Effect of freezing and frozen storage on the properties of actomyosin from pinkperch (Nemipterus japonicus)

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Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3096-3112 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 06 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.706.364 Effect of Freezing and Frozen Storage on the Properties of Actomyosin from Pinkperch (Nemipterus japonicus) K. Rathnakumar1* and B. A. Shamasundar2 1 Department of Fish Process Engineering, College of Fisheries Engineering, Tamil Nadu Fisheries University, Nagapattinam-611001 India 2 Department of Fish Processing Technology, College of Fisheries UAS, Mangalore – 575 002, India *Corresponding author ABSTRACT Keywords Actomyosin, Pink perch, SDS – PAGE, dynamic visco – elastic behaviour, setting, Ca++ ATPase Article Info Accepted: 22 May 2018 Available Online: 10 June 2018 Actomyosin from fresh pinkperch (Nemipterus japonicus) meat was isolated and its properties were assessed. The SDS-PAGE pattern indicated multiple bands in the molecular weight range of 2105 KD to 2104 KD. The dynamic viscoelastic behaviour revealed sol-gel transition at 2 temperatures, 36.7°C and 63.3°C. Setting of actomyosin at 30°C for 1 hr could increase the storage modulus values significantly. The freezing and frozen storage of actomyosin alter solubility in high ionic strength buffer by 17.9% and reduction in Ca++ ATPase activity by 41.8%. The apparent reduced viscosity as a function of protein concentration of actomyosin indicated a possible aggregation process. A reduction in myosin heavy chain concentration with storage period was revealed by SDSPAGE pattern. A progressive reduction in storage modulus values were observed with increase in frozen storage period. This pattern were also observed in set actomyosin. Introduction Fish protein represent most important class of functional ingredient because they possess range of dynamic functional properties like organoleptic, hydration, surface and rheological/textural properties. Surimi, fish flesh, waterwashed mixed with cryoprotectants and frozen is highly functional in its character to produce visco-elastic gel via protein interaction, bind water and to form cohesive and strong membrane on the surface of the fat globules in emulsion system (Xiong 1997). In fish protein it is myosin and actomyosin complex which play a dominant role in imparting various functional properties contributing to palatability or sensory perception of processed fish minced base products (Kinsella et al., 1994). The functional and rheological properties of actomyosin from fish are subjected to change during processing (Lanier et al., 1982; Chalmers et al., 1992; Sano et al., 1994). The structural changes in the natural actomyosin complex during frozen storage will alter the functional properties leading to change in eating quality. Freeze denaturation of actomyosin is highly affected by storage temperatures (Fukuda 1986; Jiang 1977; 3096 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3096-3112 Matsumoto 1980). The stability of actomyosin from cod, halibat, plaice and rose fish stored at -12°C, -18°C and -23°C was correlated with the storage temperature and -23°C was found to be the best (Dyer and Morton 1956). isolated and the effect of freezing and frozen storage at -20°C on the physico-chemical, functional and rheological properties have been studied. Materials and Methods When actomyosin is ground with salt it forms a sol and upon heating it turns to elastic gel (Noguchi 1986). The elasticity of the products prepared from surimi comes mainly from the thermal gelation characteristics of actomyosin (Sano et al., 1988). The sol gel transformation upon heating of muscle protein have been reported by many workers (Ishioroshi et al., 1979; Samejima et al., 1981; Lanier et al., 1982; Wu et al., 1985a). Both sol and gel have been considered as viscoelastic bodies. The response of the viscoelastic body when the stress is applied results in elastic deformation and viscous flow. Thus the thermal gelation of actomyosin from fish species can be understood by determining the changes in both the elastic and viscous element during sol-gel transition. Changes in rigidity and viscosity during thermal gelation of actomyosin from croaker, dynamic viscoelastic behaviour of actomyosin from carp and dynamic rheological behaviour of actomyosin from rabbit have been reported (Wu et al., 1985b; Sano et al., 1988; Ikeuchi et al., 1994). The frozen storage behaviour of actomyosin is highly species specific apart from storage temperature. A fundamental knowledge on the changes in the functional and rheologial properties especially gelling behaviour as affected by freezing and frozen storage will help in evolving a corrective measures. In the previous paper (Rathnakumar and Shamasundar 2005a) changes in the properties of total proteins from pinkperch meat during freezing and frozen storage has been reported. In the present paper actomyosin has been Fresh pink perch (Nemipterus japonicus) caught off Mangalore, west coast of India were procured on board and iced immediately in 1:1 ratio and packed in insulated container. The fishes were brought to the laboratory within 10 hrs and used for actomyosin isolation immediately. Isolation of Actomyosin Natural actomyosin was isolated according to the method described by Kawashima et al., (1973) from 100 g of mince. Actomyosin pellets thus obtained were distributed into plastic vials (5 g each) and subjected to air blast freezing at - 35°C for 45 min. and stored in deep freezer at - 20°1°C. The frozen actomyosin samples were drawn periodically and various functional and rheological properties were assessed. Proximate composition, Nitrogen solubility index (NSI), nitrogen solubility as a function of sodium chloride concentration, solubility of actomyosin in extraction buffer, viscosity, Ca2+ ATPase activity, SDS-PAGE pattern, emulsion capacity (EC), emulsion stability (ES) water absorption capacity (WAC) and were determined according to the method described in the previous paper (Rathnakumar and Shamasundar 2005a). Dynamic viscoelastic behaviour of actomyosin was carried out by using Controlled Stress Rheometer under oscillation mode. The experimental details have been described in the previous paper (Rathnakumar and Shamasundar 2005a). 3097 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3096-3112 Gel filtration Gel filtration of soluble actomyosin was performed using sepharose 6B gel packed in a column of 0.560 cm at the flow rate of 25-30 ml/hr. The bed volume (vt) of the column was 50 ml and the void volume (vo) was 12 ml. The void volume was determined using blue dextran. A known concentration of protein (45 mg) was loaded to column and eluted with extraction buffer (EB). Fractions of 3.0-3.5 ml were collected manually and measured at 280 nm using Bausch and Lomb spectronic -21, spectrophotometer. A plot of absorbance versus elution volume was obtained. Results and Discussion The composition and properties of actomyosin (AM) from fresh pink perch are given in Table 1. Bulk of the actomyosin accounted for moisture and protein constituted about 3 percent. The NPN content of actomyosin was 22.63 mg/100g which was negligible in comparison to NPN content of meat (694 mg/100g - Rathna Kumar and Shamasundar 2005a). The protein solubility of fresh actomyosin in EB was 94.11%. The solubility of fish protein in high ionic strength buffer is taken as index of denaturation by many workers (Dyer, 1951; Connell 1959; Shamasundar and Prakash, 1994a). Further, the Ca2+ ATPase enzyme activity of fresh actomyosin (0.474 g Pi/mg protein/min) indicate the native state of the molecule. The Nitrogen solubility index of actomyosin is given in Fig. 1A. The minimum solubility occurred at the range of pH 5.75 6.5. Solubility increased both on acidic and alkaline pH. These results showed that shifting of pH away from iso-electric point could solubilize more of actomyosin. Similar results reported by Kinsella (1982) Lin and park (1998). Solubility of actomyosin as a function of molar concentration of NaCl indicated salting ‘in’ phenomenon upto 1.0 M concentration and salting ‘out’ at 1.5 and 2.0 M concentration. (Fig. 1B). Higher concentration of NaCl/KCl are known to alter the solubility behaviour of myosin and actomyosin because of its interaction with water surrounding protein molecules. The solubility of myosin from salmon in KCl increased upto 1 M and then decreased at 2 and 3 M, however the salting out process was much slower (Lin and park 1998). Hence, for protein solubility studies as a function of frozen storage, 1 M NaCl concentration in phosphate buffer (0.05 M, pH 7.5) was used as solvent. The gel filtration profile of actomyosin on sepharose - 6B gel shows a single peak (Fig 4A) eluting at an volume of 32.6 ml. This single peak demonstrates a single component in the system. The SDS PAGE pattern of fresh actomyosin showed multiple bands (Fig.10 lane a) with molecular weight in the range of 2 x 105 KD to 2 x 104. The SDS-PAGE pattern indicates about 8 bands, 4 major and 4 minor bands. The concentration of myosin heavy chain (MHC) was high as evidenced by the intensity and breadth of the band. The dynamic visco-elastic behaviour (DVB) of fresh actomyosin in the temperature range of 30° - 90°C is given in Fig. 6A. The storage modulus value (G’) indicative of elastic component increased with increase in temperature upto 83.3°C. The rate of increase of G’ value was maximum in the temperature range of 36.7-43.7°C (Table 3). The loss modulus (G”) values which is indicative of viscous element during gelation process showed an increasing trend upto 83.3°C and decreased to 140.9  103 dynes /cm2 at 90°C. The increase in G” values as a function of temperature sweep is much less compared to G’ values which clearly suggests that actomyosin had ability to build up elastic component during heat processing. The 3098 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3096-3112 structure build up reaction which is indicative of elastic component is maximum between 36.7°C and 43.6°C. The sol-gel transition as indicated by tan  value occurred at two temperature viz., 36.7 and 63.3°C. Using Thermal Scanning Rigidity Monitor (TSRM) Wu et al., (1985b) observed a (Transition) peak at 38°C in Atlantic Croaker actomyosin solution with increase in viscosity and attributed to high temperature ‘setting’ phenmenon. DSC studies also confirmed a transition at 36-38°C for the above species (Wu et al., 1985a). Surimi of Alaska pollock showed elastic structure build up at 45°C when heating rate was 1°C/min (Hamann 1992). representated in the Fig.3A. The slope of the curve altered with increase in storage time indicating change in shape of the molecule. This change could be due to aggregation process of actomyosin molecule during storage as indicated by solubility and ATPase profile. The freezing and frozen storage of actomyosin altered the Ca2+ ATPase enzyme activity and protein solubility significantly (p < 0.05). The effect of freezing per se was dominant on ATPase enzyme activity as a reduction of 34% was recorded (Fig. 2). After 30 days of frozen storage there was an increase in ATPase activity to 0.472 g Pi/mg protein/min. This increase in ATPase enzyme activity was similar to that observed in total protein extract from pinkperch meat (Rathnakumar and Shamasundar 2004a). The percent protein extracted from actomyosin during different periods of frozen storage (Fig. 2) revealed a gradual decrease reaching a value of 20% at the end of 150 days. This decrease in solubility in the solvent used (EB) is mainly due to aggregation/ denaturation process brought about during frozen storage. This insolubilization of protein is mainly due to formation of larger aggregates by actomyosin complex (Shenouda, 1980; Wagner and Anon 1986) caused by the formation of disulfide bonds, and hydrophobic interactions (Jiang et al., 1988 a, b). The changes in gel filtration (GF) profile of actomyosin is depicted in Fig 4A-G. From the figure an initial aggregation at the end of 30 days of frozen storage was evident as there was a shift in the elution volume from 32.6 ml to 30.0 ml. With further increase in frozen storage period a dissociation process was observed with the shift in elution volume to 36.5, 37.5, 40.0 and 40.8 ml at the end of 50, 90, 120 and 150 days of storage respectively. The apparent reduced viscosity of actomyosin solution as a function of protein concentration at different periods of frozen storage is A derivative graph obtained (Fig. 3B) revealed that the apparent reduced viscosity at a protein concentration of 5 mg/ml decreased significantly (p < 0.05) with storage period. Similar reduction during frozen storage of actomyosin from frozen hake (Montecchia et al., 1997) and carp (Noguchi and Matsumoto, 1978) were reported. The concentration of the actomyosin peak in the GF profile decreased with increase in storage period. This reduction in concentration is mainly due to decreased solubility of actomyosin in the solvent (EB) used and/or due to aggregation/denaturation. However, the gel filtration pattern clearly demonstrate aggregation - dissociation reaction. The SDS-PAGE pattern of actomyosin as a function of frozen storage is represented in Fig.10. A reduction in MHC concentration was observed from the SDS-PAGE pattern. This suggests that the aggregates formed during storage is insolubilized even in cationic detergent like SDS. Such reports are common in literature for many fish and shell fish species (An et al., 1988, 1989, Tejada 1996). 3099 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3096-3112 Protein Extracted (% Total Protein) Fig.1 A: Nitrogen Solubility Index of total proteins from fresh actomyosin with distilled water as solvent B: Protein Solubility of fresh actomyosin as a function of molar concentration of sodium chloride in phosphate buffer (50mM; pH 7.5) 90 A 60 30 0 0 3 6 9 12 Protein Extracted (% Total Protein) pH 90 B 60 30 0 0.0 0.5 1.0 1.5 Molar Concentration of NaCl 3100 2.0 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3096-3112 Ca++ ATPase Activity (g Pi /.mg protein/min Fig.2 A: Effect of freezing and frozen storage at –20°C, of actomyosin on calcium ATPase activity of muscle extract in Tris-HCl buffer, pH 8.0, 50mM. B: Effect of freezing and frozen storage at –20°C, of actomyosin on the solubility of total proteins. The solvent used was EB and soluble protein was expressed as % solubilized of total protein content of meat 0.8 Fresh 0.6 Frozen 0.4 0.2 0 0 Protein Extracted (% Total Protein) A 50 100 150 200 250 300 50 100 150 200 250 300 100 Fresh 80 Frozen 60 40 20 0 B 0 3101 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3096-3112 Fig.3 A: A derivative graph of apparent reduced viscosity of actomyosin at 5 mg/ml protein concentration as a function of freezing and frozen storage. B: Apparent reduced viscosity of actomyosin extracted in EB, as a function of freezing and frozen storage at –20°C  reduced at 10 mg/ml 3 Fresh Frozen 2 1 0 0 50 100 150 200 250 300 Storage Period (Days) A Apparent Redcued Viscosity (ml/mg) 0.20 Fresh Imm. Frozen 50 Days 90 Days 120 Days 0.15 0.10 0.05 0.00 0 2 4 Protein Concentration (mg/ml) B. 3102 6 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3096-3112 Fig.4 Changes in gel filtration profile of total protein from actomyosin on sepharose 6B gel, as a function of freezing and frozen storage at –20°C. The eluant used was extraction buffer (phosphate buffer, 50mM, pH 7.5; containing 1M NaCl) 0.6 Fresh 0.4 0.2 0 0.6 Imm. after freezing 0.4 n m 0.2 0 0.6 30 days 2 8 0 0.4 0.2 0 0.6 90 days a t 0.4 0.2 A b s o r b a n c e 0 0.6 150 days 0.4 0.2 0 0.6 180 days 0.4 0.2 0 0.6 240 days 0.4 0.2 0 0.6 300 days 0.4 0.2 0 20 50 80 110 140 E l u t i o n V o l u m e (ml) 3103 170 200 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3096-3112 Fig.5 Changes in EC&ES of total proteins from actomyosin as a function of freezing and frozen storage at –20°C 1.0 EC 0.8 ES 9 Imm. Frozen 0.6 6 ES (min) EC (mg oil/mg protein) Fresh 0.4 3 0.2 0.0 0 0 60 120 180 240 300 Storage Period (Days) Fig.6 Changes in gel strength of set and unset meat of actomyosin as a function of freezing and frozen storage at –20°C 3104 Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 3096-3112 Fig.7 Changes in dynamic viscoelastic behaviour of actomyosin in the temperature range of 3090°C, as affected by freezing and frozen storage at –20°C. DVB was carried out under oscillatory mode 5000 A G' G'' 3000  2000 2000 1000 1000 0 0 1 0 0 3000 E G' G'' 4000 0 4000 5000 5000 F G' G'' 4000 3000 3000 2000 2000 1000 1000 0 0 D y n e s / C m 2  4000 5000 B G' G'' 5000 4000 5000 C G' G'' 4000 3000 3000 2000 2000 1000 1000 0 0 5000 D G' G'' 4000 5000 G' 4000 G'' 3000 3000 2000 2000 G G' G'' H T e m p e r a t u r e1000 ( ° C ) 1000 0 0 10 30 50 70 10 90 30 50 70 90 A) fresh actomyosin B) immediately after freezing C) 30 days D) 90 days E) 120 days F) 150 days G) 240 days H) 300 days 3105
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