Effect of Trehalose on the State of Water, Protein Denaturation and Gel-Forming Ability of Weever Surimi

Abstract

The freshness and gel-forming ability of surimi decreased during frozen storage, while trehalose inhibits biological damage at low temperatures and stabilizing protein structures during freezing and freeze-drying. This study investigated the effect of trehalose at different concentrations (2.5–10 g/100 g wet materials) on the state of water, protein denaturation, and gel-forming ability of weever surimi during frozen storage at −18°C for 90 days. The addition of trehalose increased the unfrozen water amount, regardless of level of addition differences, resulting decreased inactivation rate of calcium-adenylpyrophosphatase, and increased gel-breaking force and gel deformation of weever surimi throughout the frozen storage. Therefore, the addition of trehalose increased the gel-forming ability of weever surimi.

Keywords:

• Denaturation
• Enzyme
• Protein
• Water
• Carbohydrate

INTRODUCTION

Surimi is prepared from minced fish flesh that is washed to remove most lipids, blood, enzymes, and sarcoplasmic proteins and then frozen. The gel-forming ability of surimi is attributed to its concentrated myofibrillar proteins.^[1] However, the freshness and gel-forming ability of surimi decrease during frozen storage principally because of proteolysis and formaldehyde formation.^[2] Cryoprotectants, such as phosphate compounds, protein hydrolysate, and sugar, are used to stabilize surimi for frozen storage.^[1,3,4]

Trehalose, or α-D-glucopyranosyl-(1→1)-a-D-glucopyranoside, is a non-reducing glucose disaccharide. This carbohydrate exists in living organisms and is commonly detected at high concentrations in anhydrobiotic organisms. Trehalose has a potential biotechnological importance because of its effectiveness in stabilizing membrane structures in the dry state, inhibiting biological damage at low temperatures, and stabilizing protein structures during freezing and freeze-drying.^[5] However, no study has ever reported the use of trehalose to stabilize weever surimi for frozen storage. The present study investigated the protective effect of trehalose on weever surimi. Particularly, the effects of trehalose on the unfrozen water amount, calcium-adenylpyrophosphatase (Ca-ATPase) activity and gel-forming ability of weever surimi were examined.

MATERIALS AND METHODS

Materials

Live cultured weevers with a mean weight of 486.51 ± 48.12 g were purchased from an aquatic product market in Xinpu, China. Trehalose was purchased from Nanning Zhongnuo Biological Engineering Co. Ltd., China. All other chemicals were of reagent grade.

Surimi and Surimi Gel Preparation

Surimi was prepared following the method described by Julavittayanukul et al. with slight modifications.^[1] The fish were killed by a blow to the head, scaled, gutted, and then decapitated. The flesh was manually removed and uniformly minced. The mince was washed with cold water (5°C) at a mince/water ratio of 1:2 (w/w). The mixture was gently stirred for 3 min, and the washed mince was filtered through a layer of nylon screen. The washing process was repeated twice. Finally, the washed mince was centrifuged at 700 × g for 15 min. The pellet was mixed without or with trehalose (2.5–10 g/100 g wet materials) and then frozen and stored at –18°C to produce “surimi.”

To prepare the gel, the frozen surimi was tempered in running water at 25°C for 30 min. The surimi was cut into pieces approximately 1 cm in thickness and then placed in a mixer (National Model MKK77, Tokyo, Japan). The moisture was adjusted to 80 g/100 g wet materials, and 2.5 g/100 g wet materials salt was added. The mixture was chopped for 5 min at 4°C to obtain a homogeneous sol. The sol was stuffed into a polyvinylidine chloride casing with a diameter of 2.5 cm, and both ends of the casing were tightly sealed. Weever surimi gels were prepared by incubating the sol at 40°C for 30 min and then heating at 90°C for 20 min. All gels were cooled in iced water and stored at 4°C for 24 h prior to analyses.

Measurement of Unfrozen Water

The amount of unfrozen water considered as bound water and partially bound water in the frozen surimi was measured using a differential scanning calorimeter (model SSC-5200, Seiko Electronic Industry Inc., Tokyo, Japan) as previously described by Hossain et al.^[3] This procedure was performed to assess the changes in the state of water after the addition of trehalose and in the stability of the product during long-term freezing. The heat of fusion of distilled water (5 to 25 mg) was initially measured to establish a linear relationship between the amount of pure water and the heat of fusion. The heat of fusion of distilled water (5 to 25 mg) was 79.2 cal/g. A 20 mg surimi sample was placed in a tightly sealed aluminium cell and then accurately weighed. Meanwhile, 20 mg of Al₂O₃ was sealed in a separate aluminium cell to serve as a reference. The cells were subjected to differential scanning calorimetry wherein the heat of fusion was measured by raising the temperature from −40 to 25°C at a rate of 1°C/min. The obtained endothermic peak area reflected the heat of fusion necessary to melt the ice, which corresponded to the free water content in the surimi. For the control, the peak melting temperature was observed at −2.30 to −2.37°C. The peak points gradually shifted with the increase of added-concentration of trehalose in the surimi (−2.73 to −5.60°C). After perforation, the cells containing the surimi were dried at 105°C for 24 h to determine the total water content in the surimi. The amount of unfrozen water was determined by deducting the free water content from the total water content in the sample.

Ca-ATPase Activity Determination

Surimi samples were thawed in a cold room set at 5°C after various periods of storage at −18°C. The samples were homogenized in 30 parts of 0.1 M KCl–20 mM Tris–maleate buffer (pH 7.0), and the homogenate was centrifuged at 750 × g for 10 min. The sediment was washed with KCl–Tris–maleate buffer and then centrifuged again at 750 × g for 10 min. This procedure was repeated twice, and the obtained sediment was resuspended in the buffer.^[6]

Ca-ATPase activity was determined through the following method.^[7] Myofibril protein samples (0.2 to 0.4 mg) were incubated at 25°C with 100 mM KCl, 5 mM CaCl₂, 25 mM Tris-maleate (pH 7.0), and 1 mM adenosine triphosphate. The reaction was terminated by adding 30% (V/V) trichloroacetic acid to a final concentration of 5% (V/V). The inorganic phosphate liberated in the supernatant was measured following the method described by Fiske and Subbarow.^[8] Specific activity was expressed as micromoles of inorganic phosphate released per milligram of protein per minute. Meanwhile, the Ca-ATPase activity of the frozen myofibrils was expressed as the ratio of the specific activity before freezing (relative %).

Instrumental Texture Analysis

Texture analysis of surimi gels was performed following the method described by Julavittayanukul et al.^[1] using a texture analyzer Model TA-XT2 (Stable Micro Systems, Surrey, England). The gels were equilibrated and tested at room temperature. Five cylindrical samples 2.5 cm in length were prepared. The breaking force (gel strength) and deformation (elasticity/deformability) of the surimi gels were measured using a texture analyzer equipped with a spherical plunger (5 mm in diameter; 60 mm/min in deformation rate).

Protein Concentration Determination

Protein concentrations were determined using the biuret method.^[9] Bovine serum albumin was used as the standard.

Statistical Analysis

All data are presented as mean ± S.D. Statistical analysis was performed using Statgraphics Centurion XV version 15.1.02. Multifactor analysis of variance with posterior multiple range test was used to determine significant differences between groups.

RESULTS AND DISCUSSION

Effect of Trehalose on the Unfrozen Water Content of Weever Surimi During Frozen Storage

Figure 1a shows the changes in the amount of unfrozen water in the surimi samples containing 2.5 g/100 g wet materials to 10 g/100 g wet materials trehalose and in the control surimi samples during frozen storage. The amount of unfrozen water was higher in the surimi samples containing trehalose than in the control surimi samples. The amount of unfrozen water in the control surimi samples rapidly decreased to approximately 0.29 g H₂O/100 g dry matter after 45 days of frozen storage and then gradually decreased until the end of the frozen storage period. Conversely, the amount of unfrozen water in the surimi samples containing trehalose was consistently higher than that in the control samples during the entire frozen storage period and only slowly decreased. The optimum concentration of trehalose ranged from 7.5 g/100 g wet materials to 10 g/100 g wet materials.

FIGURE 1 Effect of trehalose on unfrozen water content A: Ca-ATPase activity; B: breaking force; C: and deformation; D: of the weever surimi during frozen storage. Data are shown as mean ± SD (n = 3).

Effect of Trehalose on the Ca-ATPase Activity of Weever Surimi During Frozen Storge

The activity of Ca-ATPase was reduced in the surimi samples added with trehalose after 24 h of frozen storage and then gradually decreased until the end of the frozen storage period (Fig. 1b). By contrast, the activity of Ca-ATPase in the control samples sharply decreased to approximately 14% of the initial value after 45 days of frozen storage and then gradually decreased until the end of the frozen storage period (remaining activity, approximately 3%). This phenomenon showed a biphasic denaturation pattern. Although the extent of denaturation of the surimi samples with trehalose was markedly retarded, the processes also showed biphasic denaturation patterns. The 3D structure of proteins, which are formed and stabilized by hydrogen bonds, hydrophobic interactions, and hydration of polar residues, can be denatured when these forces are disturbed. This denaturation disrupts the physiological activities of proteins. The freeze denaturation of fish protein is induced by high salt concentrations, pH alteration, drying, and interactions with decomposition products.^[3] Many materials, such as phosphate compounds, protein hydrolysate, and sugar, can activate proteins by stabilizing the structure of surrounding water.^[1,3,4] In the present study, trehalose constrained the water in the surimi samples and thus increased the amount of unfrozen water. This phenomenon suppressed protein denaturation in the weever surimi samples.

Effect of Trehalose on the Gel-Forming Ability of Weever Surimi During Frozen Storge

The addition of trehalose at various levels had different effects on the breaking force (Fig. 1c) and deformation (Fig. 1d) of weever surimi gels. Generally, both breaking force and deformation increased as the concentration of trehalose added increased up to 10 g/100 g wet materials. Specifically, the addition of 10 g/100 g wet materials trehalose increased the breaking force and deformation of the gels by 39.3 and 34.9%, respectively, after 90 days of frozen storage. Similarly, phosphate compounds, protein hydrolysate, sugar, starch, and protein were reported to increase gel-forming ability.^{[1,3,4,10,11]} The results of this study showed that trehalose increased the gel-forming ability of weever surimi by inhibiting biological damage at low temperatures and stabilising protein structures during freezing.^[3]

CONCLUSIONS

This study investigated the effects of trehalose on the state of water, protein denaturation, and gel-forming ability of weever myofibrillar surimi during frozen storage. Trehalose increased the amount of unfrozen water and thus suppressed protein denaturation in the weever surimi samples. Furthermore, the addition of trehalose effectively increased the gel-forming ability of weever surimi by stabilizing protein structures. Therefore, trehalose can be developed as a promising food cryoprotectant in the future.

FUNDING

This research was supported by A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Additional information

Funding

This research was supported by A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.