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Enhancement of the gelation properties of hairtail muscle protein with curdlan and transglutaminase


Food Chemistry 176 (2015) 115–122

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Enhancement of the gelation properties of hairtail (Trichiurus haumela) muscle protein with curdlan and transglutaminase
Yaqin Hu a, Wenjuan Liu a, Chunhong Yuan b, Katsuji Morioka c, Shiguo Chen a,?, Donghong Liu a, Xingqian Ye a
a Zhejiang University, College of Biosystems Engineering and Food Science, Fuli Institute of Food Science, Zhejiang Key Laboratory for Agro-Food Processing, Zhejiang R & D Center for Food Technology and Equipment, Hangzhou 310058, China b Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima 890-0056, Japan c Lab of Aquatic Product Utilization, Kochi University, Kochi 783-8502, Japan

a r t i c l e

i n f o

a b s t r a c t
The effects of curdlan in combination with microbial transglutaminase on the gelling properties of hairtail muscle protein were investigated. When curdlan of 4 g/100 g paste was combined with transglutaminase at a concentration of 0.4 units/g meat paste, the gel strength, water holding capacity and the whiteness of the heated gel were improved. Textural pro?les, such as hardness, springiness, cohesiveness, guminess and chewiness, reached their peaks as well. The increased band intensity of cross-linked proteins, accompanied by weakened myosin heavy chain, was observed from the SDS–PAGE pattern, indicating that curdlan might activate the formation of more e-(c-glutamyl) lysine cross-links induced by transglutaminase, especially at the level of 0.4 units/g paste, leading to a denser gel matrix. ? 2014 Elsevier Ltd. All rights reserved.

Article history: Received 7 July 2014 Received in revised form 13 November 2014 Accepted 3 December 2014 Available online 9 December 2014 Keywords: Hairtail Muscle protein Curdlan Microbial transglutaminase Gelation properties

1. Introduction Hairtail is one of the most popular commercial marine ?shes in the eastern Paci?c Ocean. Both economically and ecologically, the hairtail plays an important role in supporting the most valuable and largest ?shery in Asia with its abundant ?sh stocks (Chakraborty, Aranishi, & Iwatsuki, 2007). With the increasing market for ?sh meat-based gelled products and its availability and low cost, hairtail is gaining the interest of seafood manufacturers. Generally, the textural properties of gels made from whole minced pastes are less desirable than those from washed pastes due to a higher fat content. In addition, water-soluble sarcoplasmic proteins have a detrimental effect on the gelling properties of ?sh muscle proteins, mainly on water-insoluble myo?brillar proteins (An, Weerasinghe, Seymour, & Morrissey, 1994). To overcome these disadvantages, hairtail muscle protein is processed to remove the fat and sarcoplasmic proteins through washing the minced ?sh meat with cold water several times. Nevertheless, the gelling properties of hairtail muscle proteins alone are still poor and cannot meet customer expectations. Therefore, there is a need to improve the gelling properties of hairtail muscle protein,
? Corresponding author.
E-mail address: chenshiguo210@163.com (S. Chen). http://dx.doi.org/10.1016/j.foodchem.2014.12.006 0308-8146/? 2014 Elsevier Ltd. All rights reserved.

especially considering its promising market potential. At present, an array of gel enhancers, mainly carbohydrates and proteins, have been investigated to modify the mechanical and functional properties of ?sh muscle protein, but these texture-enhancing food additives failed to improve the gelling properties for hairtail and compromised other sensory parameters which hindered ?nal market acceptance. Curdlan is a thermo-gelable bacterial polysaccharide that is tasteless, colourless and odourless, and can form gels with different textural qualities, physical stabilities and water holding capacities depending on the heat treatment (Zhang & Edgar, 2014). When heated above 55 °C, neutral aqueous solutions of curdlan can form thermoreversible gels. When the heating temperature is above 80 °C, curdlan can form more stable, highset, thermoirreversible gels (Maeda, Saito, Masda, Misaki, & Harada, 1967; Nishinari & Zhang, 2004). The effect of various polysaccharides, such as starch (Hunt, Getty, & Park, 2009), konjac (Iglesias-Otero, Borderías, & Tovar, 2010), pectins (Uresti, López-Arias, González-Cabriales, Ramirez, & Vázquez, 2003) and ?bre (Debusca, Tahergorabi, Beamer, Matak, & Jaczynski, 2014), on the properties of ?sh muscle proteins have been investigated. However, published reports relevant to curdlan applied to surimi seafood are scarce, although curdlan has been well researched in meat products as an effective fat substitute, texture modi?er and

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elasticity enhancer (Funami, Yada, & Nakao, 1998). Chen et al. (2010) noted that 1% curdlan can contribute to the formation of a smooth, compact, continuous and uniform gel matrix and can increase hardness when added to duck muscle gels under high pressure. Additionally, it has been reported that curdlan can enhance the functionality of meat products as a dietary ?bre. Curdlan was approved for food usage in Korea, Taiwan and Japan as early as 1989, and registered in the United States in 1996 by the Food and Drug Administration (FDA) as a food additive (formulation aid, processing aid, stabilizer and thickener, or texturizer) (McIntosh, Stone, & Stanisich, 2005). Transglutaminases (protein-glutamine c-glutamyl-transferase, EC 2.3.2.13) originating from microbial materials are called MTGase and these enzymes induce the formation of e-(c-glutamyl) lysine cross-links in proteins via acyl transfer between the e-amino groups of a lysine residue and the c-amide group of a glutamine residue, resulting in improved gel quality (Chanarat & Benjakul, 2012). However, its enhancing effect is largely dependent on the ?sh species and other factors, such as freshness, protein quality and harvesting season (Asagami, Ogiwara, Wakameda, & Noguchi, 1995). Although much work has illustrated the enhancing effect of MTGase for gelling properties when applied alone to various ?sh species, Jiang, Hsieh, Ho, and Chung (2000) found that MTGase alone had no signi?cant effect on the gelling ability of hairtail muscle protein, even with the addition of MTGase up to 0.6 units/g. Additionally, to further understand the mechanical and functional properties of gels from ?sh muscle, the combined use of MTGase with many other texture-enhancing food additives, such as carrageenan, konjac and plant protein, has also been investigated (Cardoso, Mendes, Vaz-Pires, & Nunes, 2011; Kudre & Benjakul, 2013). However, the compatibility between MTGase and other additives, such as polysaccharides, must be considered because the combined use of these additives may lead to weaker products with inappropriate mechanical and functional properties. For example, Carmen Gómez-Guillén, Montero, Teresa Solas, and Pérez-Mateos (2005) found that gels were slightly less elastic when both chitosan and MTGase were added compared with gels that had MTGase only. A similar result was reported by Benjakul, Visessanguan, Phatchrat, and Tanaka (2003), who found that chitosan can hinder gel formation in the presence of MTGase, most likely due to chitosan particles acting as a barrier for cross-linking between myo?brils. Therefore, the combined effect of curdlan and MTGase on the gelling properties of hairtail muscle proteins was investigated in the present study. 2. Materials and methods

2.3. Breaking force and deformation The breaking force and deformation were measured using a texture analyser Model CT3 (Brook?eld, USA) based on the method of Benjakul et al. (2002). Gels held at 4 °C were equilibrated at room temperature (25–28 °C) and cut into lengths of 2.5 cm. All of the cylindrical samples were placed in a texture analyser and the cut surface of gel specimens was pressed with a spherical probe (5 mm in diameter; 1 mm/s in depression speed). The breaking force and deformation were recorded by the texture analyser. 2.4. Determination of water holding capacity The water holding capacity (WHC) of gels was determined according to the method of Benjakul et al. (2002), with slight modi?cation. A sheet of gel sample, 5 mm thick, was cut off from the intermediate portion of the cylindrical gel, weighed (m1) and placed between 3 layers of Whatman ?lter paper (upper and lower, diameter 9 cm). The sample and papers were pressed for 2 min under applied force with a 10 kg weight and then weighed again (m2). WHC was expressed as percent of sample weight and was calculated using the following equation:

WHC ?%? ? m2 =m1 ? 100:
2.5. Measurement of whiteness The whiteness of gels was tested by a colour difference metre (Model WSC-S, Tokyo, Japan). The formula for the calculation of whiteness was as follows:

W ? 100 ? ??100 ? L? ? ? a? 2 ? b ?
where L? = lightness; blueness.

2

? 2 1=2

a? = redness/greenness;

b? = yellowness/

2.6. Textural pro?le analysis A textural pro?le analysis (TPA) was conducted using a texture analyser (Model CT3, Brook?eld Lt. Co, USA) based on the method of Benjakul et al. (2002). Gel specimens (2.5 cm long) were prepared. A cylindrical plunger, with a diameter of 5 mm, was pressed into the cut surface of the gel samples perpendicularly at a constant depression speed of 1 mm/s. Hardness, springiness, cohesiveness, gumminess and chewiness were determined. 2.7. SDS–polyacrylamide gel electrophoresis (SDS–PAGE)

2.1. Materials AA grade frozen hairtail surimi muscle was obtained from Maruha Nichiro Ltd. (Tokyo, Japan). Food grade curdlan was provided by Wako Chemicals (Tokyo, Japan). Broad protein marker was purchased from Bio-Rad (USA). MTGase (activity of 100 units/ g) was purchased from Ajinomoto Ltd. (Tokyo, Japan). 2.2. Preparation of curdlan gel and ?sh muscle protein gel Curdlan powder was added to water (55 °C) and stirred for 10 min. The gel was cooled with iced water and held at 4 °C for further use. The frozen hairtail muscle protein was thawed using tap water (about 20 °C). NaCl was added to obtain a ?nal concentration of 2.5% and the ?nal moisture content was adjusted to 80%. Curdlan and MTGase were added accordingly. The resulting paste was ground at 4 °C for 30 min using a mortar and pestle, and then packed into stainless steel rings (diameter 3.0 cm) for heating Benjakul, Visessanguan, Riebroy, Ishizaki, & Tanaka, 2002). The solubilities of gels in 20 mM Tris–HCl, pH 8.0, containing 1% (w/v) SDS, 8 M urea and 2% (v/v) b-ME, were determined using the methods described by Rawdkuen, Benjakul, Visessanguan, and Lanier (2004). The sample (1 g) was homogenised in a 20 ml solution for 1 min using a homogenizer. The homogenate was heated in boiling water (100 °C) for 2 min and stirred at room temperature for 4 h. The resulting homogenate was centrifuged at 10,000?g for 30 min, using a 3K-15 centrifuge (Sigma Corp., Germany). The protein in the supernatant (10 ml) was precipitated with the addition of 50% (w/v) cold TCA to a ?nal concentration of 10%. The protein patterns of gels were analysed using SDS– polyacrylamide gel electrophoresis. The gel protein was extracted by the method of Bechtel and Parrish (Bechtel & Parrish, 1983). An aliquot of 20 ll from each sample was subjected to SDS–PAGE under reducing conditions. 2.8. Determination of protein solubility

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The mixture was kept at 4 °C for 18 h and then centrifuged at 10,000?g for 30 min. The precipitate was washed with 10% TCA and solubilised in 0.5 M NaOH. The protein content was measured using the Biuret test (Robinson & Hodgen, 1940). The solubility was expressed as percent of the total protein in gels solubilised directly in 0.5 M NaOH. 2.9. Scanning electron microscopy (SEM) SEM was performed, according to the method of Ghosh, Kumar, Chakrabarti, and Padmanabha (2010) with slight modi?cation. The microstructure of gels was determined using a Model XL 30 SEM (Philips, Holland). The gel samples were ?rst ?xed with 2.5% glutaraldehyde in phosphate buffer (pH 7.0) for more than 4 h, washed three times in the phosphate buffer (once for 15 min), and then post-?xed with 1% osmium tetroxide (OsO4) for 1 h and washed. After being dehydrated by a graded series of ethanol (50%, 70%, 80%, 90%, 95% and 100%) for approximately 15–20 min for each step, the samples were transferred to the mixture of alcohol and iso-amyl acetate (v:v = 1:1) for approximately 30 min and then were transferred to pure isoamyl acetate for approximately 1 h. As a ?nal step, the treated samples were dehydrated in a Hitachi Model HCP-2 critical point dryer with liquid CO2. After coating the samples with gold–palladium, the microstructure could be observed in the SEM and the scanned images were ampli?ed 1500 times. 2.10. Statistical analysis The experiments were run in triplicates and the data are presented as the means with standard deviations. Statistical analysis was performed using ORIGIN (Version 8.0; Microcal Software Inc., Northampton, MA) and SPSS (Version 16.0; SPSS Inc., Chicago, IL). 3. Results and discussion 3.1. Breaking force and deformation Breaking force and deformation of the hairtail muscle protein heated gels with supplemental curdlan (0–8 g/100 g meat paste) and MTGase (0–0.8 units/g paste) are depicted in Fig. 1. Without the addition of MTGase, the breaking force increased signi?cantly as the curdlan content increased (P < 0.05), especially at the levels of 2 and 4 g/100 g meat paste, and the greatest deformation was obtained at 4 g of curdlan/100 g meat paste without MTGase. Therefore, in terms of physical properties, the gel forming ability of low grade ?sh muscle protein could be improved with the addition of curdlan. The results recon?rmed that curdlan most likely contributed to the stronger gel as indicated by the noticeable increase in breaking force and deformation. Generally, the breaking force has been positively correlated with gel strength, while deformation represents the elasticity of the gels (Chanarat & Benjakul, 2013a). When MTGase was added, both the breaking force and deformation reached a peak at 4 g curdlan/100 g meat paste, irrespective of the MTGase amount. At constant levels of MTGase, ranging from 0.2 to 0.8 units/g paste, the breaking force and deformation increased signi?cantly (P < 0.05) as the curdlan amount increased at low levels (64 g/100 g meat paste) (P < 0.05). However, a slight decrease in the breaking force and deformation was observed (P < 0.05) as the curdlan content increased from 6 to 8 g/100 g paste. The disruptive effect on gel forming ability associated with higher concentrations of curdlan may be caused by the swelling of curdlan, which can absorb up to 100 times its weight in water owing to its helical molecular structure (Zhang & Edgar,

2014). The swelling of curdlan at higher levels could have a detrimental effect by competing for water molecules or simply by interfering in gel formation, although it may only be a causal factor in the decrease of breaking force and deformation, considering that the relative concentration of protein in the samples was lower with higher curdlan levels. To avoid the lower protein concentration confounding the results, a recent study used silicon dioxide as an inert ?ller, added in inverse concentrations to the ?bre forti?cation, to keep the protein/water concentrations constant (Debusca et al., 2014). On the other hand, MTGase has been shown to catalyse the acyl transfer reaction, in which the c-carboxamide groups of glutamine residues in proteins or peptides, act as acyl donors, and primary amino groups including e-amino groups of lysine residues, either as protein-bound or free lysine, act as the acyl acceptor (Greenberg, Birckbichler, & Rice, 1991; Jirawat & Penprabha, 2007). When acceptors are e-amino groups of lysine residues, the formation of e-(c-glutamyl) lysine (GL) linkages occur intra- and inter-molecularly (Joseph, Lanier, & Hamann, 1994). These bonds were stable and resistant to proteolysis. In our research, at low levels of curdlan, the breaking force and deformation of treated samples increased signi?cantly at ?rst and then decreased slightly with an increasing MTGase concentration (P < 0.05). No obvious change was observed (P > 0.05) when the amount of curdlan was above 6 g/100 g meat paste. The adverse effect on gelating properties may be caused by excessive cross-linking induced by a higher MTGase content, the substrates of which showed a saturation effect. Several previous studies have postulated that excessive cross-linking of myo?brillar protein molecules catalysed by MTGase might interfere with the ordered gel matrix and be detrimental to further increases in gel strength (Asagami et al., 1995; Tsai, Lin, & Jiang, 1996). Compared with the control (without curdlan and MTGase), a signi?cant increase in both the breaking force and deformation was found in gels with curdlan or MTGase, either separately or in combination (P < 0.05). The highest value of breaking force and deformation among all of the samples was obtained with curdlan at 4 g/100 g meat paste in combination with MTGase at 0.4 units/g meat paste. The enhancing effect of combining curdlan with MTGase surpassed the sum of their individual effects, indicating the existence of a compatibility or mutual reinforcement between curdlan and MTGase. This result also con?rmed that curdlan and hairtail muscle protein are compatible; thus, their combined use could exert a positive effect, although curdlan might dilute the muscle protein at higher rates. Therefore, there is an optimum ratio of curdlan/MTGase that will dramatically improve gelling properties. 3.2. Water holding capacity (WHC) The water holding capacity of hairtail muscle protein gels with curdlan and MTGase at different levels is shown in Table 1. Irrespective of curdlan levels, the WHC increased signi?cantly when MTGase at 0.4 units/g paste was incorporated (P < 0.05), but no further changes were observed when the MTGase level increased up to 0.8 units/g paste (P > 0.05). The addition of MTGase increases the water holding capacity of the gels but might cause a reduction or no change in the WHC at excessive levels (Chanarat & Benjakul, 2013a). There was a noticeable increase of WHC with curdlan from 0 to 4 g/100 g paste (P < 0.05), regardless of MTGase levels. The increased WHC of gels suggested that more water was bound or retained in the gel network (Chanarat & Benjakul, 2013b). Upon heating at 90 °C, curdlan can form high-set gels by imbibing a great amount of water, thus more water can be held in the gel network with the addition of curdlan. However, curdlan with more than 4 g/ 100 g paste did not cause a further increase in WHC as the curdlan concentration increased (P < 0.05). The result indicated that the

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Fig. 1. Breaking force and deformation of gels from hairtail muscle protein incorporated with curdlan and MTGase at different levels. Bars represent the standard deviation (n = 3).

Table 1 Water holding capacity (WHC) and whiteness of gels from hairtail muscle protein incorporated with curdlan (0–8 g/100 g meat paste) and MTGase (0–0.8 units/g meat paste). Curdlan (g/100 g paste) WHC 0 units MTGase/g paste 87.06 ± 1.32cA 91.78 ± 1.87bA 95.13 ± 0.26aA 95.05 ± 0.27aA 95.12 ± 0.40aA 0.4 units MTGase/g paste 93.57 ± 0.51cB 95.07 ± 0.21bB 96.25 ± 0.35aB 96.19 ± 0.39aB 96.23 ± 0.30aB 0.8 units MTGase/g paste 93.51 ± 0.45cB 95.12 ± 0.15bB 96.14 ± 0.31aB 96.14 ± 0.41aB 96.16 ± 0.47aB Whiteness 0 units MTGase/g paste 68.78 ± 0.22cA 70.04 ± 0.29bA 70.74 ± 0.22aA 70.84 ± 0.14aA 71.04 ± 0.14aA 0.4 units MTGase/g paste 68.25 ± 0.10dB 69.47 ± 0.27cAB 70.36 ± 0.21aAB 70.59 ± 0.12abAB 70.94 ± 0.19bA 0.8 units MTGase/g paste 67.82 ± 0.15dC 69.34 ± 0.12cB 70.26 ± 0.08aB 70.42 ± 0.12abB 70.78 ± 0.33bA

0 2 4 6 8

Values are mean ± SD (n = 3). Different letters in the same column denote the signi?cant difference (P < 0.05). Different capital letters in the same row under the same parameter denote the signi?cant difference (P < 0.05).

dilution effect of curdlan at higher levels might reduce the water binding sites of proteins to some degree, resulting in less binding and interaction of muscle proteins with water molecules, while curdlan at levels above 4 g/100 g paste absorbed more water in the gel network. The highest WHC was observed in gels with 4 g/ 100 g meat paste of curdlan in combination with MTGase at 0.4 units/g meat paste. The maximum WHC coincided with the greatest breaking force and deformation of the resulting gels (Fig. 1). The control, without curdlan and MTGase, showed the lowest WHC, closely associated with its weakest breaking force and deformation (Fig. 1). Moreno, Carballo and Borderías also reported changes of WHC with textural properties (Moreno, Carballo, & Borderías, 2008). 3.3. Whiteness The whiteness of gels from hairtail muscle protein with the addition of curdlan and MTGase at different levels is shown in Table 1. In the absence of MTGase, there was a signi?cant increase in whiteness with curdlan added at levels lower than 4 g/100 g paste (P < 0.05). This might be caused by the light scattering effect of curdlan. Nevertheless, such an effect did not further increase the whiteness as the curdlan concentration increased from 4 to 8 g/ 100 g paste in gels without MTGase. The colour characteristics of

gels from ?sh muscle protein are largely dependent on the types and amounts of additives added (Rawdkuen, Benjakul, Visessanguan, & Lanier, 2007). When MTGase was added, the gels with curdlan at levels lower than 8 g/100 g meat paste, showed a slight decrease as MTGase concentration increased (P < 0.05). Consistent with this result, Chanarat and Benjakul (2013b) reported that the addition of MTGase could reduce the whiteness of gels from India mackerel ?sh protein isolates and suggested that the denser gel network induced by increased MTGase possessed the higher light absorption, leading to a darker colour of gels. In contrast, Karayannakidis, Zotos, Petridis, and Taylor (2008) reported that the addition of MTGase had a positive effect on the whiteness index of heat-induced sardine ?sh muscle protein. However, gels with MTGase ranging from 0 to 0.8 units/g meat paste had a similar whiteness as gels with curdlan at the level of 8 g/100 g meat paste, indicating that whiteness-increasing effect of curdlan eclipsed the negative effect of MTGase. 3.4. Textural pro?le analysis Textural pro?les of gels from hairtail muscle protein with curdlan at various levels with and without MTGase are depicted in Table 2. In the absence of curdlan, hardness, springiness, cohesiveness, guminess and chewiness increased noticeably (P < 0.05)

Y. Hu et al. / Food Chemistry 176 (2015) 115–122 Table 2 Textural properties of gels from hairtail muscle protein with curdlan at various levels with and without MTGase at a level of 0.4 units/g meat paste. MTGase (units/g paste) 0 0.4 Curdlan (g/100 g paste) 0 0 2 4 6 8 Hardness (g) 147.17 ± 3.06
f e

119

Springness 0.46 ± 0.01
f e

Cohesiveness 0.47 ± 0.02
e d

Gumminess (g) 69.69 ± 3.71
f e

Chewiness (g) 32.28 ± 1.44f 83.51 ± 3.25e 177.56 ± 4.76d 482.52 ± 13.99a 330.66 ± 13.04b 247.54 ± 7.31c

221.50 ± 6.87 320.17 ± 5.75d 574.17 ± 13.16a 478.00 ± 7.05b 419.33 ± 7.02c

0.58 ± 0.01 0.72 ± 0.01d 0.97 ± 0.01a 0.84 ± 0.01b 0.76 ± 0.01c

0.65 ± 0.01 0.77 ± 0.01c 0.86 ± 0.01a 0.82 ± 0.01b 0.78 ± 0.01c

143.95 ± 3.49 245.48 ± 6.05d 495.74 ± 14.11a 392.01 ± 10.54b 325.69 ± 7.56c

Values are mean ± SD (n = 3). Different letters in the same column denote the signi?cant difference (P < 0.05).

when MTGase (0.4 units/g meat paste) was incorporated. Higher amounts of MTGase might improve the gel properties by inducing the formation of non-disulphide covalent bonds to a greater extent, leading to a denser gel matrix (Chanarat & Benjakul, 2013a). In the presence of 0.4 units MTGase/g meat paste, a marked increase of textural parameters was observed in all samples when compared to samples without MTGase. Moreover, all of the parameters increased signi?cantly (P < 0.05) as curdlan increased from 0 to 4 g/100 g meat paste. When the curdlan level was above 4 g/ 100 g meat paste, these parameters decreased sharply with increasing curdlan (P < 0.05). The peaks of all of the parameters occurred when curdlan was added at the level of 4 g/100 g meat paste and MTGase was added at the level of 0.4 units/g meat paste. Curdlan has been reported to enhance textural properties of edible ?lm blends by forming intermolecular hydrogen bonds when interacting synergistically with konjac glucomannan (Chunhua et al., 2012). Furthermore, the addition of MTGase into ?sh meat paste incubated at 40 °C for 30 min prior to cooking induced an increase in the penetration forces of gels (Pérez-Mateos & Lanier, 2007). Therefore, the combination of curdlan with MTGase positively affected the textural properties of hairtail muscle protein due to thermal gelling properties of curdlan and catalytic activity of MTGase. However, a negative effect was observed when curdlan was added at levels above 6 g/100 g paste. This might indicate that excessive curdlan diluted the myo?brillar proteins and, in turn, weakened the availability of those substrates catalysed by MTGase, resulting in structural disorder and weakened textural properties. 3.5. SDS–PAGE patterns The SDS–PAGE patterns of heated hairtail muscle protein gels with curdlan and MTGase alone or in combination are depicted in Fig. 2. In Fig. 2(a), the contribution of curdlan and MTGase alone to the cross-linking of myosin heavy chains (MHC) during gel formation was clari?ed. Gels contained myosin heavy chain (MHC) and actin as the major proteins, as evidenced by the higher band intensity of both MHC and actin. When curdlan was added, no changes in the MHC band intensity was observed as the curdlan concentration increased, indicating that there were only disulphide covalent bonds between curdlan and ?sh muscle protein, even though curdlan exerted enhancing effect for breaking force and deformation (Fig. 1). This result suggested that most of the inter- and intra-molecular cross-linking of ?sh muscle protein gained with curdlan was most likely mediated by weak bonds, such as hydrogen bonds or hydrophobic interactions, which could be destroyed by SDS used for solubilisation and electrophoresis. In the process of protein gelation, mainly four main types of chemical bonds are involved, hydrogen bonds, ionic linkages, hydrophobic interactions and covalent bonds (Lanier, Carvajal, & Yongsawatdigul, 2005). In this experiment, it was found that hydrogen bonds between protein molecules increased in gels with a high concentration of curdlan (data not shown). Perhaps the increasing hydrogen bonds might modify the protein–protein conjugates that are responsible for the increase of gel texture. It

has been reported that chitosan combined with endogenous MTGase could act as a gel enhancer in ?sh muscle by forming protein–protein and protein-chitosan conjugates (Benjakul et al., 2003). When compared with the control without curdlan and MTGase, a very slight decrease in the MHC band intensity was observed. It was postulated that endogenous MTGase might undergo inactivation to a higher degree during the washing process so that more MHC bands were retained or that the lower protease activity associated with the washing process caused a lower degradation of MHC. However, with the addition of MTGase, a gradual reduction of MHC and an increase of both CPI and CPII band intensity was noticeable with the increasing MTGase concentration. The disappearance of MHC and the appearance of both CPI and CPII (with higher molecular weight) indicated the formation of MHC cross-links, mainly via the formation the e-(c-glutamyl) lysine linkages induced by MTGase (Yin & Park, 2014). In Fig. 2(b), consistent with the results in Fig. 2(a), there were no signi?cant changes found when curdlan was added, but a gradual decrease of the MHC band was noticeable as the MTGase concentration increased. However, the greatest band intensity of CPI produced by the cross-linking of MHC appeared in the heated gels with curdlan and MTGase at the levels of 4 g/100 g paste, 0.4 units/ g paste, respectively. This gel exhibited the highest breaking force and deformation (Fig. 1). Lower CPI and CPII band intensities were observed, although more MHC disappeared when a higher level of MTGase (8 units/g meat paste) was added. An excess amount of MTGase could probably induce disordered cross-linking of MHC and have a detrimental effect on gel texture. Several previous studies postulated that excessive cross-linking of myo?brillar protein molecules catalysed by MTGase might be detrimental for the further increase of gel strength by interfering with the orderly gel matrix (Asagami et al., 1995; Tsai et al., 1996). In Fig. 2(c), the effect of different curdlan concentration on SDS–PAGE patterns of ?sh muscle protein is shown in the presence of MTGase at the level of 0.4 units/g paste. When MTGase was added to ?sh muscle protein, the intensity of the MHC band decreased in all of the samples regardless of the curdlan levels compared with the control without curdlan and MTGase. Although the MHC band intensity decreased markedly, actin scarcely changed in the samples. Thus, MHC appeared to be the major contributor for the gel formation when heated, while there was little involvement of other myo?brillar proteins, such as actin, tropomyosin or troponins (Careche, Alvarez, & Tejada, 1995; Montero & Gómez-Guillén, 1996). When curdlan was added at the level of 4 g/100 g meat paste, the greatest band intensity of CPI was obtained with MTGase. A similar result is depicted in Fig. 2(b). However, as the curdlan levels increased, especially at 6 and 8 g/ 100 g meat paste, MHC in gels was more retained in spite of the MTGase, which might have an adverse effect on gel texture. This result recon?rmed the disruptive effect associated with higher curdlan levels on the breaking force and deformation, as depicted in Fig. 1. Therefore, an appropriate ratio exists between curdlan and MTGase concentration. The optimum gel texture could be

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Fig. 2. SDS–PAGE patterns of gels from hairtail muscle protein incorporated with curdlan and MTGase. (a) MTGase, (b) with MTGase (0–0.8 units/g paste) in the presence of curdlan (4 g/100 g paste), (c) with curdlan (0–8 g/100 g paste) in the presence of MTGase (0.4 units/g paste). M: marker, R: raw paste, C: the control without curdlan and MTGase. Numbers designate the level of curdlan or MTGase added. All samples, except raw paste, were set at 40 °C for 90 min and then cooked at 90 °C for 25 min.

obtained when curdlan and MTGase are used in combination according at this ratio. 3.6. Non-disulphide covalent bonds Liu, Zhao, Xie, and Xiong (2011) have reported that a solution of 20 mM Tris–HCl, pH 8.0 containing 1% (w/v) SDS, 2% (v/v) b-ME and 8 M urea (S1), could be used to determine non-disulphide covalent bond measurement. The solution (S1) could destroy all of the bonds, except non-disulphide covalent bonds, particularly the e-(c-glutamyl) lysine linkage (Benjakul, Visessanguan, & Pecharat, 2004). Thus, decreased solubility indicated the formation of non-disulphide covalent bonds induced by endogenous or microbial MTGase. Non-disulphide covalent bonds play an important role in gelation of ?sh muscle protein and are the primary contributor for enhanced gel texture. The solubility of gels from hairtail muscle protein incorporated with curdlan and MTGase at different levels is shown in Fig. 3. The solubility of raw ?sh meat paste was regarded as 1. In Fig. 3, after incubation at 40 °C for 90 min followed by cooking at 90 °C for 25 min, the gels without curdlan and MTGase exhibited a solubility of approximately 0.85, which was lower than that of raw ?sh meat

Fig. 3. Solubility of gels from hairtail muscle protein incorporated with curdlan and MTGase at different levels.

Y. Hu et al. / Food Chemistry 176 (2015) 115–122

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Fig. 4. Microstructure of gels from hairtail muscle protein. (a) Control (without curdlan nor MTGase), (b) with MTGase (0.4 units/g paste) but without curdlan, (c) with curdlan (4 g/100 g paste) but without MTGase, (d) with curdlan (4 g/100 g paste) and MTGase (0.4 units/g paste).

paste. The decreased solubility indicated the formation of nondisulphide covalent bonds induced by endogenous MTGase. In the absence of MTGase, a signi?cant decrease of solubility was noticeable when curdlan at 2 g/100 g paste was incorporated (P < 0.05), suggesting an increase in the formation of non-disulphide covalent bonds, as evidenced by the increasing breaking force and deformation (Fig. 1). It can be postulated that the enhancing effect of curdlan was possibly mediated by the higher catalysing reactivity of endogenous transglutaminase induced by the interactions between curdlan and proteins. However, no further changes in solubility were observed when curdlan was added above 2 g/100 g meat paste, indicating the enhancing effect of curdlan combined with endogenous transglutaminase was limited. At the same level of curdlan, the lowest solubility was obtained when MTGase added at the level of 0.4 units/g paste. This indicated that MTGase at a suf?cient concentration of 0.4 units/g paste could fully activate the reactivity of hairtail muscle protein, leading to more crosslinking of the protein via non-disulphide covalent bonds; however, excessive amount of MTGase may have a negative effect on the formation of non-disulphide covalent bonds, as evidenced by decreased CPI band intensity in gels with MTGase at 0.8 units/g meat paste (Fig. 2(b)). Among all samples, the lowest solubility was observed in the gel with curdlan at 4 g/100 g meat paste and MTGase at 0.4 units/g meat paste, suggesting the greatest amount of nondisulphide covalent cross-linking formed through an MTGaseinduced reaction with the aid of curdlan. This combination also exhibited the greatest breaking force and deformation (Fig. 1), and showed the highest CPI band intensity (Fig. 2(c)). 3.7. Microstructure Scanning electronic microscopy (SEM) was applied to investigate the super-molecular structure, especially the surface roughness

and the macro-pore size and distribution. According to the SEM results in Fig. 4, without curdlan or MTGase, a discontinuous network with large holes and grooves was observed in the control samples (Fig. 4(a)), which was also the gel with the lowest breaking force, deformation (Fig. 1) and water holding capacity (Table 1). When MTGase alone was added (Fig. 4(b)), a more dense gel matrix was observed compared with the control. A similar well-structured and ?ne matrix was also observed in gels with curdlan (Fig. 4(c)). The addition of MTGase most likely constructed the network through intermolecular e-(c-glutamyl) lysine cross-linking in co-operation with protein aggregation via hydrophobic interaction, disulphide bonds and/or other interactions during the heating process (Benjakul, Phatcharat, Tammatinna, Visessanguan, & Kishimura, 2008), while curdlan, due to its good gelling properties, acted as an effective gel enhancer to compensate for the poor texture of the ?sh muscle protein gels. Among all of the samples, the most ordered and densest microstructure was observed in gels with both curdlan and MTGase added, as shown in Fig. 4(d). The combined use of MTGase and curdlan facilitated the formation of stronger gels with greater breaking force and deformation (Fig. 1) by inducing cross-linking of MHC (Fig. 3(c)). Therefore, curdlan and MTGase were very compatible and effectively improved the gelling properties of ?sh muscle protein when used in combination. 4. Conclusions Curdlan can enhance the gelling properties of hairtail muscle protein, as evidenced by the increased breaking force and deformation, WHC, whiteness and more acceptable textural pro?les. The enhancing effect was more pronounced when MTGase was added in combination with curdlan at optimum levels. The combined use of curdlan and MTGase improved the cross-linking of the MHC of the muscle protein and formed a denser gel matrix of

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Y. Hu et al. / Food Chemistry 176 (2015) 115–122 Ghosh Kumar, S., Chakrabarti & Padmanabha (2010). Histological and scanning electron microscopic organization and functional aspects of the surface olfactory epithelium of the freshwater minor carp, Puntius sophore (Hamilton). Proceedings of the Zoological Society (Calcutta), 63, 115–119. Greenberg, C. S., Birckbichler, P. J., & Rice, R. H. (1991). Transglutaminase: Multifunctional cross-linking enzymes that stabilize tissues. FASEB Journal, 5, 3071–3077. Hunt, A., Getty, K. J. K., & Park, J. W. (2009). Roles of starch in surimi seafood: A review. Food Reviews International, 25(4), 299–312. Iglesias-Otero, M. A., Borderías, J., & Tovar, C. A. (2010). Use of Konjac glucomannan as additive to reinforce the gels from low-quality squid surimi. Journal of Food Engineering, 101(3), 281–288. Jiang, S. T., Hsieh, J. F., Ho, M. L., & Chung, Y. C. (2000). Combination effects of microbial transglutaminase, reducing agent, and protease inhibitor on the quality of hairtail surimi. Journal of Food Science, 65, 241–245. Jirawat, Y., & Penprabha, P. (2007). Gel-enhancing effect and protein cross-linking ability of tilapia sarcoplasmic proteins. Journal of the Science of Food and Agriculture, 87(15), 2810–2816. Joseph, D., Lanier, T. C., & Hamann, D. D. (1994). Temperature and pH affect transglutaminase-catalyzed setting of crude ?sh actomyosin. Journal of Food Science, 59, 1018–1023. Karayannakidis, P. D., Zotos, A., Petridis, D., & Taylor, K. D. A. (2008). Physicochemical changes of sardines (Sardina pilchardus) at ?18°C and functional properties of kamaboko gels enhanced with Ca2+ ions and MTGase. Journal of Food Process Engineering, 31(3), 372–397. Kudre, T. G., & Benjakul, S. (2013). Combining effect of microbial transglutaminase and bambara groundnut protein isolate on gel properties of surimi from sardine (Sardinella albella). Food Biophysics, 8(4), 240–249. Lanier, T. C., Carvajal, P., & Yongsawatdigul, J. (2005). Surimi seafood: Surimi gelation chemistry. In J. W. Park (Ed.), Surimi and surimi seafood (2nd ed., pp. 435–489). Boca Raton, FL: CRC Press. Liu, R., Zhao, S. M., Xie, B. J., & Xiong, S. B. (2011). Contribution of protein conformation and intermolecular bonds to ?sh and pork gelation properties. Food Hydrocolloids, 25(5), 898–906. Maeda, I., Saito, H., Masda, M., Misaki, A., & Harada, T. (1967). Properties of gels formed by heat treatment of curdlan, a bacterial b-1, 3 glucan. Agricultural and Biological Chemistry, 31(10), 1184–1188. McIntosh, M., Stone, B. A., & Stanisich, V. A. (2005). Curdlan and other bacterial (1 ? 3)-b-D-glucans. Applied Microbiology and Biotechnology, 68(2), 163–173. Montero, P., & Gómez-Guillén, C. (1996). Thermal aggregation of sardine muscle proteins during processing. Journal of Agricultural and Food Chemistry, 44(11), 3625–3630. Moreno, H. M., Carballo, J., & Borderías, A. J. (2008). In?uence of alginate and microbial transglutaminase as binding ingredients on restructured ?sh muscle processed at low temperature. Journal of the Science of Food and Agriculture, 88(9), 1529–1536. Nishinari, K., & Zhang, H. (2004). Recent advances in the understanding of heat set gelling polysaccharides. Trends in Food Science & Technology, 15(6), 305–312. Pérez-Mateos, M., & Lanier, T. C. (2007). Comparison of Atlantic menhaden gels from surimi processed by acid or alkaline solubilization. Food Chemistry, 101(3), 1223–1229. Rawdkuen, S., Benjakul, S., Visessanguan, W., & Lanier, T. C. (2004). Chicken plasma protein affects gelation of surimi from bigeye snapper (Priacanthus tayenus). Food Hydrocolloids, 18(2), 259–270. Rawdkuen, S., Benjakul, S., Visessanguan, W., & Lanier, T. C. (2007). Effect of chicken plasma protein and some protein additives on proteolysis and gel-forming ability of sardine (Sardinella gibbosa) surimi. Journal of Food Processing and Preservation, 31(4), 492–516. Robinson, H. W., & Hodgen, C. G. (1940). The biuret reaction in the determination of serum protein. I. A study of the condition necessary for the production of the stable color which bears a quantitative relationship to the protein concentration. Journal of Biological Chemistry, 135, 707–725. Tsai, G. J., Lin, S. M., & Jiang, S. T. (1996). Transglutaminase from Streptoverticillium ladakanum and application to minced ?sh product. Journal of Food Science, 61(6), 1234–1238. Uresti, R. M., López-Arias, N., González-Cabriales, J. J., Ram?rez, J. A., & Vázquez, M. (2003). Use of amidated low methoxyl pectin to produce ?sh restructured products. Food Hydrocolloids, 17(2), 171–176. Yin, T., & Park, J. W. (2014). Effects of nano-scaled ?sh bone on the gelation properties of Alaska pollock surimi. Food Chemistry, 150, 463–468. Zhang, R., & Edgar, K. J. (2014). Properties, chemistry and applications of the bioactive polysaccharide curdlan. Biomacromolecules, 15(4), 1079–1096.

the hairtail ?sh muscle. Therefore, hairtail muscle protein may be a promising prospect for applications in the seafood industry, with improved gelling properties when a combination of curdlan and MTGase are added. Acknowledgments The authors acknowledge the ?nancial support of this study by Project 2012BAD38B09. We would like to thank the Core Facilities, Zhejiang University School of Medicine Imaging Facility for our scanning electron microscope work and we would be grateful to Ping Yang for her help of taking SEM images. References
An, H., Weerasinghe, V., Seymour, T. A., & Morrissey, M. T. (1994). Cathepsin degradation of paci?c whiting surimi proteins. Journal of Food Science, 59, 1013–1017. Asagami, T., Ogiwara, M., Wakameda, A., & Noguchi, S. F. (1995). Effect of microbial transglutaminase on the quality of frozen surimi made from various kinds of ?sh species. Fisheries Science, 61(2), 267–272. Bechtel, P. L., & Parrish, J. F. C. (1983). Effects of postmortem storage and temperature on muscle protein degradation: Analysis by SDS gel electrophoresis. Journal of Food Science, 48(1), 294–297. Benjakul, S., Phatcharat, S., Tammatinna, A., Visessanguan, W., & Kishimura, H. (2008). Improvement of gelling properties of lizard?sh mince as in?uenced by microbial transglutaminase and ?sh freshness. Journal of Food Science, 73(6), S239–S246. Benjakul, S., Visessanguan, W., & Pecharat, S. (2004). Suwari gel properties as affected by transglutaminase activator and inhibitors. Food Chemistry, 85(1), 91–99. Benjakul, S., Visessanguan, W., Phatchrat, S., & Tanaka, M. (2003). Chitosan affects transglutaminase-induced surimi gelation. Journal of Food Biochemistry, 27, 53–66. Benjakul, S., Visessanguan, W., Riebroy, W., Ishizaki, S., & Tanaka, M. (2002). Gelforming properties of bigeye snapper, Priacanthus tayenus and P. macracanthus, stored in ice. Journal of the Science of Food and Agriculture, 82, 1442–1451. Cardoso, C., Mendes, R., Vaz-Pires, P., & Nunes, M. L. (2011). Production of high quality gels from sea bass: Effect of MTGase and dietary ?bre. LWT-Food Science and Technology, 44(5), 1282–1290. Careche, M., Alvarez, C., & Tejada, M. (1995). Suwari and kamaboko sardine gels: Effect of heat treatment on solubility of networks. Journal of Agricultural and Food Chemistry, 43(4), 1002–1010. Carmen Gómez-Guillén, M., Montero, P., Teresa Solas, M., & Pérez-Mateos, M. (2005). Effect of chitosan and microbial transglutaminase on the gel forming ability of horse mackerel (Trachurus spp.) muscle under high pressure. Food Research International, 38(1), 103–110. Chakraborty, A., Aranishi, F., & Iwatsuki, Y. (2007). Polymerase chain reactionrestriction fragment length polymorphism analysis for species identi?cation of hairtail ?sh ?llets from supermarkets in Japan. Fisheries Science, 73, 197–201. Chanarat, S., & Benjakul, S. (2012). Comparative study on protein cross-linking and gel enhancing effect of microbial transglutaminase on surimi from different ?sh. Journal of the Science of Food and Agriculture, 92(4), 844–852. Chanarat, S., & Benjakul, S. (2013a). Effect of formaldehyde on protein cross-linking and gel forming ability of surimi from lizard?sh induced by microbial transglutaminase. Food Hydrocolloids, 30(2), 704–711. Chanarat, S., & Benjakul, S. (2013b). Impact of microbial transglutaminase on gelling properties of Indian mackerel ?sh protein isolates. Food Chemistry, 136(2), 929–937. Chen, C., Wang, R., Sun, G., Fang, H., Ma, D., & Yi, S. (2010). Effects of high pressure level and holding time on properties of duck muscle gels containing 1% curdlan. Innovative Food Science & Emerging Technologies, 11(4), 538–542. Chunhua, W., Shuhui, P., Chengrong, W., Xiumei, W., Linin, F., Ronghua, D., et al. (2012). Structural characterization and properties of konjac glucomannan/ curdlan blend ?lms. Carbohydrate Polymers, 89(2), 497–503. Debusca, A., Tahergorabi, R., Beamer, S. K., Matak, K. E., & Jaczynski, J. (2014). Physicochemical properties of surimi gels forti?ed with dietary ?ber. Food Chemistry, 148, 70–76. Funami, T., Yada, H., & Nakao, Y. (1998). Thermal and rheological properties of curdlan gel in minced pork gel. Food Hydrocolloids, 12(1), 55–64.


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