Effect of pH on the gel properties and secondary structure of fish myosin

Food Chemistry 121 (2010) 196–202

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Effect of pH on the gel properties and secondary structure of fish myosin Ru Liu a,b, Si-ming Zhao a,b, You-ming Liu a,b, Hong Yang a,b, Shan-bai Xiong a,b,*, Bi-jun Xie a, Li-hong Qin c a

College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei Province 430070, PR China Aquatic Product Engineering and Technology Research Center of Hubei Province, Wuhan, Hubei Province 430070, PR China c The Public Laboratory of Electron Microscope, Huazhong Agricultural University, Wuhan, Hubei Province 430070, PR China b

a r t i c l e

i n f o

Article history: Received 14 August 2009 Received in revised form 16 October 2009 Accepted 8 December 2009

Keywords: Gel property pH Myosin Secondary structure Microstructure

a b s t r a c t The relationships between gel properties and the secondary structures of silver carp myosin were investigated at pH 5.5–9.0 using dynamic rheological measurement, circular dichroism and scanning electron microscopy. The gel properties of fish myosin were strongly pH and temperature dependent. During heating at 1 °C/min, myosin formed gels in the pH range 5.5–7.5, but not at pH 8.0–9.0. a-Helix was the predominant structure at pH 7.0. The a-helix fraction declined with increasing temperature and the pH away from 7.0, whilst the other secondary structure fractions increased. The a-helix structure of myosin was more susceptive to acid-treatment than alkali-treatment. As pH increased, the gelation rate and gel strength decreased, and the water-holding capacity (WHC) showed an increasing trend followed by a plateau. High b-sheet and b-turn fractions prior to heating could improve G0 at 90 °C, but they depressed the WHC. A compact and uniform gel of fish myosin was obtained at pH 7.0. Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.

1. Introduction Gelation is an important functional property of fish protein affecting the rheological and textural properties of fish products. Gel formation involves partial denaturation of protein followed by irreversible aggregation which results in a three dimensional network (Lanier, Carvajal, & Yongsawatdigul, 2004). Myosin is abundant in muscle protein and plays a key role in gel development in fish and meat products. In our previous study, gelling of porcine myosin involved a change in myosin conformation with protein–protein and protein–water interactions. The rheological properties of salt soluble proteins were strongly pH and temperature dependent (Liu, Zhao, Xiong, Qiu, & Xie, 2008a; Liu, Zhao, Xiong, Xie, & Qin, 2008b; Raghavan & Kristinsson, 2008). It has been suggested that altering the pH of meat products could result in the attainment of the desired gel strength at a given temperature (Westphalen, Briggs, & Lonergan, 2005). For instance, textural development of fermented fish and meat products was associated with lowering pH during fermentation (Riebroy, Benjakul, Visessanguan, & Tanaka, 2007). Subjecting catfish myosin to pH 11.0–12.0 and subsequent readjustment to pH 7.3 increased the elastic modulus of thermally treated myosin (Raghavan & Kristinsson, 2008). In addition, alkaline solubilisation processing could also produce higher gelling quality than conventionally

* Corresponding author. Address: College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei Province 430070, PR China. Tel./ fax: +86 027 87288375. E-mail address: [email protected] (S.-b. Xiong).

washed surimi from Atlantic menhaden (Kristinsson & Liang, 2006; Pérez-Mateos & Lanier, 2006). Some researchers suggested that conformational changes in the myofibrillar proteins during alkaline processing may expose more functional groups for transglutaminase-induced crosslinking and other protein–protein interactions (Kristinsson & Hultin, 2003a, 2003b; Pérez-Mateos, Amato, & Lanier, 2004). Nevertheless, the initial washing at pH 5.5 was beneficial for suitable treatment for kamaboko gels from sardine (Karayannakidis, Zotos, Petridis, & Taylor, 2007). For giant squid surimi, acid washing made for better gel structure than the washing based on isoelectric precipitation (Campo-Deaño, Tovar, Pombo, Solas, & Borderías, 2009). Ikeda (2003) reported discrimination between translucent and opaque gels based on secondary structures in denatured b-lactoglobulin molecules. Choi and Ma (2007) suggested b-sheet was an important conformational component in the aggregated protein. Our previous studies also indicated that unfolding of a-helices and formation of b-sheets favored the gelation of porcine myosin (Liu et al., 2008b). There are considerable variations in gelation properties for variable muscles (Liu, Zhao, Xiong, Xie, & Liu, 2007). Moreover, gelation properties varied with fish species (Fukushima, Okazaki, Fukuda, & Watabe, 2007; Riebroy, Benjakul, Visessanguan, Erikson, & Rustad, 2009). Silver carp (Hypophthalmichthys molitrix) is one of the major species of freshwater fish in China. Specific information about the relationship between the gelation properties and secondary structures of silver carp myosin could help understand the factors affecting the quality of fish product and supply the theoretical proof for improving the quality. However, the relevant information is still scarce.

0308-8146/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.12.030

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In this paper, myosin was prepared from silver carp. Gel properties were evaluated by dynamic rheological measurement, scanning electron microscopy (SEM) and the assessment of waterholding capacity (WHC). Circular dichroism (CD) was employed to monitor the secondary structures of myosin. Subsequently, the relationships between gel properties and secondary structures of fish myosin at different pH values were studied. The aim was to provide more insight into the gelation properties of fish myosin, allowing the manipulation of processing conditions in order to obtain products with the desired structural and textural attributes.

kDa

200

MHC

130 97.4

2. Materials and methods

66.2 2.1. Materials Live silver carp ( H. molitrix) were purchased from Huazhong Agricultural University market and taken to our laboratory in a plastic bag within 30 min. The live were immediately headed, gutted and washed with water. The dorsal muscle was separated manually from skin and bone and kept at 4 °C not longer than 1 h for myosin preparation. All chemicals used were of analytical grade.

actin

43

2.2. Preparation of myosin Myosin was prepared from dorsal muscle of fresh silver carp according to the method of Park and Lanier (1989) with some modification. All solutions used for the myosin preparation were kept cold at 4 °C to minimise proteolysis and protein denaturation. The muscle was minced once through a food processor (Braun K600, Braun GmbH, Germany) and mixed with 10 volumes of solution A (0.10 M KCl, 0.02% sodium azide and 20 mM Tris–HCl buffer, pH 7.5). This was then homogenised using an inline dispersing homogeniser (Model FJ-200, Shanghai specimen and models factory, China) operating at its maximum speed setting (23,000 rpm) for 1 min. The mixture was incubated for 15 min at 4 °C and centrifuged at 3000g for 5 min in a high-speed refrigerated centrifuge (Hitachi, Japan). The sediment was suspended with 5 volumes of solution B (0.45 M KCl, 5 mM b-mercaptoethanol, 0.2 M Mg(COO)2, 1 mM EGTA and 20 mM Tris-maleate buffer, pH 6.8), mixed with adenosine 50 -triphosphate (ATP) to a final concentration of 5 mM and incubated for 60 min at 4 °C. The mixture was then centrifuged at 12,000g for 10 min. The resulting supernatant was diluted slowly with 5 volumes of 1 mM KHCO3 and kept at 4 °C for 15 min. Following this, the mixture was centrifuged at 12,000g for 10 min and the pellet was re-suspended with 2.5 volumes of solution C (0.5 M KCl, 5 mM b-mercaptoethanol and 20 mM Tris–HCl buffer, pH 7.5). The re-suspended pellet was incubated at 4 °C for 10 min and diluted with 2.5 volumes of 1 mM KHCO3. Meanwhile, MgCl2 was added to the final concentration of 10 mM. The mixture was kept overnight at 4 °C prior to centrifugation at 20,000g for 15 min. The myosin pellet was dissolved in 0.5 M NaCl–20 mM Tris–HCl buffer (pH 7.0) and then centrifuged at 5000g for 10 min following which 0.02% sodium azide was added to the supernatant for preservation. Myosin purity was checked using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (Laemmli, 1970). Fig. 1 illustrates the electrophoretic profile of the extracted myosin. The purity of the myosin samples was greater than 94% as determined by densitometry (Gel Logic 200 Imaging System, Kodak Co., New York, USA), although a little actin did remain. The samples were stored at 4 °C and used within 3 days of preparation. The storage period did not change its electrophoretic pattern. The protein content was determined by the Lowry method (Lowry, Rosebrough, & Randall, 1951) using bovine serum albumin (BSA) as a standard. The protein concentrations were adjusted to

1

2

3

Fig. 1. SDS–PAGE of extracted myosin (lane 3) from fish muscle. Lane 1 designates molecular weight markers. As a reference to monitor the myosin isolation and purification procedure, intact fish muscle was denatured and applied to the gel (lane 2). Lanes 2 and 3 were loaded with 20 lg of protein, and lane 1 with 10 lg. MHC: myosin heavy chain.

16 mg/ml, 16 mg/ml and 15 lg/ml for dynamic rheological measurement, thermal gel preparation and circular dichroism measurement, respectively. 2.3. Dynamic rheological measurement Dynamic rheological studies were performed on an AR500 dynamic rheometer (TA Instruments Co. Ltd., Manchester, England). A 40 mm parallel steel plate geometry with a 1 mm gap was used and the myosin solution (16 mg/ml) was surrounded by liquid paraffin to prevent drying. Samples were heated at a rate of 1 °C/min from 15 to 90 °C. The oscillation stress was 0.6 Pa, and the oscillation frequency was 0.1 Hz. Elastic or storage modulus (G0 ), viscous or loss modulus (G00 ) and phase angle (d) were recorded. The gelation rate was indicated by the slope of G0 versus temperature (50– 65 °C). It was estimated by the equation: 

Gelation rateðPa= CÞ ¼

G02  G01 65  50

ð1Þ

where G01 and G02 are the G0 values (Pa) at 50 and 65 °C, respectively. 2.4. Preparation of the thermal gel Myosin solutions (16 mg/ml) at various pH values (5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 9.0) were obtained by the addition of 1 M HCl or 1 M Tris using 25 ll micro syringes. The mixture was quickly homogenated and the pH value was measured using a pH metre (model 818, Orion Research Inc., Beverly, USA). Each sample (5 ml) was placed in a 10 ml capped plastic centrifuge tube. Three replicates of each treatment were used. The tubes were heated for 60 min in a water bath held at 40 °C, followed by 30 min in a water bath at 90 °C. The tubes were immediately cooled by being held in flowing tap water, and then stored overnight at 4 °C for WHC determination and SEM observations.

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2.5. Water-holding capacity Water-holding capacity (WHC, %) was measured based on the protocol described by Kocher and Foegeding (1993). Myosin thermal gels were centrifuged at 1000g for 10 min at 4 °C. The weights (g) of the centrifuge tubes, protein samples and moisture loss were all recorded. The following formula was used to determine WHC (%):

WHCð%Þ ¼

tetroxide for 2 h followed by three further washes using 0.1 M Na phosphate (pH 7.3). The washed samples were dehydrated in 10% dimethyl sulphoxide (DMSO) for 6 h and then freeze-dried. Following this they were coated with platinum–palladium in an IB-5 ion coater (EIKO Ltd., Tokyo, Japan). The specimens were observed in a JSM-6390 PLV scanning electron microscope (JEOL Ltd., Tokyo, Japan) with an accelerating voltage of 10 kV. 2.7. Circular dichroism spectrum

CG  ML  100 CG

ð2Þ

where ML is the amount (g) of moisture lost from the gel during centrifugation and CG is the weight (g) of the heated gel. All treatments were run in triplicate. 2.6. Scanning electron microscopy (SEM) The thermal gels were cut into 1–2 mm cubes, fixed with 2.5% glutaraldehyde containing 0.1 M Na phosphate (pH 7.3) at 4 °C overnight, and then washed three times using 0.1 M Na phosphate (pH 7.3). The post-fixation stage involved exposure to 1% osmium

The CD spectrum was measured using a Jasco J-810 spectropolarimeter (Jasco Co. Ltd., Tokyo, Japan). The myosin sample (15 lg/ ml) was transferred to a quartz cell with a 1 cm light-path length. Molecular ellipticity was measured in the range from 200 to 250 nm, the scan rate was 100 nm/min and the temperature was regulated with a control unit. The effect of pH was assessed at 15 °C. To measure the effects of temperature, fish myosin at pH 7.0 was heated at a rate of 2 °C/min from 5 to 90 °C then held for 2 min before measurement. Spectra were averaged over three scans and corrected for the solvent signal. A mean residue weight of 110 g/mol has been assumed. The percentages of a-helix, b-

1000

100

(a)

(b)

100 10

10 1

pH 6.0

1

pH 6.5 pH 7.0

pH 6.0 pH 6.5

0.1

pH 7.5

0.1

pH 5.5

G'' (Pa)

G' (Pa)

pH 5.5

pH 8.0

pH 7.0

pH 9.0

pH 7.5 pH 8.0

0.01

0.01

0.001

pH 9.0

0.001

15

30

45

60

75

90

15

30

45

Temperature (°C)

60

75

90

Temperature (°C)

100

(c) pH 5.5 80

pH 6.0 pH 6.5 pH 7.0 pH 7.5

60

pH 8.0 °

pH 9.0 40

20

0 15

30

45

60

75

90

Temperature (°C) Fig. 2. Dynamic rheological properties of myosin solution at various pH values. Myosin solutions containing 16 mg/ml, 0.5 M NaCl were heated at a rate of 1 °C/min from 15 to 90 °C at various pH values.

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2.8. Statistical analysis The percentage of each secondary structure type was determined using the protein secondary structure estimation programme (Yang’s method). The data were analysed using Excel and Statistical Analysis System software (SAS Institute Inc., Cary, NC, USA). Duncan’s multiple range test was used to evaluate the differences between treatments. Pearson correlation analysis was performed to explore the relationship between gel properties and secondary structures prior to heating. The differences were considered significant at P < 0.05. 3. Results and discussion 3.1. Dynamic rheological properties of fish myosin solution The dynamic rheological properties of fish myosin at pH 5.5–9.0 are illustrated in Fig. 2. The sample at pH 9.0 exhibited a decrease in G0 and G00 and an increase in d at the beginning of heating (15 °C). This may be explained by the increased molibility of myosin molecules with a rise of temperature. Upon further heating, G0 and G00 increased followed by a decrease, reaching the peak at about 39.2 °C respectively (Fig. 2a and b). d gradually decreased when heated from 22 to 40 °C (Fig. 2c). This indicated protein–protein interactions and network development in the range 20–39 °C. However, the network structure was only marginally stable and was abruptly broken upon further heating, as indicated by decreasing G0 and G00 and increasing d (40 °C). Above 50.5 °C, both G0 and G00 values were less than 0.01 Pa and hardly depended on temperature. d reached a maximum (78.4°) at 48.9 °C and then dropped down. The decreasing rate of d evidently slowed down at above 60 °C. It has been reported that dissociation of myosin occurred with high salt (>0.3 M NaCl) and high pH (>7) conditions (Godfrey & Harrington, 1970a; Godfrey & Harrington, 1970b; Weeds & Pope, 1977). High temperature might strengthen the dissociation and the mobility of the molecule. Therefore, G0 of the sample at pH 9.0 was very low above 50.5 °C. At the end of heating (at 90 °C), d of the sample at pH 9.0 reached 23.3°, significantly higher than d (2.7– 4.4°) of the samples at pH 5.5–7.5. It is obvious from the foregoing discussions that fish myosin (16 mg/ml) at pH 9.0 could not form gel when heated at a rate of 1 °C/min. Similar to the sample at pH 9.0, the G0 and G00 values at pH 8.0 were very low (<0.01 Pa) in the range 50.5–90 °C. The d value was 22.8° at 90 °C. The rheological parameters at pH 8.0 exhibited a similar pattern to those at pH 9.0 with increasing temperature. It indicated that fish myosin (16 mg/ml) at pH 8.0 could not form gel when heated at a rate of 1 °C/min. The samples at pH 5.5, 6.0, 6.5, 7.0 and 7.5 began to form gel at 31.2, 34.4, 23.2, 27.5 and 29.6 °C, respectively, as indicated by increasing G0 and G00 and decreasing d (Fig. 2). The gelation continued until the temperature reached approximately 40 °C where a weakening of gel took place. The initial increase of G0 could be related to interactions that occurred between protein molecules at low temperatures (Lanier et al., 2004). The subsequent decrease in G0 and G00 was possibly due to disentanglement and the increased molibility of the myosin molecules as a result of breaking of protein–protein bonds (Chen, Dickinson, & Edwards, 1999). Upon further heating, G0 increased again at about 45 up to 90 °C. Our previous study showed that melting temperatures of silver carp

myosin were 40 and 50 °C (Liu et al., 2007). Hydropic interactions in fish proteins increased with increasing temperature from 5 to 60 °C. Strong disulphide bondings formed at 70–80 °C (Liu, Zhao, Xie, & Xiong, 2009). Therefore, the second increase in G0 probably arose from stronger interactions between denatured myosin molecules and implied the formation of irreversible gel network. The gelation rate was indicated by the slope of G0 versus temperature (50–65 °C). The gelation rates of the samples at pH 5.5, 6.0, 6.5 and 7.0 were 11.5, 4.86, 3.31, 2.46 and 1.00 Pa/°C. In other words, the gelation rate of fish myosin decreased with increasing pH. This result was consistent with the report on porcine myosin (Liu et al., 2008b) and porcine myofibrillar protein (Westphalen et al., 2005). The isoelectric point (pI) of myosin is at about pH 5.5 (Foegeding & Lanier, 1996). However, when NaCl was added, the chloride ion preferentially binds with the positively charged amino acids to a stronger degree than the sodium ion. The system required more H+ ion reach the approximately zero net charge needed to precipitate proteins (Thawornchinsombut & Park, 2007). Thus, the pI of myosin in 0.5 M NaCl was lower than pH 5.5. As pH decreased close to the pI, the net charge is close to zero. The repulsion between protein molecules gradually decreased. The results were potentially attributable to the closer association of proteins at low pH or the increased strength of hydrophobic interactions and disulphide bonds (Riebroy, Benjakul, Visessanguan, Erikson, & Rustad, 2008; Westphalen et al., 2005). By the end of heating, G’ was seen to decline with increasing pH (Fig. 2a). The d values at pH 5.5–7.5 were lower than 5° (Fig. 2c), characterising the formation of an elastic gel structure. In conclusion, gel-forming ability of fish myosin tended to decrease with increasing pH. Besides, the samples at pH and pH 9.0 could not form gel when heated at a rate of 1 °C/min. Unlike fish myosin, porcine myosin could form gel at pH 5.5–9.0. The results further demonstrated that gelation properties of myosin differed among species. 3.2. Water-holding capacity of fish myosin gel Fig. 3 shows the water-holding capacity (WHC) of fish myosin gel at various pH values. The WHC increased significantly (P < 0.05) from 45.7% to 73.1% as pH increased from 5.5 to 8.0, and the maximum WHC (72–73.5%) was achieved at pH 8.0–9.0. It was reported that the WHC of porcine protein gel significantly increased with increasing pH from 5.5 to 7.0 (Bertram, Kristensen, & Andersen, 2004; Liu et al., 2008b). Similarly, chicken-breast mus-

80 73.1 a

72.3a

8.0

9 .0

69.1a 61.0b 60

53.8c 49.5d

WHC (%)

sheet, b-turn and random coil structures were determined using the protein secondary structure estimation programme (Yang’s method) provided with a Jasco J-810 spectropolarimeter. All treatments were tested in triplicate; mean values from these replicates are represented in the data reported.

45.7e 40

20

0

5.5

6.0

6.5

7.0

7.5

pH Fig. 3. Water-holding capacity of myosin gel at various pH values. Error column indicates the mean values ± standard deviations of three replicates. (a–e) Mean values with different letters on the top of each column differ significantly (P < 0.05).

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cle gels have been found to retain water better at higher pH values (7.0–7.4) than at lower pH values (6.4–6.8) (Kristinsson & Hultin, 2003c). Generally, water was classified into bound water and bulk water. Bound water associates primarily with ionic groups. Bulk water condenses into the clefts and crevices of protein molecules, or in the capillaries of insoluble protein systems (Damodaran, 1996). In denatured proteins, –CO– and –NH became positive and negative polarisation centres at polypeptide chains, respectively. These centres could form multilayer water system. Upon cooling, hydrogen bonds between proteins contributed to the structure entrapping free water (Damodaran, 1996). Above the pI, myosin swelled and bound a large volume of water because of many charged groups and repulsive forces. Shifting pH close to pI, proteins tended to coagulate due to increased protein–protein interactions. Meanwhile, protein hydration gradually became weak due to the changes in the hydration states of charged amino acids. So WHC of myosin gel decreased with decreasing pH. 3.3. Microstructure of fish myosin gel Scanning electron micrographs of myosin gels were observed at pH 5.5, 6.5, 7.0 and 8.0. Myosin solutions at pH 5.5 formed a coarse and disordered gel network with some big pores (Fig. 4a). It was possible that the least charged groups of myosin at pH 5.5 made the rate of aggregation faster than denaturation. The faster aggregation rate led to a coarse gel network and a low WHC (45.7%). A compact and uniform gel was obtained at pH 7.0 (Fig. 4c). Although myosin gel at pH 8.0 was compact too, there were some particles with different sizes on the surface (Fig. 4d). Myosin gel at pH 6.5 was coarse, which consisted of some particles with various sizes (Fig. 4b). Hermansson (1979) suggested that gel properties in proteins depend on the relative speeds of unfolding and aggregation. It could be presumed that the relative speed of unfolding and aggregation of fish myosin at pH 7.0 was beneficial for formation of a fine gel network. Comparatively, fine gel network was formed at pH 6.5 for porcine myosin (Liu et al., 2008b).

3.4. Circular dichroism of fish myosin solution In order to gain insight into the gelation mechanisms of fish myosin at different pH values, myosin conformation were measured using circular dichroism. The secondary structure of fish myosin was significantly affected by pH (Fig. 5a and b). At neutral pH (pH 7.0), the CD spectrum exhibited two minima at around 208 and 222 nm (Fig. 5a), suggesting the predominant presence of a-helix structures (Greenfield, 1999). Shifting pH away from neutrality obviously induced a loss of a-helix structures. The ahelix structure is mainly stabilized by hydrogen bonds between the carbonyl oxygen (–CO) and amino hydrogen (NH–) of a polypeptide chain (Damodaran, 1996; Sano, Ohno, Otsuka-Fuchino, Matsumoto, & Tsuchiya, 1994). Electrostatic interactions between amino acids also contribute to stability of secondary structures (Damodaran, 1996; Satoh, Nakaya, Ochiai, & Watabe, 2006). According to Dumetz, Chockla, Kaler, and Lenhoff (2008), the role of pH is due to its effect on the protonation state of charged amino acids and a-carboxyl and a-amino terminal groups at the surface of proteins. By changing the protonation state of charged residues, the pH affects the detailed nature of protein interactions. As pH decreased from 7.0 to 5.5, a-helix content markedly decreased from 91.8% to 25.5%. As adjusting to pH 9.0, the a-helix content decreased to 65.7% (Fig. 5b). By comparison, a-helix content of porcine myosin declined from 87.4% to 15.8% and 64.7% as adjusting the pH from 7.0 to 5.5 and 9.0, respectively (Liu et al., 2008b). It indicated that the decrease in a-helix content by acid-treatment was more pronounced than by alkali-treatment. The result was consistent with the observation that myofibrillar protein degradation was significantly more pronounced after acidification than alkalinisation (Kim, Park, & Choi, 2003; Underland, Kellerher, & Hultin, 2002). Beyond this, fish myosin was more sensitive to alkaline pH than porcine myosin. The difference may contribute to diverse gelation properties of fish and porcine myosin as mentioned earlier. It can be seen from Fig. 5b that the b-sheet fraction increased after acid- and alkali-treatment. The b-

Fig. 4. Scanning electron micrographs of myosin gel at various pH values. (a) pH 5.5; (b) pH 6.5; (c) pH 7.0; (d) pH 8.0.

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200

(a)

210

220

230

240

250

pH 9.0

70

pH 8.0 pH 7.5 pH 7.0

2

[ ] (×10 deg.cm /dmol)

50

30

3

pH 6.5

pH 6.0 10

pH 5.5 -10

-Helix

-Sheet

-Turn

(c)

Random coil

100

100

80

80

Secondary structure (%)

Secondary structure (%)

(b)

Wavelength (nm)

60

40

-Sheet

-Turn

Random coil

60

40

20

20

0

-Helix

5.5

6.0

6.5

7.0

7.5

8.0

0

9.0

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

Temperature ( )

pH

Fig. 5. CD spectra (a) and secondary structures (b) of myosin. (a) CD spectra of myosin at various pHs (15 °C); (b) secondary structures of myosin at various pHs (15 °C); (c) secondary structures of myosin at various temperatures (pH 7.0).

sheet fraction in the acid range was significantly higher than that in the alkaline range. Heating could induce denaturation and aggregation of proteins followed by gel formation at a sufficiently high protein concentration. It is a very important process for heat-induced gelation of proteins. Therefore, the effect of temperature on the secondary structures of myosin at pH 7.0 was investigated by CD. As shown in Fig. 5c, the a-helix fraction gradually decreased from 94% to 34.5% with increasing temperature from 5 to 90 °C (Fig. 5c). The b-sheet and b-turn fractions were considerably higher in the range 35–90 °C than those in the range 5–30 °C. In general, the random coil fraction tended to increase with increasing temperature.

correlation between b-sheet and G0 at 15 °C was not significant (0.74a), either. The correlations only became significant at 90 °C (P < 0.01). It was suggested that a-helix and b-sheet had no considerable effect on G0 at 15 °C. However, the presence of fewer a-helices and more b-sheets and b-turns prior to heating would increase G0 at 90 °C. It has been reported that b-sheets are important conformational component of aggregated-state (Choi & Ma, 2007; Liu et al., 2008b). It was possible that the reduced hydration of bsheets compared to a-heliced promoted protein–protein interacTable 1 Correlation coefficient between secondary structure and gel properties at different pH values (n = 21).

3.5. Relationship between secondary structures and gel properties Table 1 shows the relationship between gel properties and the secondary structures of fish myosin prior to heating. a-Helices exhibited a significant and negative effect on G0 at 90 °C (Table 1), whereas b-sheets and b-turns exhibited significant and positive effects on G0 at 90 °C. However, the negative correlation between a-helix and G0 at 15 °C was not significant (0.67a). The positive

a b c

Gel properties

a-Helix (%)

b-Sheet (%)

b-Turn (%)

Random coil (%)

G0 at 15 °C d at 15 °C G0 at 90 °C d at 90 °C

0.67a 0.50a 0.87b 0.15a

0.74a 0.58a 0.93c 0.26a

0.76b 0.46a 0.87b 0.18a

0.44a 0.34a 0.68a 0.01a

NS, not significant. P < 0.05. P < 0.01.

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tions. The correlation coefficients between the WHC and secondary structures also confirmed the assumption. As shown in Table 1, the WHC of myosin gel was positively correlated with the a-helix fraction prior to heating, but negatively correlated with the other secondary structure fractions. Namely, plenty of a-helices prior to heating were beneficial for the WHC of fish myosin gel. In addition, b-turn fraction exhibited a significant and positive effect on G0 at 15 °C (P < 0.05). The results were consistent with the observation on porcine myosin. It was revealed that secondary structures of myosin played a central role in gel properties. 4. Conclusion During heating at 1 °C/min, fish myosin formed gel only at pH 5.5–7.5, but did not at pH 8.0 and pH 9.0. As pH increased, the gelation rate and gel strength decreased and the WHC increased. CD analysis showed that fish myosin possessed a-helix, b-sheet, bturn and random coil structures. a-Helix was the predominant structure of fish myosin at pH 7.0. The a-helix fraction declined with movement of the pH away from 7.0, whilst the other secondary structure fractions increased. Additionally, b-sheet fraction was higher at acid pH than that at alkaline pH. High b-sheet and b-turn fractions prior to heating decreased the WHC of the thermal gel, although they could improve gel strength. Heating caused the partial transformation of a-helices into b-sheets and b-turns. The transformation played an important role in the gelation process. A compact and uniform gel was obtained at pH 7.0 whilst there was a decrease in three dimensional ordering when pH was moved away from 7.0. Acknowledgements The authors are grateful to Qun-ying Xu (Wuhan Polytechnic University) for her technical advice and support. The research is supported by ‘‘the earmarked fund for Modern Agro-industry Technology Research System (No. NYCYTX-49)”, ‘‘the Scientific Research Foundation for the Talents by Huazhong Agricultural University (No. 52204-08075)” and ‘‘The Natural Science Foundation of Hubei Province of China (No. 2007ABA350)”. References Bertram, H. C., Kristensen, M., & Andersen, H. J. (2004). Functionality of myofibrillar proteins as affected by pH, ionic strength and heat treatment – a low-field NMR study. Meat Science, 68, 249–256. Campo-Deaño, L., Tovar, C. A., Pombo, M. J., Solas, M. T., & Borderías, A. J. (2009). Rheological study of giant squid surimi (Dosidicus gigas) made by two methods with different cryoprotectants added. Journal of Food Engineering, 94, 26–33. Chen, J., Dickinson, E., & Edwards, M. (1999). Rheology of acid-induced sodium caseinate stabilized emulsion gels. Journal of Texture Studies, 30, 377–396. Choi, S. M., & Ma, C. Y. (2007). Structural characterization of globulin from common buckwheat (Fagopyrum esculentum Moench) using circular dichroism and Raman spectroscopy. Food Chemistry, 102, 150–160. Damodaran, S. (1996). Amino acids, peptides, and proteins. In O. R. Fennema (Ed.), Food chemistry (3rd ed., pp. 321–429). New York: Marcel Dekker. Dumetz, A. C., Chockla, A. M., Kaler, E. W., & Lenhoff, A. M. (2008). Effects of pH on protein–protein interactions and implications for protein phase behavior. Biochimica et Biophysica Acta, 1784, 600–610. Foegeding, E. A., & Lanier, T. C. (1996). Characteristics of edible muscle tissues. In O. R. Fennema (Ed.), Food chemistry (3rd ed., pp. 879–942). New York: Marcel Dekker. Fukushima, H., Okazaki, E., Fukuda, Y., & Watabe, S. (2007). Rheological properties of selected fish paste at selected temperature pertaining to shaping of surimibased products. Journal of Food Engineering, 81, 492–499. Godfrey, J. E., & Harrington, W. F. (1970a). Self-association in the myosin system at high ionic strength. I. Sensitivity of the interaction to pH and ionic environment. Biochemistry, 9, 886–893. Godfrey, J. E., & Harrington, W. F. (1970b). Self-association in the myosin system at high ionic strength. II. Evidence for the presence of monomer M dimer equilibrium. Biochemistry, 9, 894–908. Greenfield, N. J. (1999). Applications of circular dichroism in protein and peptide analysis. Trends in Analytical Chemistry, 18(4), 236–244.

Hermansson, A. M. (1979). Aggregation and denaturation involved in gel formation. In A. Pour-EI (Ed.). Functionally and protein structure, ACS symposium series (Vol. 92, pp. 81–103). Washington, DC: American Chemical Society. Ikeda, S. (2003). Heat-induced gelation of whey proteins observed by rheology, atomic force microscopy, and Raman scattering spectroscopy. Food Hydrocolloids, 17, 399–406. Karayannakidis, P. D., Zotos, A., Petridis, D., & Taylor, K. D. A. (2007). The effect of initial wash at acidic and alkaline pHs on the properties of protein concentrate (kamaboko) products from sardine (Sardina pilchardus) samples. Journal of Food Engineering, 83, 510–520. Kim, Y. S., Park, J. W., & Choi, Y. J. (2003). New approaches for the effective recovery of fish proteins and their physicochemical characteristics. Fisheries Science, 69, 1231–1239. Kocher, P. N., & Foegeding, E. A. (1993). Microcentrifuge-based method for measuring water-holding of protein gels. Journal of Food Science, 58, 1040–1046. Kristinsson, H. G., & Hultin, H. O. (2003a). Effect of low and high pH treatment on the functional properties of cod muscle proteins. Journal of Agricultural and Food Chemistry, 51(17), 5103–5110. Kristinsson, H. G., & Hultin, H. O. (2003b). Changes in conformation and subunit assembly of cod myosin at low and high pH and after subsequent refolding. Journal of Agricultural and Food Chemistry, 51(24), 7187–7196. Kristinsson, H. G., & Hultin, H. O. (2003c). Role of pH and ionic strength on water relationships in washed minced chicken-breast muscle gels. Journal of Food Science, 68(3), 917–922. Kristinsson, H. G., & Liang, Y. (2006). Effect of pH-shift processing and surimi processing on Atlantic croaker (Micropogonias undulates) muscle proteins. Journal of Food Science, 71(5), C304–C312. Laemmli, U. K. (1970). Cleavage of structure protein during the assembly of head bacteriophage T4. Nature, 277, 680–685. Lanier, T. C., Carvajal, P., & Yongsawatdigul, J. (2004). Surimi gelation chemistry. In J. W. Park (Ed.), Surimi and surimi seafood (2nd ed., pp. 451–470). New York: Marcel Dekker. Liu, R., Zhao, S. M., Xiong, S. B., Xie, B. J., & Liu, H. M. (2007). Studies on fish and pork paste gelation by dynamic rheology and circular dichroism. Journal of Food Science, 72(7), E399–E403. Liu, R., Zhao, S. M., Xiong, S. B., Qiu, C. G., & Xie, B. J. (2008a). Rheological properties of fish actomyosin and pork actomyosin solutions. Journal of Food Engineering, 85, 173–179. Liu, R., Zhao, S. M., Xiong, S. B., Xie, B. J., & Qin, L. H. (2008b). Role of secondary structures in the gelation of porcine myosin at different pH values. Meat Science, 80, 632–639. Liu, R., Zhao, S. M., Xie, B. J., & Xiong, S. B. (2009). Contribution of protein conformation and intermolecular bonds to fish and pork gelation properties. Food Hydrocolloids, FOODHYD-D-08-00042R3 (manuscript draft). Lowry, O. H., Rosebrough, N. J., & Randall, R. J. (1951). Protein measurement with Folin phenol reagent. Journal of Biological Chemistry, 193, 256–275. Park, J. W., & Lanier, T. C. (1989). Scanning calorimetric behavior of tilapia myosin and actin due to processing of muscle and protein purification. Journal of Food Science, 54(1), 49–51. Pérez-Mateos, M., & Lanier, T. C. (2006). Comparison of Atlantic menhaden gels from surimi processed by acid or alkaline solubilization. Food Chemistry, 101, 1223–1229. Pérez-Mateos, M., Amato, P. M., & Lanier, T. C. (2004). Gelling properties of Atlantic croaker surimi processed by acid or alkaline solubilization. Journal of Food Science, 69(4), 328–333. Raghavan, S., & Kristinsson, H. G. (2008). Conformational and rheological changes in catfish myosin during alkali-induced unfolding and refolding. Food Chemistry, 107, 385–398. Riebroy, S., Benjakul, S., Visessanguan, W., & Tanaka, M. (2007). Changes during fermentation of Som-fug produced from different marine fish. Journal of Food Processing and Preservation, 31, 751–770. Riebroy, S., Benjakul, S., Visessanguan, W., Erikson, U., & Rustad, T. (2008). Comparative study on acid-induced gelation of myosin from Atlantic cod (Gardus morhua) and burbot (Lota lota). Food Chemistry, 109, 42–53. Riebroy, S., Benjakul, S., Visessanguan, W., Erikson, U., & Rustad, T. (2009). Acidinduced gelation of natural actomyosin from Atlantic cod (Gadus morhua) and burbot (Lota lota). Food Hydrocolloids, 23, 26–39. Sano, T., Ohno, T., Otsuka-Fuchino, H., Matsumoto, J. J., & Tsuchiya, T. (1994). Carp natural actomyosin: Thermal denaturation mechanism. Journal of Food Science, 59, 1002–1008. Satoh, Y., Nakaya, M., Ochiai, Y., & Watabe, S. (2006). Characterization of fast skeletal myosin from white croaker in comparison with that from walleye pollack. Fisheries Science, 72, 646–655. Thawornchinsombut, S., & Park, J. W. (2007). Effect of NaCl on gelation characteristics of acid- and alkali-treated pacific whiting fish protein isolates. Journal of Food Biochemistry, 31, 427–455. Underland, I., Kellerher, S. D., & Hultin, H. O. (2002). Recovery of functional proteins from herring (Clupea harengus) light muscle by an acid or alkaline solubilization process. Journal of Agricultural and Food Chemistry, 50, 7371–7379. Weeds, A. G., & Pope, B. (1977). Studies on the chymotryptic digestion of myosin. Effect of divalent cations on proteolytic susceptibility. Journal of Molecular Biology, 111, 129–157. Westphalen, A. D., Briggs, J. L., & Lonergan, S. M. (2005). Influence of pH on rheological properties of porcine myofibrillar protein during heat induced gelation. Meat Science, 70, 293–299.