凝膠化時間和卵清蛋白添加量對竹莢魚魚糜凝膠特性的影響

王雨生，陳海華*

(1.青島農(nóng)業(yè)大學(xué)食品科學(xué)與工程學(xué)院，山東 青島 266109；2.青島農(nóng)業(yè)大學(xué)學(xué)報編輯部，山東 青島 266109)

Proteolytic degradation of myofibrillar proteins has an adverse effect on gel-forming properties of surimi. The breakdown of myofibrillar proteins inhibits the development of three-dimensional gel network[1]. The gel-softening phenomenon or “modori” observed at temperatures above 50―70 ℃ was attributed to myosin hydrolysis by some heatactivated proteinases[2-3]. Gel softening varies with species, but it is generally caused by two major groups of proteinase, including cathepsin and heat-stable alkaline proteinases[4]. To alleviate the problems associated with protein degradation caused by endogenous proteinases, inhibitors and other additives have been used in surimi to improve the physical properties of surimi gels. Egg white protein (EWP), beef plasma proteins (BPP), porcine plasma protein (PPP), whey proteins concentrate, potato powder, etc. could be used as food-grade inhibitors[5-11]. These additives are often used to improve gelling characteristics of surimi with a high activity of endogenous heat-activated proteinase, such as from arrowtooth flounder, Pacific whiting, Alaska pollock, Bigeye snapper and Lizardfish[5,10]. Among all proteinase inhibitors, BPP is the most effective surimi gel enhancer resulting from both proteinase inhibitory activity and protein cross-linking activity[12]. However, the discoloration in surimi gel[13]and the association of bovine spongiform enceohalopathy (BSE) or mad cow disease has led to the prohibition of BPP in surimi. Due to the differences in proteinases among fish species, different proteinase inhibtors are needed to alleviate the proteolysis.

Since the access to the Alaska pollack sources has been limited in China, relative effort has been made to produce acceptable surimi from pelagic fishes, such as mackerel and sardine. Horse-mackerel (Trachurus trachurus) is one of the major fish consumed in China, but it still has not been commercialized to surimi-based products (eg. kamaboko) due to high lipid content, pigment and low gel-forming ability[14]. In our previous studies, horse-mackerel surimi showed the highest gel properties when setting at 30 ℃ for 5 h or 40 ℃ for 4 h, which was higher than that of setting at 20 ℃ for 36 h[15]. Horsemackerel surimi showed gel weakening properties when heated from 50 ℃ to 70 ℃[15]. Effects of whey protein concentration, soybean protein isolate, gluten powder, peanut protein concentration on the gel properties of horse-mackerel was studied by Chen Haihua et al.[16-17]. Moreover, less information regarding the inhibitory activity of EWP on the horse-mackerel surimi has been reported previously. The objective of this research was to investigate the effect of setting condition and egg white protein content on the gel properties of surimi from frozen horse-mackerel (Trachurus trachurus).

1 Materials and Methods

1.1 Materials

Frozen horse-mackerel (weight of 215 g, K-value of 12%, total volatile basic nitrogen (TVB-N) of 13 mg/100 g) was obtained from Zhoushan Xingye Frozen Food Co. Ltd., (Zhejiang, China), which was kept at ―30 ℃ not more than 2 weeks. Egg white protein (EWP, protein content of 88%, Food grade) was gifted from Qingdao Tianxin Food Additive Co. Ltd., (Qingdao, China).

1.2 Frozen surimi preparation

Surimi was prepared according to the method of Benjakul et al.[8]. Frozen horse-mackerel were thawed in running water (20 ℃) until the core temperature reached 0―2 ℃. Fish were headed, gutted and washed in turn. The flesh was removed manually and minced into the uniformity with the diameter of 1 mm. The mince was then washed with cold water (4 ℃, containing 0.3% NaHCO3) at a mince and water ratio of 1∶3 (m/m). The mixture was stirred slowly for 10 min and washed mince was filtered with a layer of nylon screen. The washing process was repeated twice with cold water only. Finally the washed mince was dewatered by centrifuging at 4500 × g at 4 ℃ for 15 min using a TDL-5-A centrifuge (Shanghai Anting, China). Sorbitol (4%) and sucrose (4%) were added to the dewatered mince as cryprotective agents and then mixed for 5 min in a silent cutter below 10 ℃. These surimi were quickly frozen at ―30 ℃ for 5 h and were kept frozen at ―20 ℃ until used in the experiments. Moisture and protein contents of horse-mackerel surimi were detected to 78% and 13%, respectively.

1.3 Effect of EWP on gel properties of surimi

1.3.1 Surimi gel preparation

Frozen surimi was thawed at 20 ℃ for 1 h and then was cut into small pieces (about 2.5 cm cubes). The surimi cubes were chopped at 2400 r/min for 5 min in a Stephan vacuum cutter (Model UMC-5, Germany) with a circulating chiller to maintain the temperature less than 10 ℃. Salt (2.5%, m/m) and ice water (final protein concentration of surimi pastes was adjusted by adding ice water according to the experimental design) was added, while chopping was continued for another 3 min. EWP was added at different concentrations (0%, 0.5%, 1%, 5% and 10%, m/m, respectively). The mixture was chopped for 4 min. Then, the surimi paste was stuffed into polyvinylidine casing with a diameter of 25 mm and both ends were sealed tightly. Kamaboko gel was prepared by incubating surimi paste in a temperature-controlled water bath (Guohua Electrical Appliance Co. Ltd., China) at 30 ℃ for 0, 1, 3, 5, 7 h and 9 h, respectively, followed by heating at 90 ℃ for 20 min. Modori gel was prepared by incubating surimi paste at 50 ℃ for 0, 0.5, 1, 2, 3, 5, 7 h and 9 h, respectively, followed by heating at 90 ℃ for 20 min. After heating, all gels were immediately cooled in iced water for 30 min and stored at 4 ℃ overnight prior to the next analysis.

1.3.2 Texture analysis

Texture analysis of surimi gels was carried out using a TA-XT2i texture analyser (Stable Micro System, Surrey, UK). Gels were equilibrated at room temperature (25―30 ℃) before analysis. 5 cylindrical samples (25 mm in length) were prepared and tested. Breaking force (strength) and deformation (cohesiveness/elasticity) were measured by the texture analyser equipped with a spherical plunger (5 mm diameter), with a depression speed of 60 mm/min and 60% compression. All measurements were repeated 5 times. Results were reported as (±s).

1.3.3 Determination of whiteness

5 Gel samples from each treatment were subjected to whiteness measurement using a WSC-5 color difference meter (Shanghai Presision & Scientific Instrument Co. Ltd., China). L* (lightness), a* (redness/greenness) and b* (yellowness/blueness) were measured and whiteness was calculated using the following equation (1)：

All measurements were repeated 5 times. Results were reported as (±s).

1.3.4 Determination of expressible water

Expressible water was measured as follows： cylindrical gel samples were cut to a thickness of 5 mm, weighed (m1) and placed between three pieces of Whatman paper (No.1) at the bottom and two pieces of paper on the top. A standard weight (5 kg) was placed on the top of the sample for 2 min, then the sample was removed from the papers and weighed again (m2). Expressible water was calculated with the following equation (2) and expressed as percentage of sample weight：

All measurements were repeated 10 times. Results were reported as (±s).

1.3.5 Microstructure of surimi gels

Surimi gels were subjected to microstructure determination. Surimi gels (5 mm×5 mm× 3 mm) were fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.2 for 2 h at room temperature. Fixed specimens were dehydrated in graded ethanol solutions with serial concentrations of 50%, 60%, 70%, 80%, 90% and 100%, respectively. Specimens were coated with a gold layer and observed with KYKY-2800B scanning electronic microscope (Chinese Academy of Sciences, Beijing, China).

1.4 Statistical analysis

Data were subjected to analysis of variance (ANOVA) by SAS8.2 software. Comparison of means was carried out by Duncan’s multiple-range test.

2 Results and Ana1ysis

2.1 Effect of EWP on textural properties of surimi gels

Regardless of heating conditions, breaking force and deformation increased with the addition of EWP increased (Fig.1A and 1B). For the control gels (without EWP), the highest values were found in kamaboko gel (30 ℃/90 ℃), while the lowest values of breaking force and deformation were observed with modori gel (50 ℃/90 ℃). It indicated gel strengthening in kamaboko gel and weakening in modori gel, respectively. The reason might be that when setting at 40 ℃, endogenous transglutaminase induced the cross-linking of MHC via nondisulfide bond[16], whereas endogenous heat-stable proteinase caused the degradation of MHC, especially at 60 ℃[18].

Fig.1 Effect of EWP on breaking force and deformation of horsemackerel surimi

With the same amount of EWP added, breaking force and deformation of kamaboko gels increased as the setting time prolonged to some extent (P＜0.05) (Fig.1A). The highest breaking force and deformation of kamaboko gels with addition of 0.5%, 1%, 5% and 10% EWP were observed with settings at 5 h and 3 h, respectively. However, the breaking force and deformation decreased with settings more than the above setting time. Dominated action of endogenous transglutaminase was ascribed to the increasing effect of gel properties as setting at 30 ℃. But endogenous heat-stable proteinase might be activated even setting at 30 ℃ for prolonging setting time more than 5 h. For the same setting time, breaking force of kamaboko gels increased as the amount of EWP added increased (P＜0.05) (Fig.1A), and the highest value was observed at a level of 10% EWP. Deformation of kamaboko gels also increased as the amount of EWP less than 5%, and the highest value was observed at a level of 1% EWP, respectively. However, deformation decreased with the addition of more than 5% EWP. At a level of 1% EWP, breaking force and deformation of kamaboko gels were 665 g and 9.94 mm, which increased by 24% and 37% compared to the control (P＜0.05) (kamaboko gel without EWP), respectively.

Breaking force and deformation of modori gels with 0.5% or 1% EWP setting for 30 min reached the maximum, then decreased with setting time prolonged (P＜0.05) (Fig.1B). Breaking force and deformation of modori gels with 5% or 10% EWP setting for 30 min was also observed the highest value, remaining stable from 30 min to 5 h (P＞0.05), then decreased for more than 5 h (P＜0.05). These results indicated that setting time less than 30 min had positive effect on the gel properties of horse-mackerel surimi with setting at 50 ℃. The reason might be that endogenous transglutaminase still showed activity and endogenous heat-stable proteinase was inactivated due to the setting time was too short when setting at 50 ℃. Whereas the gel properties decreased because that endogenous transglutaminase was inactivated and endogenous heat-stable proteinase was activated with the setting time prolonged when setting at 50 ℃. At a level of 10% EWP and setting for 30 min, breaking force and deformation of modori gels was 753 g and 6.90 mm, respectively, increased by 168% and 15% compared to those of the control (modori gel without EWP). They indicated that EWP could inhibit the modori of horse-mackerel surimi setting at 50 ℃ to some extent. EWP showed the inhibition efficacy with increasing addition of EWP. The result was in agreement with those reported by others. Yuwathida[19]reported that the breaking force and deformation of surimi gels from bigeye snapper increased as the amount of EWP increased. Morrissey et al.[1]found that EWP inhibited proteolysis in Pacific whiting surimi gel. Benjakul et al.[8]reported that addition of EWP improved gel properties of lizardfish. Addition of 2% EWP increased the gel strength of arrowtooth flounder and pollock surimi[9]. EWP showed inhibitory effect on preventing myosin degradation in Pacific whiting muscle[20]. Lu et al.[21]found that EWP was functioned as binder in meat. Thus, the addition of EWP presumably contributed to gel strengthening via proteinase inhibition and its binding effect.

2.2 Effect of EWP on whiteness of surimi gel

Table 1 Whiteness of kamaboko gel and modori gel with the addition EWP setting for different time (±s)

Table 1 Whiteness of kamaboko gel and modori gel with the addition EWP setting for different time (±s)

Note： Capital letters indicated significant differences in the same column (P＜0.05), while lowercase letters indicated significant differences in the same line (P＜0.05).

Setting temperature/℃Setting time/h Addition of EWP/%0.00.51.05.010.0 30 1.0 82.54±0.12Aa 81.69±0.13ABab 81.16±0.07ABb 78.35±0.15Ac 76.94±0.03Ad 3.0 82.32±0.01Aa 81.69±0.11ABab 81.49±0.10ABb 77.66±0.05Ac 77.14±0.24Ac 5.0 81.68±0.25ABab 81.88±0.15ABa 81.37±0.21ABb 77.72±0.24Av 76.89±0.05Ac 7.0 81.67±0.05ABa 81.34±0.11Ba 81.08±0.09Aa 77.45±0.08ABb 76.89±0.08Ab 9.0 81.63±0.03ABab 82.38±0.07Aa 81.10±0.15Ab 77.38±0.16ABc 76.79±0.12Ac 0.5 80.53±0.15Ba80.10±0.14Ca 79.63±0.18Ba 77.83±0.16Ab 75.65±0.15Bc 1.0 80.14±0.11Ba80.22±0.23Ca 79.63±0.07Ba 77.35±0.08ABb 76.15±0.11ABc 2.0 80.91±0.12Ba 80.36±0.11BCab 79.54±0.08Bb 77.17±0.18ABc 75.69±0.05Bd 3.0 80.37±0.12Bab 81.06±0.08BCa 79.48±0.11Bb 76.63±0.09Bc 76.23±0.13ABc 5.0 81.83±0.11ABa 81.11±0.21Bab 79.38±0.13Bb 76.13±0.20Bc 75.74±0.11Bc 7.0 81.48±0.06ABa 81.35±0.15Ba 79.58±0.15Bb 76.15±0.15Bc 74.91±0.06BCd 9.0 81.17±0.12Ba81.23±0.11Ba 79.22±0.23Bb 76.83±0.11Bc 74.39±0.03Cd 50

The whiteness of kamaboko gels and modori gels with EWP at different levels is shown in Table 1. Regardless of heating condition for surimi gel preparation, no changes in whiteness were observed with the same amount of EWP (P＞0.05). However, the whiteness of kamaboko gels and modori gels with EWP decreased to some extent, compared with the control (P＜0.05) (without EWP). For the gels of surimi from horse-mackerel, the addition of less than 1% EWP showed no effect on whiteness (P＞0.05), while addition of more than 1% EWP resulted in a decrease in whiteness (P＜0.05). Since EWP is predominantly light cream-colored in nature and higher amount of EWP resulted in the decrease of gel properties, it might reduce the whiteness of surimi gel slightly, especially when a higher amount is used. Thus, EWP at a low level (adding amount is less than 1%) can be used as a protein additive in surimi to improve the gel strength without causing marked changes in whiteness. The result was not in agreement with that reported by Benjakul et al.[8], who found that EWP adding had no effect on whiteness of lizardfish surimi gels. We guess the disagreement might be caused by different sources of EWP or the different species of the fish used in the experiment.

2.3 Effect of EWP on expressible water of surimi gel

Fig.2 Effect of EWP on expressible water of kamaboko gel (A) and modori gel (B) from horse-mackerel

Expressible water of kamaboko gels was lower than those of modori gels under the same condition (P＜0.05) (Fig.2). It indicated that protein matrix was formed and water was imbibed regularly throughout gel network when setting at 30 ℃, while poor gel matrix with low water holding capacity when setting at 50 ℃.

With the same amount of EWP, the expressible water of kamaboko gels from horse-mackerel decreased slightly with setting time prolonged (P＞0.05) (Fig.2A). While at the same setting time, the expressible water of kamaboko gels decreased significantly as addition of EWP increased (P＜0.05) (Fig.2A). Highest expressible water was found in modori gels without EWP addition (Fig.2B). With the same amount of EWP, the expressible water of modori gels increased as setting time increased (P＜0.05). Modori gels with EWP added exhibited expressible water lower than those without EWP. Expressible water of modori gels decreased to a higher extent as higher amount of EWP was added. Expressible water for kamaboko gels without EWP was 14.1% and 29.7%, respectively, higher than modori gels with 1% and 10% EWP added, respectively. The result indicated that dried EWP was able to absorb and retain water effectively. The lower expressible water content obtained in the samples added with EWP is indicative for the higher water binding property of protein gel matrix. Therefore, EWP could prevent the degradation of muscle proteins, leading to the well-ordered network with high water holding capacity.

2.4 Effect of EWP on microstructure of surimi gels

Fig.3 Electron microscopic image of surimi gel from horse-mackerel with or without EWP(×1500)

The microstructures of kamaboko gels and modori gels from horse-mackerel, without and with EWP, were visualized by SEM with magnification of 1500×, as shown in Fig.3. The microstructure of kamaboko gel without EWP showed a wellstructured matrix with highly interconnected network of strands and fine three-dimensional protein network (Fig.3A). That may cause more resistance to applied stress and greater water-holding capacity. The protein network of kamaboko gel containing 1% EWP seemed to be more compact with smaller clusters of aggregated protein than that of the control gel (Fig.3B). Thus, the breaking force, deformation and water holding capacity of kamaboko gel with 1% EWP were better than those of the control samples. Modori gels without EWP showed a structure with aggregates of sparse packed spherical proteins, arranged in clusters (Fig.3C). This was caused by the cross-linking or aggregation of protein fragments resulting from activity of proteinases. Large cavities were observed at the surface of the gel structure, probably caused by the haphazard globule aggregation. When 10% EWP was added, the network fibers became more clearly defined (Fig.3D). The regularly ordered and fine fibrillar structures in such surimi gels are more likely to be responsible for the higher breaking force and deformation.

3 Conclusion

Regardless of heating conditions, the addition of EWP within some extent resulted in the increased breaking force, deformation and gel strength of surimi gels. No changes in whiteness were observed with gels added with EWP less than 1%, while addition of EWP more than 5% resulted in a lower whiteness. Expressible water of kamaboko gels was lower than those of modori gels under the same condition. Expressible water of modori gels decreased to higher extent as higher amount of EWP was added. The microstructure of surimi gels generally became denser with the addition of EWP.

[1] MORRISSEY M T, WU J W, LIN D D, et al. Effect of food grade protease inhibitor on autolysis and gel strength of surimi[J]. Journal of Food Science, 1993, 58： 1050-1054.

[2] JIANG S T. Enzymes and their effects on seafood texture[M]. New York： Marcel Dekke, 2000： 411-450.

[3] VISESSANGUAN W, AN H. Effects of proteolysis and mechanism of gel weakening in heat-induced gelation of fish myosin[J]. Journal of Agricultural and Food Chemistry, 2000, 48： 1024-1032.

[4] AN H, PETERS M Y, SEYMOURS T A. Roles of endogenous enzymes on surimi gelation[J]. Trends in Food Science and Technology, 1996, 7： 321-327.

[5] AKAZAWA H, MIYAUCHI Y, SAKURADA K, et al. Evaluation of protease inhibitors in Pacific whiting surimi[J]. Journal of Aquatic Food Product Technology, 1993, 2： 79-95.

[6] BENJAKUL S, VISESSANGUAN W. Pig plasma protein： potential use as proteinase inhibitor for surimi manufacture; inhibitory activity and the active components[J]. Journal of the Science of Food and Agriculture, 2000, 80： 1351-1356.

[7] BENJAKUL S, VISESSANGUAN W, SRIVILAI C. Gel properties of bigeye snapper (Priacanthus tayenus) surimi as affected by setting and porcine plasma proteins[J]. Journal of Food Quality, 2001, 24： 453-472.

[8] BENJAKUL S, VISESSANGUAN W, TUEKSUBAN J, et al. Effect of some protein additives on proteolysis and gel-forming ability of lizardfish (Saurida tumbil)[J]. Food Hydrocolloids, 2004, 18： 395-401.

[9] LEE J J, TZENG S S, WU J, et al. Inhibition of thermal degradation of mackerel surimi by pig plasma protein and L-kininogen[J]. Journal of Food Science, 2000, 65： 1124-1129.

[10] RAWDKUEN S, BENJAKUL S. Whey protein concentrate： autolysis inhibition and effects on the gel properties of surimi prepared from tropical fish[J]. Food Chemistry, 2008, 106： 1077-1084.

[11] RAWDKUEN S, BENJAKUL S, VISESSANGUAN W, et al. Chicken plasma protein： proteinase inhibitory activity and its effect on surimi gel properties[J]. Food Research International, 2004, 37： 156-165.

[12] KANG I S, LANIER T C. Bovine plasma protein functions in surimi gelation compared with cysteine protease inhibitors[J]. Journal of Food Science, 1999, 64： 842-846.

[13] REPPOND K D, BABBITT J K. Protease inhibitors affect physical properties of arrowtooth flounder and walleye pollock surimi[J]. Journal of Food Science, 1993, 58： 96-98.

[14] CHEN H H. Decoloration and gel-forming ability of horse mackerel mince by air-flotation washing[J]. Journal of Food Science, 2002, 67： 2970-2975.

[15] CHEN Haihua, XUE Changhu. Effects of heating conditions on gel properties of horse mackerel surimi[J]. Food Science, 2010, 31(1)： 6-13.

[16] CHEN Haihua, XUE Changhu. Effect of whey protein concentration on suwari and modori phenomena of horse-mackerel surimi[J]. Food Science, 2010, 31(11)： 25-31.

[17] CHEN Haihua, XUE Changhu. Effects of three kinds of non-muscle proteins on the gel properties of horse-mackerel surimi[J]. Food Science, 2010, 31(13)： 31-35.

[18] BENJAKUL S, VISESSANGUAN W, LEELAPONGWATTANA K. Purification and characterization of heat-stable alkaline proteinase from bigeye snapper (Priacanthus macracanthus) muscle[J]. Comparative Biochemistry and Physiology： Part B, 2003, 134： 579-591.

[19] YUWATHIDA K. Improvement of gel quality of surimi from bigeye snapper[D]. Songkla： Prince of Songkla University, 2001.

[20] CHANG-LEE M V, PACHECO-AGUILAR R, CRAWFORD D L, et al. Proteolytic activity of surimi from Pacific whiting (Merluccius productus) and heat-set gel texture[J]. Journal of Food Science, 1989, 54： 1116-1119.

[21] LU G H, CHEN T C. Application of egg white and plasma powders as muscle food binding agents[J]. Journal of Food Engineering, 1999, 42： 147-151.