Effect of several cryoprotectants on the physicochemical and rheological properties of suwari gels from frozen squid surimi made by two methods

Journal of Food Engineering 97 (2010) 457–464

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Effect of several cryoprotectants on the physicochemical and rheological properties of suwari gels from frozen squid surimi made by two methods Laura Campo-Deaño a, Clara A. Tovar a,*, Javier Borderías b a b

Department of Applied Physics, Faculty of Sciences, University of Vigo, As Lagoas, 32004 Ourense, Spain Instituto del Frío (CSIC), José Antonio Nováis 10, 28040 Madrid, Spain

a r t i c l e

i n f o

Article history: Received 22 June 2009 Received in revised form 2 November 2009 Accepted 3 November 2009 Available online 10 November 2009 Keywords: Squid surimi Suwari gels Acid washing Isoelectric precipitation Frozen storage Cryoprotectants

a b s t r a c t Functionality of squid surimi (Dosidicus gigas) made by two methods (isoelectric precipitation A, and acid washing, B) and stored for 6 months at 15 °C, was analysed as a function of several cryoprotectants. The cryoprotectant effect was studied in terms of the ability to form suwari gels (SA and SB) from the two kinds of surimi. Chemical analyses to detect protein aggregation, dynamic oscillatory tests at constant temperature (10 °C) and temperature sweep tests from 10 to 90 °C were performed. Frozen storage of surimi samples produced two different effects on the thermo-rheological properties of SA and SB suwari gels: in SB samples there were no significant differences over frozen storage. Of the SA samples, the ones with trehalose retained their initial viscoelastic properties best at 10 °C; moreover, trehalose significantly altered the pattern of the thermal gelation profile in SA samples, making it similar to SB samples. All these thermo-rheological results are consistent with protein solubility and electrophoresis (SDS–PAGE) data evidencing protein aggregation. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Surimi, a refined fish protein concentrate manufactured by successive washings of fish mince, is an intermediate product involved in the production of seafood analogues such as crab sticks (Okada, 1992) and other new products. Surimi is usually produced from relatively lean fish species such as Alaska Pollock (Theragra chalcogramma). However, overfishing of lean species has prompted the industry to process more abundant seafood species such us giant squid (Dosidicus gigas) to produce surimi (Sánchez-Alonso et al., 2007; Cortés-Ruiz et al., 2008). When making surimi the washing procedure is particularly important for quality, not only to remove fat and unwanted matter, but more importantly to increase the concentration of myofibrillar protein, thereby improving gel forming ability (Rawdkuen et al., 2009). When cephalopod mantle is washed after mincing, as in the traditional surimi making method, much of the myofibrillar protein is washed away because it solubilizes at very low ionic strength (Tsuchiya et al., 1978). Two new methods were designed for making surimi from giant squid (D. gigas) muscle (CSIC, 2005, 2006). The first involves protein precipitation at isoelectric point (type A), and the second washing with an acid solution (type B). Campo-Deaño et al. (2009) reported that the myofibrillar proteins retain their functionality better with

* Corresponding author. Tel.: +34 988 387241; fax: +34 988 387001. E-mail address: [email protected] (C.A. Tovar). 0260-8774/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2009.11.001

method B, so that its viscoelastic magnitudes indicate better gel properties, than when method A is used. They also reported that the presence of trehalose favours less initial protein aggregation and therefore a more thermorheologically stable structure. The rheological and textural characteristics of surimi gels depend on the temperature that is applied to the minced muscle after it has been mixed with NaCl to solubilize the proteins. When it is subjected to temperatures <40 °C, the resulting gel, called ‘‘suwari”, is elastic and not very cohesive (Niwa, 1992). This kind of gel preserves the flavour and colour of raw fish muscle. During setting in this temperature range, myosin heavy chain (MHC) becomes polymerized through the formation of non-disulphide covalent crosslinks, catalysed by an endogenous transglutaminase (TGase) (Park, 2004). TGase has been known to catalyse acyl transfer reactions between the c-carboxamide groups of glutamine residues in suitable protein A acceptors, usually primary amines. Formation of a e-(c-glutamyl) lysine isopeptide has been reported to serve for gel strengthening (Benjakul et al., 2004). The recent increase in demand for ‘‘fresh products” has encouraged studies on restructured or other new products based on ‘‘suwari gels” that present a similar appearance and eating characteristics to cuts from raw muscle, instead of the cooked appearance and flavour of most products of this kind currently on the market. These cold-set products may be cooked in the same way as fish fillets or processed in a variety of ways, for instance as marinated products, ‘‘sushi”, carpaccio, or as a ready-to-cook fish fillet analogue.

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The rheological properties of surimi and its gels are affected by freezing temperature and time in storage (Howell, 2000). It is therefore essential to use cryoprotectants like sugars and polyols to preserve the biological activity of proteins in surimi during frozen storage (Matsumoto and Noguchi, 1992). Such additives protect against dehydration stress through a stress-specific stabilization scheme (Prestrelski et al., 1993). A mix of sorbitol and sucrose is the most widely used cryoprotectant in the surimi industry (Park, 2004); however, a new alternative could be trehalose, which is an odourless white powder with 45% less relative sweetness than sucrose. It is a nonreducing homodisaccharide in which two glucose units are bonded together in a a-1,1-glycosidic linkage (a-D-glucopyranosil a-D-glucopyranoside). It is abundant in yeast and mushrooms. The structure of trehalose is thought to contribute to the formation of a stable hydrating water sphere and hence to restrain the motion of water most strongly among the oligosaccharides (Osako et al., 2005). The objective of the present work was to study the physicochemical and rheological properties of suwari gels made with giant squid surimi processed in two different ways (following CampoDeaño et al., 2009), with different cryoprotectants added. 2. Materials and methods 2.1. Preparation of samples 2.1.1. Preparation of surimi A Raw material was frozen mantles from giant squid ( D. gigas) transported from west coast of Mexico. The major constituent of the raw squid muscle used were: moisture 84.3 ± 0.3%, crude protein 14.5 ± 0.2%, crude fat 0.6 ± 0.1% and ash 0.60 ± 0.05%. Some parts of the mantle, free of inner and outer skin, were cut into pieces and homogenized with a Stephan (Stephan und Shone GmbH and Co., Hameln, Germany) model micro-cut, in a solution of H3PO4 (1:4) so as to produce a final pH of around 3. This solution was passed through a 100 lm filter to remove fasciae. Sodium bicarbonate was added to this filtered solution to bring the pH up to 5. The solution was then centrifuged in a decanter at 5000g (Alfa Laval Corporation AB, Lund, Sweden). Cryoprotectants and 0.5% of sodium tripolyphosphate were added to the protein precipitate and the sample was processed in a vacuum cutter–mixer mod. CUTMIX 120l STL (K + G Wetter GmbH, Biedenkopf, Germany). The temperature should not exceed 10 °C throughout the process. 2.1.2. Preparation of surimi B Pieces of mantle were homogenized with water (1:4) and the suspension was passed through a 100 lm filter to remove fasciae. A solution of 1 N H3PO4 was added to bring the pH up to 5. The suspension was stirred for 15 min then centrifuged in a decanter (Alfa Laval Corporation AB, Lund, Sweden), and the precipitate was removed. Cryoprotectants and 0.5% sodium tripolyphosphate were added to the precipitate and the whole was mixed in a vacuum mixer. The following cryoprotectants were added to both surimis: Lots A1 and B1: 4% sorbitol + 4% sucrose. Lots A2 and B2: 4% sorbitol + 4% trehalose. Lots A3 and B3: 8% trehalose. Mix of 4% sorbitol + 4% sucrose is use very often in fish surimi. The other two formulae are in order to test the trehalose. 0.5% Sodium tripolyphosphate was added to all lots. The final moisture of all surimi lots was supposed to be around 80%. The final pH of this surimi was around 5.2. It was then placed in trays

and frozen in a plate freezer until the core reached 20 °C. Afterwards it was packed in plastic bags and stored at 15 °C. This temperature is close to the standard of frozen storage for surimi (20 °C) but not so low; this was in order to accelerate the possible denaturation process, and to be able to evaluate the influence of storage time on the functionality of surimi according to the type of cryoprotectant. All chemical reagents are from Panreac Quimica SA, Barcelona, Spain. Food grade ingredients, except trehalose were got in Manuel Riesgo S.A., Madrid, Spain. Trehalose was got in Cerestar Food and Pharma Specialities, Mechelen, Belgium. 2.1.3. Preparation of suwari gel samples The preparation sequence from the different lots described above was: chopped semi-thawed surimi was homogenized in a cutter for 5 min with 0.65% Ca(OH)2 until the pH reached 6.5–7, then 3% NaCl of total batter, was added and the mixture was homogenized for another 5 min. The raw paste was placed in a cylindrical steel cell 2 cm in diameter and 6 cm long, then the cells were placed in a water-bath (Memmert WB 10, from Memmert GmbH + Co. KG, Schwabach, Germany) at 40 °C for 30 min. Afterwards the cylinders with the sample inside were placed in a water–ice slurry and finally kept refrigerated at 7 °C for one day. Surimi Lots A1, A2, A3, B1, B2 and B3 result in samples SA1, SA2, SA3, SB1, SB2 and SB3 after suwari gelation. 2.2. Analyses 2.2.1. Protein solubility This was determined in triplicate essentially according to the Ironside and Love procedure (1958), by analysing the amount of soluble protein in a chilled aqueous solution of 5% NaCl (Panreac Quimica SA, Barcelona, Spain). The protein was analysed in a LECO FP2000 (Leco Corporation, St. Joseph, M1) analyzer, and the results were expressed as a percentage of soluble protein over the total protein. 2.2.2. Sodium dodecyl sulphate–polyacridamide gel electrophoresis (SDS–PAGE) Protein samples were treated with a denatured solution composed of 5% 2-b-mercaptoethanol, 2.5% sodium dodecyl sulphate (SDS), 10 mM Tris–HCl, 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.002% bromophenol blue (all reagents from Panreac Quimica SA, Barcelona, Spain), following Hames (1985), and the final average concentration was adjusted to 2 mg/ml. Sample was heated for 5 min at 100 °C. Electrophoresis assays were performed on a PhastSystem apparatus (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) using 12.5% polyacrylamide gels supplied by Pharmacia. Electrophoretic conditions were 10 mA, 250 V and 3.0 W, temperature 15 °C. Protein bands were stained with Coomassie brilliant blue, commercialized by Pharmacia LKB Biotechnology as ‘‘PhastGel Blue R” tablets. An aqueous solution of 30% methanol and 10% acetic acid was used for de-staining, and a solution of 5% glycerol and 10% acetic acid was used as a preservative. The reference standard used for molecular weights was a commercial HMW (High Molecular Weight) calibration kit from Pharmacia, composed of: Myosin (220 kDa), a2-Macroglobulin (170 kDa), bGalactosidase (116 kDa), Transferrin (76 kDa) and Glutamic dehydrogenase (53 kDa). The density of the Myosin Heavy Chain band (MHC) was analysed by the programme ID-Manager v.2.0 (TDI S.A., Madrid, Spain) on a computer with a scanner (hp Scanjet 4470c). To measure the myosin heavy chain at 220 kDa degradation during frozen storage, the percentage of density decreasing of MHC band of all samples after 24 weeks of storage was calculated with reference to the density at the beginning of storage.

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2.2.3. Dynamic rheological measurements All rheological measurements were performed using a Bohlin CVO controlled-stress rheometer (Bohlin Instruments, Inc. Cranbury, NJ) and a RS600 Haake rheometer (Thermo Electron Corporation Karlsruhe, Germany), the measurement system used was a 20 mm parallel-plate. Suwari gels were cut into cylindrical samples 20 mm in diameter and approximately 1 mm thick. Samples were placed between the parallel plates of the rheometer; the upper plate was lowered and stopped at a final gap of 1.0 mm from the lower plate. Any excess sample protruding beyond the upper plate was carefully removed. Samples were allowed to rest for 15 min before analysis to ensure both thermal and mechanical equilibrium at the time of measurement. A thin film of Vaseline oil (Codex purissimum) was gently applied to the edge of each exposed sample to prevent moisture losses. No evidence of specimen slippage at the bottom plate was detected in any case. The temperature of the lower plate was kept at (10.0 ± 0.1)°C using a Bohlin Rheology fluid circulating bath (also from Bohlin Instruments, Inc.) and controlled via a computer. To determine the linear viscoelastic (LVE) region, a stress sweep (angular frequency 6.28 rad/s) was performed in the range of 10– 4000 Pa at 10 °C. Changes in storage (G0 ) and loss modulus (G00 ) were recorded. The procedure for determining the limit values of stress (rmax) and strain (cmax), was explained in a previous work using creep and recovery tests to corroborate the amplitude of LVE range (Campo-Deaño and Tovar, 2009). Frequency sweeps were carried out on the samples in the 0.1–10 Hz range at 0.5% strain and changes in G0 and G0 0 moduli were recorded at 10 °C. For temperature sweeps, oscillation temperature ramp mode (1 °C/min) was used to heat surimi samples from 10 to 90 °C. Frequency (0.1 Hz) and strain (c = 0.5%) were fixed. 2.3. Statistical analyses

over the course of frozen storage (Table 1). This suggests that trehalose produces a good cryoprotective effect during frozen storage when protein has been damaged by low-pH, as happened in type A surimi made by isoelectric precipitation after acid solubilization. On the other hand, it had no effect when a milder procedure was used, as in the case of type B surimi, where there were no cryoprotectant-related differences. 3.2. Electrophoresis (SDS–PAGE) Table 2 shows the percentage of the density decrease in the MHC band at 22 kDa in all samples after 24 weeks of frozen storage, with reference to the density at the beginning of storage. The decrease in this band is much smaller in both types of surimis with 8% trehalose as cryoprotectant, which is consistent with the fact that protein solubility was higher in sample A3 than in A1 or A2 in the eight first weeks of frozen storage. This means that much less non-disulphide bonds form during frozen storage when trehalose is added to either type of surimi (bearing in mind that a buffer with mercaptoethanol is used). Therefore, because hydrogen and ionic bonds are cleaved by SDS, the bonds that trehalose interferes with are mainly hydrophobic bonds. Zhou et al. (2006) reported that actomyosin isolated from tilapia surimi with trehalose as cryoprotectant contained less covalent bonds than if sucrose plus sorbitol was added. The evident reduction of the MHC band in samples B1 and B2 is in apparent contradiction with the fact that the protein solubility was not significantly different (although it did tend to be lower) at the beginning and at the end of frozen storage in the same samples (Table 1). The fact that the hydrophobic bonds are not cleaved with the electrophoretic buffer suggests that these bonds are formed largely in the aggregates. 3.3. Rheology of suwari gels after frozen storage of surimi

At least five independent batches were tested for each experiment and data are presented as averages. Statistical analysis was carried out using Microsoft Excel software. Trends were considered significant when means of compared sets differed at P < 0.05 (Student’s t-test). 3. Results and discussion 3.1. Protein solubility Table 1 shows that A surimi samples, which were exposed to pH 3 in the solubilization step during preparation of the surimi, were less soluble than B samples (P < 0.05), which were only exposed to pH 5 during surimi preparation. The decrease in protein solubility is an indicator of protein denaturation resulting from the formation of hydrophobic, hydrogen and disulphide bonds (Benjakul and Bauer, 2000; Park and Lin, 2004). Shikha et al. (2006) reported that irreversible changes take place in Walleye Pollack surimi during low-pH processing. Statistical analysis distinguished between surimi A and surimi B, and within surimi A, sample with trehalose,

3.3.1. Oscillatory tests at small strain and constant temperature The stress sweep tests show that suwari gels SA presented higher values of G* (high rigidity) than SB (P < 0.05) throughout the 6 months of frozen storage (Fig. 1). Moreover, the evolution of G* in SA samples underwent significant changes, particularly in SA2 during the first eight weeks and to a lesser extent in SA1 between weeks 8 and 16. This high variability in samples SA1 and SA2 after gelation of frozen stored surimi lots A1 and A2, could be related to the major initial protein aggregation observed in surimi A (see Table 1), where the lowest protein solubility values were recorded in surimi samples A1 and A2 from start to finish. This effect produces a random structure with less water holding capacity which favours the formation of ice crystals with consequent structural damage (Shenouda, 1980), particularly in the first 2 months. This effect has also been observed in surimi from Tilapia (Sarotherodon nilotica) (Zhou et al., 2006). On the other hand, of the SA suwari gels, SA3 was the sample that presented the lowest G* values and differed least from G* in SB samples (Fig. 1a and b). Also G* values in SA3 remained almost constant throughout frozen storage (Fig. 1a). This result is

Table 1 Soluble protein/total protein in different A and B surimi during frozen storage at 15 °C. Type A surimi with 4% sorbitol + 4% sucrose (A1), 4% sorbitol and 4% trehalose (A2) and 8% trehalose (A3), respectively as cryoprotectants. Type B surimi with the same cryoprotectants as in type A: B1, B2 and B3.

a–j

t (Weeks)

A1

A2

A3

B1

B2

B3

0 8 16 24

22.3abc ± 3.4 23.0b ± 1.3 19.0a ± 1.3 17.5a ± 2.2

30.0de ± 3.0 26.6cd ± 1.8 21.0ab ± 2.5 17.2a ± 1.8

40.3fj ± 5.4 34.6ef ± 2.6 27.0bcd ± 3.0 24.7bcd ± 3.1

52.6fgh ± 8.4 59.1h ± 2.3 50.0fg ± 4.8 45.4fi ± 8.9

49.0gi ± 2.2 55.0fgh ± 4.5 45.5fg ± 9.6 42.4f ± 0.9

46.1gij ± 6.9 50.3gi ± 2.8 43.3fg ± 7.6 38.3efg ± 6.3

Different letters indicate significant differences at P < 0.05.

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consistent with the electrophoresis data, which show that trehalose retained the initial state of the MHC band better in both kinds of surimi (Table 2). Trehalose has been classified as a kosmotrope or water-structure maker, that is, the interaction between trehalose/ water is much stronger than the water/water interaction. Trehalose orders water molecules around itself, thus preventing the formation of ice (Jain and Roy, 2009), which would explain why surimi A presented lower rigidity values and was better preserved during frozen storage. This result is consistent with the fact that soluble protein content was higher in surimi A3 than in A1 or A2 after 24 weeks of frozen storage (Table 1). However G* in SB samples was more stable and uniform in the case of three cryoprotectants, with no significant differences (P < 0.05) among them (Fig. 1b). This result is consistent with their high soluble protein content (Table 1).

The strain amplitude of the LVE range changes according to the cryoprotectant for each kind of suwari gel. In SA, sorbitol + sucrose added to surimi type A retained the initial cmax value (P < 0.05) throughout the 6 months (Fig. 2a), while in SB it decreased during the eight first weeks then increased strongly in the following eight (Fig. 2b). Trehalose was responsible for a similar tendency in cmax in both SA and SB samples during surimi frozen storage (Fig. 2a and b). Frequency sweep tests were used to fit G0 (storage modulus) and G00 (loss modulus) to the power law (Ec.1 and 2). G0 is a measure of the deformation energy stored by the sample during the shear process and represents the elastic behavior of a material. G00 is a measure of the deformation energy used up by the sample during the shear process, and so lost by the sample; it represents the viscous behavior of the material (Mezger, 2006).

G0 ¼ G00 xn Table 2 Percentage of MHC density band reduction in electrophoretic analyses of surimi at the end of the experiment, with respect to the initial value. Difference (%) of MHC density band A1 A2 A3

40.8 ± 3.0 40.7 ± 3.1 13.2 ± 1.0

B1 B2 B3

40.0 ± 3.0 68.2 ± 3.1 4.0 ± 1.0

0

G00 ¼ G000 xn

ð1Þ 00

ð2Þ

The difference between the strong (true) gel and the weak gel can be established using mechanical spectroscopy (Clark and Ross-Murphy, 1987). In the former, the molecular rearrangements within the network were reduced over the time scales analysed; G0 is higher than G00 (data not shown here) and almost independent of x (low

Fig. 1. Effect of cryoprotectants on complex modulus G* of suwari gels SA and SB after frozen storage of A and B surimi, (a) SA gels, and (b) SB gels.

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and similar n0 and n00 parameters) (Rao, 2007). Suwari gels SA1 and SA2 were weaker than SA3 and the other SB since they were more dependent on x (Table 3). These higher n0 and n00 values show that SA1 and SA2 possessed protein networks which were transient in time and involved specific interactions between denser and less flexible particles, such as aggregated proteins (Rao, 2007). These results could be related to the fact that G* values were higher in SA1 and SA2 (Fig. 1a) and soluble protein content lower in its correspondent surimi A and B (Table 1).

In general the three cryoprotectants produced similar effects on surimi B, since n0 and n00 values in SB samples are practically undistinguishable, not only at the start but also after 24 weeks of frozen storage (Table 3). However, in surimi A trehalose gave the best gel characteristics, (low n0 values) at the start and after 16 weeks, given that a low n0 value was maintained in sample SA3 (Table 3). The striking structural differences between suwari gels SA and SB are quantifiable in terms of quality factor Q, a term frequently used in mechanical oscillatory systems. It is a dimensionless quantity and represents the degree of damping of an oscillator. Q is defined as 2p times the ratio of energy stored to the average energy loss per period (Arya, 1990). Taking into account the oscillatory character of frequency sweeps, and the structural information that mechanical spectra provide, (Ferry, 1980), the following Eq. (3) is proposed to calculate the value of Q. In this way we can identify the energy stored with G0 (Eq. (1)) and the energy loss with G00 (Eq. (2)) in a sinusoidal strain.

Q ¼ 2p

G00 ðn0 n00 Þ x G000

ð3Þ

Q factor unifies parameters which provide structural information of different kinds: G00 and G000 are related to the strength of the intermolecular interactions, and n0 and n00 to the extent and stability of the protein network (Gabriele et al., 2001; Moresi et al., 2004). As we can see in Table 4, SB samples presented significantly higher Q values after surimi type B frozen storage, confirming that myofibrillar protein setting capacity was best in surimi B. This is consistent with the higher soluble protein content in surimi B (Table 1). The structure of SB suwari gels, then, is cohesive and deformable since the myofibrillar protein is more functional in surimi B (Campo-Deaño et al., 2009). As a result, the protein chains Table 4 Influence of frozen storage time of surimis A and B, with different cryoprotectants, on quality factor of Eq. (3) for suwari gels SA and SB, at frequency 1 Hz and 10 °C.

Fig. 2. Evolution of cmax of suwari gels SA and SB after frozen storage of A and B surimi, (a) SA gels, and (b) SB gels.

t (Weeks)

SA1

SA2

SA3

0 8 16 24

27.69c ± 0.18 26.83a ± 0.12 30.26f ± 0.16 31.73h ± 0.19

27.22b ± 0.23 26.75a ± 0.14 27.98c ± 0.16 28.97d ± 0.18

29.62e ± 0.15 31.01g ± 0.31 31.52gh ± 0.29 28.76d ± 0.15

0 8 16 24

SB1 30.77g ± 0.24 31.02g ± 0.27 31.90h ± 0.24 32.74i ± 0.23

SB2 30.23f ± 0.21 34.14j ± 0.38 33.73j ± 0.27 33.15i ± 0.27

SB3 35.82k ± 0.33 35.46k ± 0.25 32.73i ± 0.26 33.16i ± 0.23

a–k

Different letters indicate significant differences at P < 0.05.

Table 3 Evolution of power law exponents of Eqs. (1) and (2) of suwari gels SA1 and SB1 (4% sorbitol + 4% sucrose), SA2 and SB2 (4% sorbitol + 4% trehalose), SA3 and SB3 (8% trehalose) at 10 °C, after frozen storage of surimi A and B. t (Weeks)

n0 ± S.D. SA1

n0 0 ± S.D. SA1

n0 ± S.D. SB1

n0 0 ± S.D. SB1

0 8 16 24

0.1340 ± 0.0004 0.1384 ± 0.0005 0.1227 ± 0.0005 0.1156 ± 0.0005

0.1399 ± 0.0035 0.1439 ± 0.0019 0.1403 ± 0.0029 0.140 ± 0.003

0.1147 ± 0.0010 0.1138 ± 0.0017 0.1118 ± 0.0013 0.1084 ± 0.0031

0.116 ± 0.004 0.121 ± 0.007 0.120 ± 0.006 0.122 ± 0.013

0 8 16 24

SA2 0.1354 ± 0.0027 0.1386 ± 0.0004 0.1341 ± 0.0010 0.1269 ± 0.0004

SA2 0.138 ± 0.017 0.149 ± 0.003 0.149 ± 0.004 0.144 ± 0.003

SB2 0.1157 ± 0.0009 0.1038 ± 0.0013 0.1067 ± 0.0008 0.1080 ± 0.0009

SB2 0.121 ± 0.004 0.111 ± 0.009 0.111 ± 0.004 0.114 ± 0.005

0 8 16 24

SA3 0.1231 ± 0.0005 0.1176 ± 0.0008 0.1167 ± 0.0007 0.1267 ± 0.0005

SA3 0.133 ± 0.003 0.141 ± 0.008 0.142 ± 0.007 0.152 ± 0.002

SB3 0.0978 ± 0.0008 0.0997 ± 0.0015 0.1085 ± 0.0013 0.1074 ± 0.0009

SB3 0.111 ± 0.005 0.116 ± 0.005 0.119 ± 0.007 0.120 ± 0.004

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in SB samples are more flexible and less dense; they are less viscous than in SA samples, and so Q increases (Table 4). This particular behaviour of SB samples was maintained throughout the 6 months of frozen storage regardless of the cryoprotectant. 3.4. Temperature sweep test for suwari gels during frozen storage of surimi Figs. 3 and 4 show the thermal gelation profiles of suwari gels SA and SB in terms of the thermal behaviour of G0 and G00 moduli

from 10 to 90 °C. It can be seen that in surimi type B, the G0 and G00 values of its gelated samples SB1, SB2 and SB3 show the characteristic stages in the gelation profile (Fig. 4a–f): (1) gel weakening; G0 and G0 0 moduli decrease between 45 and 50 °C, reflecting an increase in fluidity of the semi-gel, and some of the protein network already formed may be disrupted so that gel elasticity decreases. (2) Gel strengthening from 50 to 65 °C; involves denaturation of actin and can be ascribed both to an increase in the number of cross-links between protein aggregates or strands, and to deposition of additional denatured proteins in the existing protein

Fig. 3. Effect of cryoprotectant added to surimi A on the thermal gelation profile of suwari gels SA: (a) elastic moduli SA1 samples; (b) viscous moduli SA1 samples; (c) elastic moduli SA2 samples; (d) viscous moduli SA2 samples; (e) elastic moduli SA3 samples; and (f) viscous moduli SA3 samples.

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463

Fig. 4. Effect of cryoprotectant added to surimi B on the thermal gelation profile of suwari gels SB: (a) elastic moduli SB1 samples; (b) viscous moduli SB1 samples; (c) elastic moduli SB2 samples; (d) viscous moduli SB2 samples; (e) elastic moduli SB3 samples; and (f) viscous moduli SB3 samples.

networks, thus reinforcing the gel matrix (Damodaran and Paraf, 1997). From that point up to T > 70 °C, G0 continues increasing whereas G00 remains almost constant, indicating the formation of a highly elastic myofibrillar protein gel. The setting characteristic of SB samples at around 40 °C was maintained after surimi type B frozen storage; this means that any of the three cryoprotectants will preserve the initial setting induced by endogenous TGase, and therefore any cryoprotectant will maintain the myofibrillar protein intact in surimi B. Moreover, the

G0 and G00 moduli values from 10 to 90 °C were still maintained (with due allowance for experimental uncertainty) after 6 months of surimi frozen storage. However in SA samples there were some differences among the different cryoprotectants: sorbitol + trehalose in SA2 sample caused a significant increase in G0 and G00 moduli, after eight weeks of storage of surimi A2, throughout the temperature range. This thermal behaviour is consistent with the fact that the sample presented the highest rigidity (Fig. 1a), and the lowest cmax (Fig. 2a) in

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the same time interval at 10 °C. Sorbitol plus trehalose had a weak cryopreservative effect on surimi A; in this case the cryoprotectant may have increased the internal mechanical stress produced by freezing-induced dehydration. These stresses and strains are anisotropic, producing thickening and contraction in two normal space directions (Yoon et al., 1998), which could be a contributing factor to the freezing-induced damage in surimi A2. This effect was more intense in the first 2 months of frozen storage. On the other hand, trehalose (sample SA3) retained its initially good thermal profile during surimi A3 frozen storage. This cryoprotectant significantly altered the pattern of the gelation profile in surimi A (Fig. 3a–d). Sample SA3 (Fig. 3e and f) presented a thermo-rheological profile similar to the SB suwari gels (Fig. 4a–f), including two phases of thermal transitions mentioned above. This thermo-rheological behaviour is typical of samples with more native protein content (Campo-Deaño et al., 2009). Moreover this peculiar behaviour of trehalose in surimi A, is consistent with the fact that its cryoprotectant functionality is greater than that of other sugars such as sucrose, fructose or glucose (Wang and Haymet, 1998). The structural properties of trehalose and its particular interaction with water, may lead to the formation of a trehalose– water complex through linkage by intermolecular hydrogen bonds, involving all the hydroxyl groups in the system. The geometry of this molecular complex produces a symmetric building block which enhances water holding capacity (Ballone et al., 2000) and may be capable of minimizing freezing-induced dehydration.

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