Insights into Cryoprotective Roles of Carrageenan Oligosaccharides

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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

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Corresponding

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[email protected],

13

E-mail:[email protected]

or

ZHANG,

Tel: +(86)-0580-255-4781, E-mail:

[email protected];

Shang-gui

DENG,

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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

1

ACS Paragon Plus Environment


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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.

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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.

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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

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of approximately 95°C.

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Determination of myofibrillar protein content. Myofibrillar proteins were 10

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prepared and analyzed as described previously

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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,

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re-suspended in the same buffer, and extracted again. After two repeated cycles of

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homogenization and centrifugation, the resulting sediment was added to 10-volumes

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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

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solution, the concentration of which was determined after proper dilution using the

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method described by Lowry et al. 11.

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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

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inorganic phosphate liberated in the supernatant was assayed according to the method

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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

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sequence of myosin in whiteleg shrimp has not been reported to date. Besides, the

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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

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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).

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MD simulation. MD simulations of myosin in water and oligosaccharide systems were performed by using Amber 12 Software

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. An Amber ff03 force field

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and

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17

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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

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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

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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

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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

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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.

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Therefore,

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oligosaccharide-treated

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Ca2+-ATPase activity is commonly used as a measure of actomyosin integrity as well

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as to monitor post-mortem changes in marine species during ice shipment or frozen

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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

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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

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interactions caused by decreased water-retention ability might contribute to the

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reduced Ca2+-ATPase activity. Nevertheless, the oligosaccharide-treated samples

248

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

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processing.

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Texture analysis and histology staining. Fig. 2 shows the texture parameters of

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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

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storage, the shrimp treated with oligosaccharides showed springiness and chewiness

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similar to that of shrimp treated with Na4P2O7. As a comparison, the samples treated

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with fresh water (control) exhibited significantly lower springiness and chewiness

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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

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presence of the saccharides and Na4P2O7. In this study, the superior stability of the

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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

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the control group (Fig. 3D) were seriously disordered, forming numerous small

270

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

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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

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cryoprotective effects were still less pronounced than in the carrageenan

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oligosaccharide treatment groups. Previous studies reported that the freezing and

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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

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environments. This increased the solute concentration in the extracellular solution and

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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

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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.

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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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331

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

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(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

17



Page 18 of 41

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

18


Page 19 of 41


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

19



Page 20 of 41

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

.

20


Page 21 of 41


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

21



Page 22 of 41

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

REFERENCES

481

(1) Ma, L. K.; Zhang, B.; Deng, S. G.; Xie, C. Comparison of the cryoprotective

482

effects of trehalose, alginate, and its oligosaccharides on peeled shrimp

483

(Litopenaeus vannamei) during frozen storage. J. Food Sci. 2015, 80, 540-546.

484

(Grant

No.

2016C32081

and

2016C32080).

We

thank

LetPub

(2) Chantarasataporn, P.; Yoksan, R.; Visessanguan, W.; Chirachanchai, S.

22


Page 23 of 41


485

Water-based nano-sized chitin and chitosan as seafood additive through a case

486

study of Pacific white shrimp (Litopenaeus vannamei). Food Hydrocolloid. 2013,

487

32, 341-348.

488

(3) Somjit, K.; Ruttanapornwareesakul, Y.; Hara, K.; Nozaki, Y. The cryoprotectant

489

effect of shrimp chitin and shrimp chitin hydrolysate on denaturation and

490

unfrozen water of lizardfish surimi during frozen storage. Food Res. Int. 2005, 38,

491

345-355.

492

(4) Boonsumrej, S.; Chaiwanichsiri, S.; Tantratian, S.; Suzuki, T.; Takai, R. Effects of

493

freezing and thawing on the quality changes of tiger shrimp (Penaeus monodon)

494

frozen by air-blast and cryogenic freezing. J. Food Eng. 2007, 80, 292-299.

495 496

(5) Arakawa, T.; Timasheff, S.N. Stabilization of protein structure by sugars. Biochemistry 1982, 21, 6536-6544.

497

(6) Matsumotro, J. J.; Noguchi, S. F. Cryostabilization of protein in surimi. In Surimi

498

Technology, edited by Lanier, T. C. and Lee, C. M. New York/Basel/Hong Kong:

499

Marcel Dekker, 1992.

500

(7) Benjakul, S.; Visessanguan, W.; Thongkaew, C.; Tanaka, M. Comparative study

501

on physicochemical changes of muscle proteins from some tropical fish during

502

frozen storage. Food Res. Int. 2003, 36, 787-795.

503 504

(8) Yuan, H.; Song, J.; Li, X.; Li, N.; Dai, J. Immunomodulation and antitumor activity of κ-carrageenan oligosaccharides. Cancer lett. 2006, 243, 228-234.

505

(9) Campo, V. L.; Kawano, D. F.; da Silva, D. B.; Carvalho, I. Carrageenans:

506

Biological properties, chemical modifications and structural analysis-A review.

23



507

Page 24 of 41

Carbohyd. Polym. 2009, 77, 167-180.

508

(10) Xia, X.; Kong, B.; Liu, Q.; Liu, J. Physicochemical change and protein oxidation

509

in porcine longissimus dorsi as influenced by different freeze-thaw cycles. Meat

510

Sci. 2009, 83, 239-245.

511 512

(11) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265-275.

513

(12) Ooizumi, T.; Xiong, Y. L. Biochemical susceptibility of myosin in chicken

514

myofibrils subjected to hydroxyl radical oxidizing systems. J. Agr. Food Chem.

515

2004, 52, 4303-4307.

516 517

(13) Fiske, C. H.; Subbarow, Y. The colorimetric determination of phosphorus. J. Biol. Chem. 1925, 66, 375-400.

518

(14) Koyama, H.; Akolkar, D. B.; Shiokai, T.; Nakaya, M.; Piyapattanakorn, S.;

519

Watabe, S. The occurrence of two types of fast skeletal myosin heavy chains from

520

abdominal muscle of kuruma shrimp Marsupenaeus japonicus and their different

521

tissue distribution. J. Exp. Biol. 2012, 215, 14-21.

522

(15) Case, D. A.; Darden, T. A.; Cheatham, III. T. E.; Simmerling, C. L.; Wang, J.;

523

Duke, R. E.; et al. AMBER 12. University of California, San Francisco, 2012.

524

(16) Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; et al. A

525

point-charge force field for molecular mechanics simulations of proteins based on

526

condensed-phase quantum mechanical calculations. J. Comput. Chem. 2003, 24,

527

1999-2012.

528

(17) Wang, J. M.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A.

24


Page 25 of 41


529

Development and testing of a general amber force field. J. Comput. Chem. 2004,

530

25, 1157-1174.

531

(18) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J.

532

R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 1984,

533

81, 3684-3690.

534 535 536 537

(19) Leygonie, C.; Britz, T. J.; Hoffman, L. C. Impact of freezing and thawing on the quality of meat: Review. Meat Sci. 2012, 91, 93-98. (20) Li, B.; Sun, D. W. Novel methods for rapid freezing and thawing of foods-a review. J. Food Eng. 2002, 54, 175-182.

538

(21) Reza, M.; Bapary, M. A. J.; Ahasan, C. T.; Islam, M. N.; Kamal, M. Shelf life of

539

several marine fish species of Bangladesh during ice storage. Int. J. Food Sci.

540

Technol. 2009, 44, 1485-1494.

541

(22) Sriket, P.; Benjakul, S.; Visessanguan, W.; Kijroongrojana, K. Comparative

542

studies on the effect of the freeze-thawing process on the physicochemical

543

properties and microstructures of black tiger shrimp (Penaeus monodon) and

544

white shrimp (Penaeus vannamei) muscle. Food Chem. 2007, 104, 113-121.

545

(23) Chen, Y. L.; Pan, B.S. Morphological changes in tilapia muscle following

546

freezing by airblast and liquid nitrogen methods. Int. J. Food Sci. Technol. 1997,

547

32, 159-168.

548

(24) Kuwajima, K.; Goto, Y.; Hirata, F.; Terazima, M.; Kataoka, M. Water and

549

biomolecules: physical chemistry of life phenomena (Biological and Medical

550

hysics, Biomedical Engineering). Springer-Verlag Berlin Heidelberg, 2009.

25



Page 26 of 41

551

(25) Hossain, M. A.; Alikhan, M. A.; Ishihara, T.; Hara, K.; Osatomi, K.; Osaka, K.;

552

Nazaki, Y. Effect of proteolytic squid protein hydrolysate on the state of water

553

and denaturation of lizardfish (Saurida wanieso) myofibrillar protein during

554

freezing. Innov. Food Sci. Emerg. 2004, 5, 73-79.

555 556

(26) Cottone, G.; Ciccotti, G.; Cordone, L. Protein-trehalose-water structures in trehalose coated carboxy-myoglobin. J. Chem. Phys. 2002, 117, 9862-9866.

557

(27) Sola-Penna, M.; Meyer-Fernandes, J. R. Stabilization against thermal inactivation

558

promoted by sugars on enzyme structure and function: why is trehalose more

559

effective than other sugars. Arch. Biochem. Biophys. 1998, 360, 10-14.

560 561

(28) Lins, R. D.; Pereira, C. S.; Hünenberger, P. H. Trehalose-protein interaction in aqueous solution. Proteins. 2004, 55, 177-186.

562

(29) Tadanori, S.; Takehiko, G.; Yoshiyasu, A. Growth rate and morphology of ice

563

crystals growing in a solution of trehalose and water. J. Cryst. Growth 2002, 240,

564

218-229.

565

(30) Kan, Z.; Yan, X.; Ma, J. Conformation dynamics and polarization effect of α,

566

α-trehalose in a vacuum and in aqueous and salt solutions. J. Phys. Chem. A 2014,

567

119, 1573-1589.

568 569

(31) Crowe, J. H.; Carpenter, J. F.; Crowe, L. M. The role of vitrification in anhydrobiosis. Ann. Rev. Physiol. 1998, 60, 73-103.

570

(32) Nayeem, A.; Sitkoff, D.; Krystek, S. A. Comparative study of available software

571

for high-accuracy homology modeling: From sequence alignments to structural

572

models. Protein Sci. 2006, 15, 808-824.

26


Page 27 of 41


573

(33) Bernacki, K.; Kalyanaraman, C.; Chorny, I.; Jacobson, M. P. Improving the

574

quality of virtual ligand screening against homology models. In Abstracts of

575

Papers, 229th ACS ational Meeting, San Diego, CA. 2005, 13-17.

576

(34) Edelman, R.; Kusner, I.; Kisiliak, R.; Srebnik, S.; Livney, Y. D. Sugar

577

stereochemistry effects on water structure and on protein stability: The templating

578

concept. Food Hydrocolloid. 2015, 48, 27-37.

579

(35) Tao, Y.; Rao, Z. H.; Liu, S. Q. Insight derived from molecular dynamics

580

simulation into substrate-induced changes in protein motions of proteinase K. J.

581

Biomol. Structure Dynam. 2010, 28, 143-157.

582

(36) Shrivastava, I. H.; Sansom, M. S. P. Simulations of ion permeation through a

583

potassium channel: molecular dynamics of KcsA in a phospholipid bilayer.

584

Biophys. J. 2000, 78, 557-570.

585

(37) Alizadeh-Rahrovi, J.; Shayesteh, A.; Ebrahim-Habibi, A. Structural stability of

586

myoglobin and glycomyoglobin: a comparative molecular dynamics simulation

587

study. J. Biolog. Phys. 2015, 41, 349-366.

588

(38) Bahrami, H.; Zahedi, M. Comparison of the effects of sucrose molecules on

589

alcohol dehydrogenase folding with those of sorbitol molecules on alcohol

590

dehydrogenase folding using molecular dynamics simulation. J. Iran. Chem. Soc.

591

2015, 12, 1973-1982.

592 593 594

(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

27



595 596 597 598 599

Page 28 of 41

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.

600

(43) Allison, S. D.; Chang, B.; Randolph, T. W.; Carpenter, J. F. Hydrogen bonding

601

between sugar and protein is responsible for inhibition of dehydration-induced

602

protein unfolding. Arch. Biochem. Biophys. 1999, 365, 289-298.

603

(44) Xie, C.; Zhang, B.; Ma, L. K.; Sun, J. P. Cryoprotective effects of trehalose,

604

alginate, and its oligosaccharide on quality of cooked-shrimp (Litopenaeus

605

vannamei)

606

DOI:10.1111/jfpp.12825.

during

frozen

storage.

J.

Food

Process.

Pres.

2016,

607 608 609 610 611 612 613 614 615 616

28


Page 29 of 41


617

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

29



Page 30 of 41

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.

30


Page 31 of 41

661


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.

31



Page 32 of 41

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

32


Page 33 of 41


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)



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)


Page 34 of 41

Page 35 of 41


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)



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)


Page 36 of 41

Page 37 of 41


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)



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)


Page 38 of 41

Page 39 of 41


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)



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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TOC Graphic 210x71mm (300 x 300 DPI)


Insights into Cryoprotective Roles of Carrageenan Oligosaccharides

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