Effect of superchilling storage on quality characterizes and physical properties of surimi from Silver carp (Hypophthalmichthys molitrix) as compared with cooling freshness preservation
Liu Qian, Han Jian-chun*, Li Shuang-mei, Li Jing, Chen Qian
College of Food Science
Northeast Agricultural University
email@example.com (J. Han)
Abstract—Quality characterizes of surimi of silver carp at two different conditions of superchilling and cooling freshness preservation. The pH value, TBARS value, protein soubility and ATPase activity were studied. The result showed that pH value and TBARS value increased with the increasing of the storage time (P < 0.05), protein soubility and both ATPase activity decreased with the increasing of the storage time (P < 0.05). In general, superchilling is a good way to preserve freshness of fresh products and the raw material before processing, and also could have great effect on improving the quality characterizes of surimi and prolong its shelf life.
In China, freshwater fishes account for about 45% of the total aquacultural production, and have reached more than 18 million tons . Silver carp (Hypophthalmichthys molitrix) is one of the main freshwater fish species, and it is the lowest-cost freshwater fish produced commercially. Surimi is stabilised myofibrillar proteins concentrated through heading, gutting, mincing, washing, and dewatering . Their distribution sphere and storage period are very limited. Subsequently, although there is high production, the commercial value of their fresh fish is low. It is therefore essential that adequate preservation technologies are applied to maintain its safety and quality .
Superchilling is one method that can be used to maintain food products at a low temperature. Superchilling implies temperatures in the borderline between chilling and freezing. Superchilling is a process by which the temperature of a food product is lowered to 1-2 ℃ below the initial freezing point of the product . The advantages of temperatures below the freezing point were in prolonging the useful storage life of surimi and in discouraging microbial . At superchilling temperatures, microbial activity is reduced and most bacteria are unable to grow. In addition to microbial growth, enzymatic activities are important for the determination of shelf -life and quality of surimi . Superchilling gives the food product an internal ice reservoir so that there is no need for external ice around the product during transportation or storage for shorter periods, however, for long term superchilled storage, refrigerated storage at superchilled temperatures will be needed . Studies on superchilling have shown extended shelf life of food products compared to conventional chilling [8-10]. And superchilling results in better quality, and extends shelf life of stored food 1.5 to 4 times compared to conventional chilling.
Superchilling processing technology has shown several advantages on the quality of food products, for example had lower bacterial counts compared to the corresponding chilled fillets . Superchilling was found to be a promising method for storing raw material before salting, slowing down biochemical quality degradation while at the same time the degree of protein denaturation was low and the degree of structural damage was less than in frozen storage .
E-mail address: firstname.lastname@example.org (J C. Han).
This study was supported by Program for Innovative Research Team of Northeast Agricultural University (Grant No: CXZ011-4) Generally, superchilling has emerged as a potential method for extending shelf life, most of the studies on superchilling have focused on microbiology, sensory analysis and spoilage indicators and how they influence quality parameters, such as loss of juiciness and negative textural changes. But still there is a need to know more about how the degree of superchilling affects biochemical changes, such as protein denaturation, enzymatic activity and liquid retention. The aim of this study was to investigate the effects of a superchilling process on selected quality parameters of surimi in order to prolong shelf life without freezing treatment.
Ⅱ. MATERIALS & METHODS
Silver carp (Hypophthalmichthys molitri) was purchased at DaRunFa market in Haerbin, and was transported to the laboratory alive. The beheaded and gutted silver carp was washed with water, then filleted (only white muscle was used) by hand and washed. Fillets were then minced. The washing of the minced meat was performed in wash tanks below 10℃ water using a fish mince to solution ratio of 1:4 (w/v) 4 times (twice with distilled water and then twice with 0.3% NaCl water solution, each time for 15 min). Final dewatering was carried out in a pressing machine. And then the surimi was stored in two different temperatures, which are -1℃and 4℃. Moisture and total nitrogen contents of surimi were determined using standard oven and Kjeldahl procedures, and three replications were taken. Moisture and protein contents of silver carp surimi were 74.10% and 14.58%.
Preparation of myofibrillar protein
Myofibrillar protein was prepared from surimi according to the procedure of Xia, with some modification . The final pellet (myofibrillar protein) was stored in a tightly capped bottle, kept on ice, and utilized within 24 h.
The pH was determined using a pH meter (pHS-25, Shanghai, China) on 10g homogenated surimi samples in 100 ml of distilled water. Three measurements were performed for each lot.
Thiobarbituric acid-reactive substances (TBARS)
Lipid oxidation was evaluated by TBARS according to the method of Sinnhuber and Yu, with slight modification . After reaction with TBA, the sample solution was mixed with chloroform (1:1 ratio, v/v), vortexed, and subsequently centrifuged at 1800g for 10 min. The TBARS value, expressed as mg of malonaldehyde/kg of muscle sample, was calculated using the following equation:
TBARS(mg/kg) = (A532/Ws) * 9:48
where A532 was the absorbance (532 nm) of the assay solution, Ws was the meat sample weight (g), and “9.48” was a constant derived from the dilution factor and the molar extinction coefficient of the red, TBA reaction product.
Protein solubility was determined as described by Benjakul, Visessanguan, Thongkaew, and Tanaka, with some modification . To a 2 g sample, 18 ml of 0.6 M KCl was added and the mixture was homogenized for 30 s at a speed of 13,000 rpm. The homogenate was stirred at room temperature for 4 h using a magnetic stirrer (IKA-18, WERKE, Staufen, Germany), followed by centrifuging at 8500g for 30 min at 4℃ using a refrigerated centrifugation (Sorvall, Norwalk, CT, USA). To 10 ml of the supernatant, cold 50% (w/v) TCA was added to obtain the final concentration of 10%. Theprecipitate was washed with 10% TCA and solubilized in 10 ml of 0.5 M NaOH. The muscle was also solubilized using 0.5 M NaOH to obtain total protein amount. Protein content was determined using the Biuret method. Protein solubility was expressed as percentage of soluble protein relative to total proteins in the muscle excluding connective tissues.
ATPase activities of MPI were determined according to the method of Wells, Werber, and Yount, with slight modification . Briefly, MPI samples were diluted to a 3.0 mg/mL protein concentration. Aliquots of 0.2 mL of the protein suspension were mixed with 2.0 mL of the reaction solution (for Ca-ATPase: 7.6 mM ATP, 15 mM CaCl2, 150 mM KCl, 180 mM Tris-HCl, pH 7.4; for K-ATPase: 7.6 mM ATP, 300 mM KCl, 5.0 mM EDTA, 180 mM Tris-HCl, pH 7.4). After reaction at 25 ℃ for 10 min, 1.0 mL of 10% trichloroacetic acid was added to stop the reaction. The mixture was subsequently centrifuged at 2500g for 5 min, and 1 mL of the supernatant was reacted with 3.0 mL of 0.66% ammonium molybdate in 0.75M sulfuric acid. One half-milliliter of freshly prepared 10% FeSO4 in 0.15M sulfuric acid was then added, and the mixture was allowed to react for 2 min for color development. The absorbance of the liberated inorganic phosphate was read at 700 nm to determine the Ca2+-ATPase and K+-ATPase activities. Results were expressed as lmol phosphate/mg protein. A series of NaH2PO4 solutions (0.0-1.0mM) were used to prepare the standard curve for phosphate calculation.
G. Statistical analysis
The experiment was replicated twice with at least triplicate analyses. Data were analyzed by using the General Linear Models procedure of Statistix 8.1 software package (Analytical Software, St. Paul, MN) for microcomputer. Analysis of variance (ANOVA) was done to determine the significance of the main effects. Significant differences (P < 0.05) among means were identified using Turkey procedures.
In our research, we concluded that the pH of all sample were increased with the storage times extend (P < 0.05). During superchilling storage the pH value of surimi increased from the initial 5.90–6.40 to 5.90–7.75 (Fig. 1), which is in agreement with results from many studies. However, this increase was delayed in superchilled (day 16–20) compared to cooling freshness preservation (day 8–12). Fig. 2 shows the results of TBARS value of all samples increased during 20 days of chilling storage at 4℃ and supperchilling storage at -1℃ (P <0.05). However, Lipid oxidation could have taken place since higher amounts of MDA (mg/kg) were found in meat stored under chilling conditions for 12 days than in meat stored under supperchilling conditions for 20 days (p < 0.05). TBARS
value in this portion tended to increase to a higher extent during storage, which indicated an increase in lipid oxidation and protein oxidation. This was possibly due to the release of oxidative enzymes and prooxidants from various ruptured cellular organelles .
Fig. 2 Influence of TBARS value of silver carp surimi under different storage conditions
Solubility of myofibrillar protein
Fig. 3 Influence of protein solubility of silver carp surimi under different storage conditions
Solubility of myofibrillar proteins is of a primary importance for the manufacture of processed muscle foods, including comminuted, restructured and formed meats. Changes in protein solubility were observed throughout the storage as shown in Fig. 3. Protein solubility of all samples decreased continuously throughout times of both storage conditions (P < 0.05). However, the protein solubility of cooling freshness preservation samples was more lower than supperchilling samples at the same stored time (P < 0.05), and the lowest solubility was found at the end of storage, which was 55.38﹪for 12 days stored under cooling freshness preservation condition and 79.32﹪ for 12 days under supperchilling condition, and for 20 days, Its decerased to 58.14﹪. This could be an indication that superchilling does not result in a high degree of protein denaturation compare to cooling freshness preservation during the same storage time. Jiang, Wang, and Chen reported that denaturation and aggregation of muscle proteins are associated with the formation of disulfide bond .
Changes of ATPase activity
Fig. 4 Influence of Ca2+-ATPase (a) and K+-ATPase (b) of myofibrillar proteins of silver carp surimi under different storage conditions
The activities of both Ca2+-ATPase and K+-ATPase of myofibrillar proteins extracted from surimi subjected to different condictions are depicted in Fig. 4a and Fig. 4b. ATPase activity was gradually lowered with increasing storage times under both conditions (P < 0.05). Ca2+-ATPase and K+-ATPase decreased 44.0% and 77.3%, respectively after 12 days storage times under cooling freshness preservation conditions ,but under supperchilling conditions, Ca2+-ATPase and K+-ATPase decreased 44.0% and 77.3%, respectively after 20 days storage times. And K+-ATPase changes proceeded more rapidly than the Ca2+-ATPase decay. The results indicated that myosin underwent some changes in native conformation after chilling and supperchilling stored. Benjakul, Visessanguan, Thongkaew, and Tanaka reported that Ca2+-ATPase activity is used as an indicator for the integrity of myosin molecules . The loss in ATPase activity was possibly associated with the oxidation of sulfhydryl groups on the myosin globular head .
Results from this study indicated that superchilling at -1℃ could significantly extended shelf life compared to traditional cooling freshness preservation at 4℃. Supperchilling could decreased the protein oxidation, discoloration, and changed structure of myofibrillar protein, loss of myofibrillar protein functionality. Further research is required to design optimal process and storage conditions, thus, we can find an effective superchilling process to satisfied commercial need for different products with various shape and size, and at the same time preserve the premium quality of the product.
This study was supported by Program for Innovative Research Team of Northeast Agricultural University (Grant No: CXZ011-1).
 Y. Q. Luo, H. X. Shen, D. D. Pan, and G. H. Bu, “Gel properties of surimi from silver carp (Hypophthalmichthys molitrix) as affected by heat treatment and soy protein isolate,” FoodHydrocolloids, vol. 22, pp. 1513-1519, 2008.
 J. W. Park, and T. M. Lin, “Surimi: Manufacturing and evaluation,” In J. W. Park (Ed.), Surimi and surimi seafood (pp. 33-106), 2005. Boca Raton: CRC Press Taylor and Francis Group.
 T. Aymerich, P. A. Picouet, and J. M. Monfort, “Decontamination technologies for meat products,”Meat Science, vol. 78, pp. 114-129, 2008.
 A. S. Duun, and T. Rustad, “Quality changes during superchilled storage of cod (Gadusmorhua) fillets,” Food Chemistry, vol. 105, pp. 1067-1107, 2007.
 T. Morkore, A. A. Hansen, E. Unander, and O. Einen, “Composition, liquid leakage, and mechanical properties of farmed rainbow trout: Variation between fillet sections and the impact of ice and frozen storage,” Journal of Food Science, vol. 67, pp. 1933-1938, 2002.
 L. Gram, and H. H. Huss, “Microbiological spoilage of fish and fish products,” InternationalJournal of Food Microbiology, vol. 33, pp. 121-137, 1996.
 F. Bin, and P. L. Theodore, “Shelf-life prediction: theory and application,” Food Control, vol.4, pp. 125-133, 1993.
 A. S. Duun, A. K. T. Hemmingsen, A. Haugland and T. Rustad, “Quality changes during superchilled storage of pork roast,” LWT-Food Science and Technology, vol. 41, pp. 2136-2143, 2008.
 B. H. N. Duy, S. Arason, K. A. Thorarinsdóttir, “Effects of dry ice and superchilling on the quality and shelf life of arctic charr (Salvelinus alpinus) fillets,” International Journal of Food Engineering, vol. 3, pp. 1-27, 2007.
 K. Fernández, E. Aspé, and M. Roeckel, “Shelf-life extension on fillets of Atlantic salmon (Salmo salar) using natural additives, superchilling and modified atmosphere packaging,” Food Control, vol. 20, pp. 1036-1042, 2009.
 L. D. Kaale, T. M. Eikevik, T. Rustad and K. Kolsaker, “Superchilling of food: A review,” International Journal of Food Engineering, vol. 107, pp. 141-146, 2011.
 L. Gallart-Jornet, T. Rustad, J. M. Barat, P. Fito, and I. Escriche, “Effect of superchilled storage on the freshness and salting behavior of Atlantic salmon (Salmo salar) fillets,” Food Chemistry, vol. 103, pp. 1268-1281, 2007.
 X. F. Xia, B. H. Kong, Q. Liu, and J. Liu, “Physicochemical change and protein oxidation in porcine longissimus dorsi as influenced by different freeze–thaw cycles,” Meat Science, vol. 83, pp. 239-245, 2009.
 R. O. Sinnhuber, and T. C. Yu, “The 2-thiobarbituric acid reaction, an objective measure of the oxidative deterioration occurring in fats and oils,” Journal of Japan Oil Chemists Society, vol. 25, pp. 259-267, 1977.
 S. Benjakul, W. Visessanguan, C. Thongkaew, and M. Tanaka, “Comparative study on physicochemical changes of muscle proteins from some tropical fish during frozen storage,” FoodResearch International, vol. 36, pp.787-795, 2003.
 J. A. Wells, M. M. Werber, and R. G.Yount, “Inactivation of myosin subfragment one by cobalt(II)/cobalt(III) phenanthroline complexes 2. Cobalt chelation of two critical SH groups,” Biochemistry, vol. 18, pp. 4800-4805, 1979.
 L. H. Yu, E. S. Lee, and J. Y. Jong, “Effects of thawing temperature on the physicochemical properties of pre-rigor frozen chicken breast and leg muscles,” Meat Science, vol. 71, pp. 375-382, 2005.
 S. T. Jiang, F. J. Wang, and C. S. Chen, “Properties of actin and stability of the actomyosin reconstituted from milkfish (Chanos chanos) actin and myosin,” Journal of Agriculture and FoodChemistry, vol. 37, pp. 1232-1235, 198