Application of Antifreeze Protein for Food Preservation: Effect of Type III Antifreeze Protein for Preservation of Gel-forming of Frozen and Chilled Actomyosin

Type III antifreeze protein (AFP) remarkably preserved Ca²⁺ ATPase activity of actomyosin during frozen and chilled storage. Under frozen conditions, AFP helped to retain the Ca²⁺ ATPase activity of actomyosin much higher than that of conventional cryoprotectants (sucrose‐sorbitol mixture). The Ca²⁺ ATPase activity increased with increasing AFP concentration and reached a maximum at 50g/L AFP. After 3‐d chilled storage, the Ca²⁺ ATPase activity of control and sucrose‐sorbitol samples had lost 80% and 50%, respectively, while the AFP samples remained unchanged. A Type III AFP mechanism based on freezing temperature depression (more unfrozen water) and inhibition of ice recrystallization that protects against the freezing of muscle proteins in chilled or frozen conditions is proposed.     

Comparison of gilthead sea bream (Sparus aurata) and hake (Merluccius merluccius) muscle proteins during iced and frozen storage

Gilthead sea bream (Sparus aurata) and hake (Merluccius merluccius) muscle behave differently during storage, whether in ice or deep frozen. Rapid changes have been observed in the texture of hake muscle during frozen storage, while gilthead sea bream has proved to be more stable. In order to ascertain the role of muscle proteins in the changes observed during storage, parameters related to protein functionality and the properties of extracted natural actomyosin (NAM) were studied initially and during storage in ice or at ?20 °C. Initially, the parameters related to functionality had higher values in hake muscle and extracted NAM than in gilthead sea bream. At the end of iced storage (22 days), less myosin heavy chain (MHC) and actin were extracted from hake, but there was practically no change in gilthead sea bream. This decrease was not accompanied by lower Ca²⁺‐ATPase activity. Freezing produced no drastic changes, with lower values for gilthead sea bream. However, this species was more stable after 1 year, except for the Ca²⁺‐ATPase activity of NAM. This suggests that the changes that hake proteins underwent during storage particularly affected properties related to aggregation, whereas in gilthead sea bream the changes hardly affected the formation of soluble or insoluble aggregates but did affect the active sites of myosin. © 2002 Society of Chemical Industry     

Effect of Calcium Salts on the Properties of Proteins from Oil Sardine (Sardinella longiceps) During Frozen Storage

ABSTRACT:  The effect of calcium sulfate and calcium chloride on the enzymatic and structural properties of actomyosin isolated from sardine was investigated. Mince was prepared from sardine and different concentrations of calcium chloride and calcium sulfate were added to the mince and kept in frozen condition at −20 °C. The physico-chemical and functional properties of proteins from mince were analyzed as a function of time. The solubility of proteins decreased during storage. The reduction in solubility was less for samples treated with calcium chloride. However, sardine mince showed better functionality during storage in the presence of calcium compounds. The ATPase enzyme activity of actomyosin increased with increase in concentration of calcium and decreased after reaching the maximum value. ATPase activity of proteins from mince decreased during storage at low temperature. The reduction in ATPase activity did not correlate with the loss of functionality of proteins. SDS-PAGE did not reveal any major changes in the protein profile during storage as well as in the presence of different concentration of calcium compounds. The secondary structural content of actomyosin was not altered in the presence of both calcium chloride and calcium sulfate as evident from circular dichroic measurements.     

Effect of frozen storage on the structure and enzymatic activities of myofibrillar proteins of rabbit skeletal muscle

The effect of frozen storage on the biochemical properties of myofibrils, and of their major constituents, actin and myosin, was investigated. Extractability of myofibrillar proteins increased slightly for 3 weeks during frozen storage of muscle, decreasing thereafter. The change in myofibrillar ATPase activity during frozen storage was consistent with that of a reconstituted acto-heavy meromyosin (HMM) complex prepared from frozen stored muscle at the same weight ratio of actin to myosin as in situ. However, myosin ATPase activity showed a different pattern of change when compared with myofibrillar ATPase activity. The maximum velocity of acto-HMM ATPase activity and the apparent dissociation constant of the acto-HMM complex decreased for 1 week during frozen storage, increasing thereafter, indicating that the affinity of actin for myosin was greatest in muscle which had been frozen for 1 week.     

Comparison of the Cryoprotective Effects of Trehalose,Alginate, and Its Oligosaccharides on Peeled Shrimp (Litopenaeus Vannamei) During Frozen Storage

The cryoprotective effects of trehalose, alginate, and its oligosaccharides on peeled shrimp (Litopenaeus vannamei) during frozen storage was investigated by monitoring thawing loss, color, texture, myofibrillar protein content, Ca²⁺‐ATPase activity, and performing microscopic structural analysis. Data revealed significant (p < 0.05) inhibitory effects on thawing loss and textural variables (springiness and chewiness) in trehalose‐, alginate oligosaccharides‐, and sodium pyrophosphate‐treated shrimp compared with the control and alginate‐treated batches. L* values revealed that these saccharides had a positive effect on color stability during frozen storage. In addition, the results of chemical analyses showed that trehalose and alginate oligosaccharide treatments effectively maintained an increased myofibrillar protein content and Ca²⁺‐ATPase activity in frozen shrimp. In addition, hematoxylin & eosin staining and SDS‐PAGE confirmed that these cryoprotective saccharides slowed the degradation of muscle proteins and the damage to muscle tissue structures. Overall, the application of trehalose and alginate oligosaccharides to peeled frozen shrimp might maintain better quality and extend the commercialization of these refrigerated products.     

Cryoprotective additives and cryostabilisation effects on muscle fillets of the freshwater teleost fish Rohu carp (Labeo rohita) during prolonged frozen storage

The effects of various cryoprotective additives separately and in combination were studied on the myofibrillar protein integrity, biochemical enzyme activity levels and muscle ultrastructure in the freshwater teleost fish Rohu carp (Labeo rohita). Fish muscle samples were divided into eight groups and immersed in different mixtures of cryoprotective additives (S1–S8), then frozen at ? 20 or ? 30 °C for 24 months. Electrophoretic studies revealed early (within 6 months) alteration of the myofibrillar proteins myosin light chain, α‐actinin and tropomyosin. Reduction of the storage temperature from ? 20 to ? 30 °C slowed down the degradative processes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that fish muscle treated with cryoprotective mixture S8 (40 g L^?1 sorbitol/3 g L^?1 sodium tripolyphosphate/4 g L^?1 sodium alginate) showed minimal post mortem changes in myofibrillar proteins. Ultrastructural results also revealed post mortem damage to the muscle, seen earliest (within 6 months) in the sample frozen‐stored without additives (S2), as compared with the normal, unfrozen muscle (S1). The influence of cryoprotectants alone and in combination on fish muscle structural proteins, myosin and actin filaments (A and I bands), during prolonged frozen storage was investigated. After 12 months, samples frozen‐stored with various cryoprotective additives (S2‐S7), except S8, showed signs of myofibrillar disintegration. Beyond that time the degradative processes started showing up in all samples, with minimal muscle ultrastructural damage in sample S8. Again, reducing the storage temperature from ? 20 to ? 30 °C slowed down the degradative processes. Ultrastructural results correlated well with levels of biochemical enzymes (Ca²⁺ myofibrillar ATPase and succinic dehydrogenase) during frozen storage. This is the first report of the cryoprotective effects of these additives on this popular edible fish species. Of the various combinations of additives tested, cryoprotective mixture S8 was found to preserve the muscle structure longest under frozen storage conditions. However, even this mixture was only effective for 18 months at ? 30 °C. Beyond that time the myofibrillar degradative processes were apparent with correlative electrophoretic, biochemical and ultrastructural studies. Copyright © 2006 Society of Chemical Industry     

CHARACTERISTICS OF MUSCLE FROM TWO SPECIES OF BIGEYE SNAPPER, PRIACANTHUS TAYENUS AND PRIACANTHUS MACRACANTHUS

Composition and some properties of muscle from two species of bigeye snapper, P. tayenus and P. macracanthus, were investigated. Both species had a similar composition with the same myofibrillar protein content. However, muscle proteins from P. tayenus had higher thermal stability than those from P. macracanthus, as indicated by the higher enthalpy for transitions as well as the lower inactivation rate constant (K_D). Upon 15 days of iced storage, natural actomyosin Ca^2*‐ATP ase and Mg²⁺‐Ca²⁺‐ATPase activities decreased, whereas Mg²⁺‐EGTA‐ATPase activity increased, suggesting the denaturation of myosin, actomyosin and troponin/tropomyosin complexes, respectively. Increased surface hydrophobicity and decreased sulfhydryl groups indicated the denaturation possibly occurred via hydrophobic interaction and disulfide formation. Heading and eviscerating offish retarded the denaturation and physicochemical changes of proteins during iced storage. The results indicated that a rapid and proper post harvest handling was of importance to maintain the muscle quality of bigeye snapper.     

Effect of reducing agents on physicochemical properties and gel-forming ability of surimi produced from frozen fish

Effects of different reducing agents (cysteine, ascorbic acid and sodium bisulfite) at various levels on physicochemical properties of protein, transglutaminase activity and gel properties of surimi produced from frozen croaker, lizardfish, threadfin bream and bigeye snapper were studied. Addition of cysteine resulted in the highest increase in the breaking force and the deformation of surimi gels, compared with other reducing agents. The optimum levels of cysteine were 0.05, 0.1, 0.05 and 0.1% (w/w) for surimi from frozen croaker, lizardfish, threadfin bream and bigeye snapper, respectively. Surimi from frozen croaker with cysteine added showed a similar breaking force and deformation to that produced from fresh fish. With addition of cysteine, an increase in sulfhydryl content with a concomitant decrease in disulfide bond content was generally observed. Ca²⁺ ATPase activity also increased, indicating the renaturation of the myosin molecule. T_max of peak 1 (myosin peak) of all surimi sols in the presence of cysteine was shifted to higher temperature. The increased transglutaminase activity was observed with addition of cysteine. Therefore, reducing agents, especially cysteine, recovered the denatured muscle proteins and activated the transglutaminase in the muscle, leading to the increased gel-forming ability of surimi produced from frozen fish.     

Effect of Electrical Stimulation on Postmortem Property Change of Myofibrillar Proteins

Changes in biochemical properties of myofibrillar proteins of rabbit muscle, which had been subjected to electrical stimulation soon after slaughter, during postmortem storage at 0°C were investigated. Myofibrillar ATPase activity and the ATPase activity of acto-heavy meromyosin (HMM) complex, reconstituted from actin and HMM which had been prepared from at-death and postmortem muscles, decreased at first and then increased slightly during 7 days storage. In addition, the change of the dissociation constant of acto-HMM complex of electrically stimulated muscle during postmortem storage was quite small, i.e., 1.59 ± 10^?4M for at-death muscle, 1.70 ± 10^?4M for muscle stored for 1 day and 1.49 ± 10^?4M for muscle stored for 7 days. This indicates that electrical stimulation treatment minimized the postmortem change of actin-myosin interaction.     

Investigation of the activity of cathepsin B in red shrimp (Solenocera crassicornis) and its relation to the quality of muscle proteins during chilled and frozen storage

Cathepsin B is a cysteine protease that has important effects on the quality of muscle products. In this study, the changes of cathepsin B activity and its relation to muscle proteins were investigated in intact and beheaded shrimp during chilled and frozen storage. The obtained results indicated that the water holding capacity (WHC), shear force, hardness, and myofibrillar protein (MP) content all significantly decreased in both the intact and beheaded shrimp samples with increasing storage period (p < 0.05). Specifically, beheading shrimp exhibited much more stable characteristics than intact shrimp samples during both chilled and frozen storage. The enzyme activity results suggested that cold temperature and storage induced the release of cathepsin B from the lysosomes to the mitochondria, sarcoplasm, and myofibrils in the muscle tissues. Furthermore, SDS-PAGE and transmission electron microscopy (TEM) analysis revealed that beheading the shrimp greatly inhibited the dissociation of shrimp muscle proteins during storage. The current findings suggest that cathepsin B located in the head of shrimp was likely transferred to the muscle through the first abdominal segment during storage, accelerating the dissociation of the muscle proteins. Therefore, beheading the shrimp was conducive to prolonging the shelf-life of stored shrimp products.     

Effect of Slurry Ice on the Functional Properties of Proteins Related to Quality Loss during Skipjack Tuna (Katsuwonus pelamis) Chilled Storage

The effect of slurry ice on the quality of Skipjack tuna (Katsuwonus pelamis) during chilling storage was investigated and compared to flake ice. Slurry ice‐treated samples showed significantly higher springiness and chewiness variables than the blank and flake ice‐treated samples (P < 0.05). The growth of microorganisms in tuna muscle treated with slurry ice was also down significantly (P < 0.05), and the total aerobic counts didn't reach higher scores than 5.0 log CFU/g during the whole chilling storage. Additionally, the myofibrillar protein, Ca²⁺‐ATPase activity, and total sulfydryl (SH) content in muscle treated with slurry ice were all significantly higher than the blank and flake‐iced samples (P < 0.05). This was probably due to the faster cooling, subzero final‐temperature, and larger heat exchange derived from slurry ice. Standard error of mean and sodium dodecyl sulfate–polyacrylamide gel electrophoresis results also confirmed that slurry ice treatment could effectively retard the degradation of myofibrillar proteins and showed a positive effect on the stability of tissue structures.     

Effect of lipid oxidation and frozen storage on muscle proteins of Atlantic mackerel (Scomber scombrus)

The effect of storage on the lipids and proteins in Atlantic mackerel stored for up to 24 months at ?20 and ?30 °C was studied. Traditional methods including the peroxide value, thiobarbituric acid‐reactive substances (TBARS) and a reverse phase HPLC method were used to determine the primary and secondary lipid oxidation products. All tests showed an increase in lipid oxidation products with storage time and at a higher storage temperature of ?20 °C compared with samples stored at ?30 °C. Antioxidants had a significant effect (P < 0.01) on the inhibition of lipid oxidation, as shown by the reduction in peroxide value and hydroxides, and malondialdehyde formation. Similarly, deterioration of protein structure and functionality in mackerel stored for 3, 6, 12 and 24 months was greater at ?20 than ?30 °C. ATPase activity in the myosin extract of Atlantic mackerel showed a significant decrease (P < 0.01) with progressive frozen storage. Protein solubility in high salt concentration (0.6 M NaCl) decreased (P < 0.01) during storage at both ?20 and ?30 °C but was greater at ?20 °C. Interestingly, antioxidants BHT, vitamin C and vitamin E protected the proteins against complete loss of ATPase activity and protein solubility to a significant level (P < 0.01) for up to 1 year at ?20 °C compared with samples stored without antioxidants. This study confirms the deleterious effect of lipid oxidation products on protein structure and function in frozen fatty fish. © 2002 Society of Chemical Industry     

Enzyme stability of microencapsulated Bifidobacterium animalis ssp. lactis Bb12 after freeze drying and during storage in low water activity at room temperature

Abstract: Stability of enzymes such as β‐galactosidase (β‐gal), β‐glucosidase (β‐glu), lactate dehydrogenase (LDH), pyruvate kinase (PK), hexokinase (HK), and ATPase of microencapsulated Bifidobacterium animalis ssp. lactis Bb12 after freeze‐drying and after 10 wk of storage at low water activity (a_w) at room temperature was studied. Bacteria were microencapsulated using alginate formulation with or without mannitol fortification (sodium alginate and mannitol [SAM] and sodium alginate [SA], respectively) by creating gel beads followed by freeze drying. Two types of dried gel beads were then stored at low a_w, such as 0.07, 0.1, and 0.2; storage in an aluminum foil was used as control. All storage was carried out at room temperature of 25 °C for 10 wk. Measurement of β‐gal, β‐glu, LDH, PK, HK, and ATPase (with or without exposure to pH 2.0 for 2 h) activities was carried out before freeze drying, after freeze drying, and after 10 wk of storage. There was a significant decrease in almost all enzyme activities, except that of PK. SAM and SA showed no different effect on maintaining enzyme activities during freeze drying. Storage for 10 wk at room temperature at various low a_w using SAM and SA system had a significant effect on retention of most enzymes studied, except that of PK and LDH. Storage at a_w of 0.07 and 0.1 was more effective in maintaining enzyme activities than storage at a_w of 0.2 and in an aluminum foil. However, mannitol fortification into alginate system did not significantly improve retention of enzymes during 10 wk of storage.     

Assessment of protein changes in farmed giant catfish (Pangasianodon gigas) muscles during refrigerated storage

Farmed giant catfish (Pangasianodon gigas) muscles (dorsal and ventral sites) were stored in a refrigerator (at 4 °C) for 14 days to determine the effect of refrigerated storage on biochemical and physical changes. The analyses were carried out at 0, 2, 4, 7, 10 and 14 days of storage. At day 14, Ca²⁺ ‐ATPase activity markedly decreased when compared to its value at day 1 (>90%), while a small decrease was observed for surface hydrophobicity and reactive sulfhydryls content. Total volatile basic nitrogen and trichloroacetic‐soluble peptide content gradually increased when the storage period was extended. The myosin heavy chain decreased slightly on SDS‐PAGE for both meat cuts with increased storage time. Expressible drip and cooking loss were highest during the first day of storage and slightly decreased with storage time. Instrumental hardness was significantly higher in the ventral compared to the dorsal muscle, while the toughness was the highest at the second day of storage. The muscle bundles with scanning electron microscopy were less attached, resulting in the observed big gaps over increasing storage time. Results indicated that changes of proteins have detrimental effects on the quality attributes of farmed giant catfish muscles during refrigerated storage, particularly physical and biochemical properties.     

Comparative study on physicochemical changes of muscle proteins from some tropical fish during frozen storage

Physicochemical changes of muscle from croaker, lizardfish, threadfin bream and bigeye snapper during storage at −18 °C were investigated for up to 24 weeks. Ca²⁺–ATPase activity decreased, whereas Mg²⁺–EGTA–ATPase activity increased throughout the storage. However, no marked changes in Mg²⁺–Ca²⁺–ATPase and Mg²⁺–ATPase activities were observed. Among all species tested, lizardfish muscle was most susceptible to those changes. Disulfide bond formation with the concomitant decrease in sulfhydryl group was found in all species. However, croaker and lizardfish contained higher disulfide bonds as storage time increased, compared to other species. Surface hydrophobicity increased in all species with the sharp reduction observed in lizardfish after 12 weeks. For all species, α-glucosidase and β-N-acetyl-glucosaminidase activities increased in association with the increased expressible drip. Therefore, extended frozen storage caused the denaturation of protein as well as the cell disruption in all species, but the degree of changes was dependent upon species.     

Superchilled,chilled and frozen storage of Atlantic mackerel (Scomber scombrus) fillets – changes in texture,drip loss,protein solubility and oxidation

Changes in quality characteristics in relation to protease activity and protein oxidation in chilled, superchilled and frozen mackerel fillets during storage were studied. The solubility of sarcoplasmic proteins was quite stable in mackerel samples for all storage experiments, whereas the solubility of myofibrillar proteins decreased in both superchilled and frozen samples. A significant correlation (r = 0.983, P < 0.05) between the increased activity of cathepsin B+L in chilled fillets and softening of the fish flesh during storage was revealed. Contrary with chilled samples, the texture of superchilled mackerel fillets became tougher along the storage period, which can be explained by a higher rate of myofibrillar oxidation (r = 0.940, P < 0.05). The hardness and drip loss decreased slightly at the end of frozen storage. Superchilling preserved the quality of mackerel fillets with the least side effects in relation to protein solubility, drip loss and softening of the fish tissue as compared to chilled and frozen storage.     

Gel‐forming properties of surimi produced from bigeye snapper,Priacanthus tayenus and P macracanthus,stored in ice

The influence of iced storage of two species of bigeye snapper, Priacanthus tayenus and P macracanthus, on the gel‐forming ability of the resulting surimi was investigated. Upon iced storage, whole fish underwent deterioration faster than beheaded/eviscerated fish. Total volatile base and trimethylamine contents of whole fish were higher than those of beheaded/eviscerated fish, particularly after 9 days of storage (P < 0.05). P macracanthus muscle was more susceptible to proteolytic degradation than P tayenus muscle. Ca²⁺‐ATPase activity decreased as the storage time increased (P < 0.05), indicating the denaturation of myosin. A marked decrease in Ca²⁺‐ATPase activity was found in whole fish kept for more than 6 days in ice (P < 0.05). Breaking force and deformation of surimi gels from both species decreased, with a concomitant decrease in whiteness, as the storage time increased (P < 0.05). Beheading and evisceration of fish retarded the deterioration. However, the gel‐forming ability of surimi produced from both species decreased continuously throughout iced storage (P < 0.05), probably owing to the denaturation and degradation of myofibrillar proteins. © 2002 Society of Chemical Industry     

Muscle changes in hard and soft shell crabs during frozen storage

Chemical and physicochemical changes of muscles from hard and soft shell mud crabs (Scylla serrata) were monitored during 12 weeks of storage at −20 °C. Ca²⁺-ATPase activity of natural actomyosin (NAM) from both crabs decreased continuously during storage, regardless of muscle types. After 8 weeks of storage, Ca²⁺-ATPase activity of NAM from lump muscle of soft shell crab decreased to a greater extent than that of hard shell crab (P < 0.05). An increase in disulfide bonds was observed with the coincidental decrease in sulfhydryl group content during extended storage (P < 0.05). Surface hydrophobicity of all samples increased up to 8 weeks, being this sampling time followed by a gradual decrease. Formaldehyde content of all samples increased throughout the storage (P < 0.05). Slightly higher formaldehyde content was found in soft shell crab muscle, compared with hard shell counterpart (P < 0.05). Claw muscle generally contained a greater amount of formaldehyde than lump counterpart (P < 0.05). Protein solubility of all samples decreased continuously throughout the 12 weeks of frozen storage (P < 0.05). Lipid oxidation took place during the extended storage as evidenced by the increase in thiobarbituric acid reactive substances. The pH of all samples generally decreased during frozen storage. Cooking loss of all crab muscles increased as storage time increased (P < 0.05) and was more pronounced in claw muscle, particularly from soft shell crab.     

Changes in functional properties of common carp (Cyprinus carpio) myofibrillar protein as affected by ultrasound-assisted freezing

Ultrasound-assisted freezing (UF) has proven to be a method that can effectively increase the freezing rate of frozen food and improve its quality. The functional properties of myofibrillar proteins (MP) are important factors that affect the further processing quality of meat products. At present, the effect of UF on the functional properties of frozen MP is not yet clear. Therefore, in the present study, changes in the functional properties (emulsifying and gel properties) of MP in common carps (Cyprinus carpio) frozen with UF at different power levels were investigated. The results revealed that, compared with immersion freezing (IF), UF at 175 W (UF-175) effectively inhibited the decrease in protein solubility, absolute Zeta potential, emulsion activity index, storage modulus (G'), and loss modulus (G'') caused by freezing. UF-175 sample had lower protein turbidity, and smaller protein particle size than any other frozen samples (P < 0.05), which suggested that UF-175 inhibited protein aggregation induced by freezing. In addition, shorter T₂₁ and T₂₂ relaxation times were obtained in UF-175 sample than other frozen samples, indicating that UF-175 reduced the mobility of immobilized and free water. Accordingly, UF-175 sample had higher gel strength and water holding capacity than other frozen samples (P < 0.05). A denser and more uniform gel network structure was also found in UF-175 sample than other frozen samples. In general, improved functional properties of common carp MP can be achieved by optimal UF.     

PIGMENTS MODIFICATIONS DURING FREEZING and FROZEN STORAGE of PACKAGED BEEF

Muscle pigment modifications during freezing and frozen storage of beef wrapped in high (polyethylene) and low (EVA/SARAN/EVA) gaseous permeability films were analyzed. Myo (Mb), oxy (MbO²) and metmyoglobin (MetMb) concentrations were determined using reflectance spectrophotometry. Results indicated that in polyethylene wrapped samples myoglobin oxygenation took place during freezing and the only reaction observed throughout frozen storage was MetMb production from MbO₂. Autoxidation was interpreted as a first order kinetic reaction. Storage temperature effect on kinetic and equilibrium constants was analyzed; reaction enthalpy and activation energy were calculated. MbO₂ and MetMb production during beef oxygenation was represented as a consecutive first order reaction system determining previous vacuum storage time and temperature influence on oxygenation capacity. Vacuum packaging of frozen beef increased color stability maintaining MetMb concentration levels lower than those corresponding to just frozen samples wrapped in polyethylene.