrimp muscle tissue treated with the
624
control, sodium pyrophosphate, and carrageenan oligosaccharides during frozen
625
storage. Mean values of triplicate samples are shown
626
; vertical bars denote standard deviation.
627 628
Figure 3. Micrographs of transverse-sections of shrimp muscle tissue from the second
629
abdominal segment treated with the control, sodium pyrophosphate, and carrageenan
630
oligosaccharides after six weeks of frozen storage. A: fresh shrimp muscle tissue (0 d);
631
B: shrimp muscle tissue treated with 3% carrageenan oligosaccharides; C: shrimp
632
muscle tissue treated with 3% sodium pyrophosphate; D: control (shrimp muscle
633
tissue treated with water); magnification was 200× original size, bar length = 100 µm.
634 635
Figure 4. Amino acid sequence and homology model. A: alignment of amino acid
636
sequence of the target (myosin, top) and the template protein (1C1G, bottom); B: 3D
637
representation of myosin by homology modeling.
638
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639
Figure 5. Comparison of atom root-mean-squared deviation (RMSD, A) and
640
backbone atom root-mean-square fluctuation (RMSF, B) for simulations of myosin in
641
water and carrageenan oligosaccharide systems.
642 643
Figure 6. Representative regions with large flexibility (ball-and-stick models, top)
644
and its electrostatic potentials at the surfaces of myosin (bottom). A: the regions of
645
amino acids 99-105 (KRKLEGE); B: amino acids 198-204 (LDEEVRR).
646 647
Figure 7. The calculated cross-correlations between the homologous residues of the
648
two myosin chains in water (A) and carrageenan oligosaccharide (B) systems.
649 650
Figure 8. Comparison of RMSD for simulations of each myosin chain in water (A)
651
and carrageenan oligosaccharides (B) systems. Number of hydrogen bonds formed
652
between myosin and water molecules in water and carrageenan oligosaccharide (C)
653
systems.
654 655
TABLE CAPTIONS
656
Table 1. Effect of sodium pyrophosphate and carrageenan oligosaccharides on
657
water-holding capacity of shrimp during frozen storage.
658
Table 2. Interaction energies (kJ/mol) between two chains of myosin, myosin and
659
water, and myosin and saccharide molecules in different solutions after MD
660
simulations.
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661
Journal of Agricultural and Food Chemistry
Table 1 Effect of sodium pyrophosphate and carrageenan oligosaccharides on water-holding capacity of shrimp during frozen storage.
WHC
Storage time (week)
Sodium pyrophosphate (%)
Carrageenan oligosaccharides (%)
1%
3%
1%
3%
Fresh water (%)
0
6.08 ± 0.18Ab
5.02 ± 0.23Aa
4.85 ± 0.13Aa
4.95 ± 0.16Aa
4.88 ± 0.20Aa
2
6.71 ± 0.21Bb
5.22 ± 0.19ABa
4.98 ± 0.15ABa
5.18 ± 0.26ABa
4.93 ± 0.17ABa
4
7.16 ± 0.24Cc
5.52 ± 0.24BCb
5.16 ± 0.13BCa
5.60 ± 0.37Bb
5.22 ± 0.33Bab
6
8.85 ± 0.17Dc
5.85 ± 0.36Cb
5.33 ± 0.20Cab
5.72 ± 0.34Bb
5.23 ± 0.23Ba
0
8.50 ± 0.38Ab
5.36 ± 0.25Aa
5.11 ± 0.21Aa
5.31 ± 0.22Aa
5.14 ± 0.19Aa
2
9.07 ± 0.47Ab
5.45 ± 0.19ABa
5.32 ± 0.28ABa
5.38 ± 0.31Aa
5.28 ± 0.23ABa
4
10.05 ± 0.51Bb
5.84 ± 0.30Ba
5.78 ± 0.31BCa
5.71 ± 0.18Ba
5.56 ± 0.27BCa
6
12.28 ± 0.49Cd
6.62 ± 0.36Cc
6.29 ± 0.34Cbc
6.08 ± 0.24Bab
5.70 ± 0.31Ca
Thawing loss (%)
Cooking loss (%)
662
Data represent the means ± S.D. of measurement for three replicates. Duncan’s test was used to determine the significance, and the means with
663
different capital and lowercase letters in the same column and row for the thawing loss and cooking loss respectively, were both significantly
664
different at p < 0.05.
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665
Table 2 Interaction energies (kJ/mol) between two chains of myosin, myosin and water, and myosin and saccharide molecules in different
666
solutions after MD simulations. Simulations
Between two chains within myosin
Myosin interacted with water
Myosin interacted with saccharides
Water system
Carrageenan oligosaccharide system
Van der Waals force
-3085.55 ± 63.25
-3092.71 ± 60.08
Electrostatic interaction
-1347.99 ± 217.33
-910.94 ± 145.64
Subtotal
-4433.54 ± 217.77
-4003.65 ± 147.18
Van der Waals force
-5626.84 ± 221.90
-3945.74 ± 362.87
Electrostatic interaction
-69244.20 ± 1037.16
-52424.34 ± 2202.06
Subtotal
-74871.10 ± 1009.26
-56370.09 ± 2403.37
Van der Waals force
‒
-7266.24 ± 300.81
667
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Figure 1. Myofibrillar protein content (A) and Ca2+-ATPase activity (B) in shrimp muscle tissue treated with the control, sodium pyrophosphate, and carrageenan oligosaccharides during frozen storage. Mean values of triplicate samples are shown; vertical bars denote standard deviation. 338x128mm (150 x 150 DPI)
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Journal of Agricultural and Food Chemistry
Figure 2. Springiness (A) and chewiness (B) of shrimp muscle tissue treated with the control, sodium pyrophosphate, and carrageenan oligosaccharides during frozen storage. Mean values of triplicate samples are shown ; vertical bars denote standard deviation. 338x125mm (150 x 150 DPI)
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Journal of Agricultural and Food Chemistry
Figure 3. Micrographs of transverse-sections of shrimp muscle tissue from the second abdominal segment treated with the control, sodium pyrophosphate, and carrageenan oligosaccharides after six weeks of frozen storage. A: fresh shrimp muscle tissue (0 d); B: shrimp muscle tissue treated with 3% carrageenan oligosaccharides; C: shrimp muscle tissue treated with 3% sodium pyrophosphate; D: control (shrimp muscle tissue treated with water); magnification was 200× original size, bar length = 100 µm. 169x130mm (300 x 300 DPI)
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Journal of Agricultural and Food Chemistry
Figure 4. Amino acid sequence and homology model. A: alignment of amino acid sequence of the target (myosin, top) and the template protein (1C1G, bottom); B: 3D representation of myosin by homology modeling. 66x25mm (300 x 300 DPI)
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Journal of Agricultural and Food Chemistry
Figure 5. Comparison of atom root-mean-squared deviation (RMSD, A) and backbone atom root-meansquare fluctuation (RMSF, B) for simulations of myosin in water and carrageenan oligosaccharide systems. 169x64mm (300 x 300 DPI)
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Journal of Agricultural and Food Chemistry
Figure 6. Representative regions with large flexibility (ball-and-stick models, top) and its electrostatic potentials at the surfaces of myosin (bottom). A: the regions of amino acids 99-105 (KRKLEGE); B: amino acids 198-204 (LDEEVRR). 127x106mm (300 x 300 DPI)
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Journal of Agricultural and Food Chemistry
Figure 7. The calculated cross-correlations between the homologous residues of the two myosin chains in water (A) and carrageenan oligosaccharide (B) systems. 118x38mm (300 x 300 DPI)
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Journal of Agricultural and Food Chemistry
Figure 8. Comparison of RMSD for simulations of each myosin chain in water (A) and carrageenan oligosaccharides (B) systems. Number of hydrogen bonds formed between myosin and water molecules in water and carrageenan oligosaccharide (C) systems. 254x63mm (300 x 300 DPI)
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Journal of Agricultural and Food Chemistry
TOC Graphic 210x71mm (300 x 300 DPI)
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