Contribution of Retained Organ Tissues to the Alkaline Protease Content of Mechanically Separated Atlantic Croaker (Micropogon undulatus)

The greater texture breakdown observed in gel-type products prepared from mechanically separated tissue as opposed to manually separated tissue can to some extent be attributed to the significantly higher protease activity measured in mechanically separated tissue. Failure to thoroughly wash eviscerated fish prior to mincing appears to result in the retention of tissue from internal organs as evidenced by a concommitant increase in protease activity in the minced tissue. Alkaline protease activity in croaker kidney and liver is several thousand fold higher than in skeletal muscle. Addition of kidney and liver tissue to deboned tissue from thoroughly washed fish results in increased protease activity and degradation of the fish tissue upon comminuting and cooking at 60°C. Isoelectric focusing provided evidence that the increased alkaline protease activity observed in mechanically separated fish tissue resulted to some degree from retention of kidney tissue in the fish mince. Isolation of Ca⁺⁺-dependent protease fractions from liver also implicated contamination from this source as an additional contribution to the total protease activity of minced fish.     

Studies on fish muscle protein. Nutritional consequences of the changes occurring during frozen storage

Samples of cod, whiting, herring, lemon sole and skate muscle were frozen and stored at ? 8°C for up to 20 months. Samples of cod muscle were also stored at ? 30°C. During storage at ?8°C the decrease in the solubility of the muscle proteins in 5% NaCl (denaturation) was most rapid in whiting followed in order by skate, cod, herring and lemon sole. Little change was detected in the NaCl solubility of cod muscle protein stored at ? 30°C. An increase in the pH was found to occur in all the species examined. The relationship between these changes and the toughening that occurs in fish muscle during frozen storage is considered. No nutritionally significant decrease in the lysine availability or the in vitro digestibility of the muscle proteins was found to occur in any of the species examined. In whiting, lemon sole and skate very small, statistically significant, increases in the available lysine content were detected after 7 months of storage at ?8°C and this may indicate that unfolding of the protein chains had occurred.     

Effects of Heat-Stable Alakaline Protease Activity of Atlantic Menhaden (Brevootii tyrannus) on Surimi Gels

Heat stable protease isolated from the sarcoplasmic fraction of menhaden (Brevoorti tyrannus) muscle tissue was characterized as to optimum temperature and pH against casein substrate and its degradative action on actomyosin and surimi during heating. The optimum conditions for activity were 60°C at a pH of 7.5 to 8.0. Activity dropped off remarkably at temperatures below 45°C or above 70°C and when pH was below 7.0 or above 8.0. The enzyme(s) was capable of degrading actomyosin as observed by sodium dodecyl sulfate electrophoresis. This implicated a causative role for this protease system in the texture degradation observed during thermal processing of menhaden surimi at temperatures of 50-70°C.     

Purification and Characterization of a Halotolerant Alkaline Serine Protease from Penicillium citrinum YL-1 Isolated from Traditional Chinese Fish Sauce

A halotolerant alkaline serine protease from Penicillium citrinum YL-1 which was isolated from traditional Chinese fish sauce was purified by ammonium sulfate precipitation, dialysis, and DEAE 52-Cellulose column, thereby resulting in a 4.66-fold increase in specific activity (110.68 U/mg). The molecular weight (MW) was estimated to be 32.27 kDa using SDS-PAGE analysis. The protease exhibited optimal activity toward the substrate casein at pH 8.0 at 40°C and was stable at pH 6.0–8.0 and 4–30°C. Activity was inhibited by NaCl and retained at 28.3, 21.4 and 18.1% of the initial activity after incubation for 6 h at 20, 25 and 30% NaCl concentrations, respectively. The enzyme was stimulated by Mn²⁺ and inhibited by K⁺, Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, and Fe³⁺. K_m and V_max of the protease for casein were 1.93 mg/ml and 56.81 μg/(min·ml), respectively. Protease activity was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), which confirmed the serine protease nature of the enzyme. The protease can hydrolyze tilapia protein in the absence or presence of NaCl (5–30%), thus suggesting that this protease is more halotolerant than the protease from other bacteria with high salinity resistance based on the current literature. These properties make the halotolerant alkaline serine protease a suitable candidate enzyme for fish protein hydrolysis during fish sauce fermentation.     

Biochemical and nutritional changes in fish proteins during drying

Biochemical and nutritional changes in the muscle proteins of a lean marine fish Nempiterus japonicus during drying at 50, 60 and 70°C were investigated. Solubility of proteins in water, 0.6 M NaCl, 1.5 M urea, 8 M urea and 10 g litre^?1 sodium dodecyl sulphate (SDS) decreased as drying progressed at all three temperatures; most of the decrease occurring in the initial 4 h of drying SDS-polyacrylamide gel electrophoresis of 1.5 M urea, 8 M urea and SDS extracts showed that higher molecular weight (MW) protein fractions were more sensitive to drying and disappeared much earlier from electropherograms than the lower MW protein fractions. Residual solubility of proteins near the pH range of 4–6 was found to increase during drying, but solubility at acid and alkaline pH was adversely affected. Decrease of solubility by drying was more affected at acid pH, especially at higher temperatures than at alkaline pH. Sulphydryl groups registered a regular and sharp decrease with drying except at 50°C, where initially an increase was observed. Apart from disulphide and hydrophobic bonds, free amino groups also appear to be involved in denaturation reactions during drying. Pepsin digestibility of fish muscle decreased slightly during drying but a clear relationship with drying temperature was not evident. Highly significant differences in proteins between protein efficiency ratio (PER), net protein utilisation (NPU) and biological value were observed between the drying temperatures. The PER and NPU of fish dried at 60°C were significantly higher than those dried at 50 or 70°C.     

PROPERTIES OF AN ALKALINE PROTEASE FROM THE SKELETAL MUSCLE OF ATLANTIC CROAKER1

An alkaline protease was partially purified from the skeletal muscle of Atlantic croaker. The protease is a cytoplasmic enzyme and heat stable. The enzyme preparation was shown to degrade fish actomyosin in vitro between 50–60°C. The enzyme is a sulfhydryl protease and does not require Ca⁺⁺ ions for its activity. Preparations of the enzyme do not hydrolyze TAME, BTEE or denatured hemoglobin. Column chromatographic analyses suggest an apparent molecular weight of 80,000 ± 4,000 and the isoelectric point is 6.0 ± 0.2.     

ATPase and Lactate Dehydrogenase Activities in Frozen Stored Fish Muscle as Indices of Cold Storage Deterioration

The effect of prolonged cold storage on muscle adenosine triphosphatase (ATPase) and lactate dehydrogenase (LDH) activities was studied in a variety of fresh water and brackish water fish. Decrease in enzyme activity was observed in all samples stored frozen (- 20°C) over a period of 180 days. Highly significant negative correlation was observed between enzyme activity and frozen storage period with mullet, pearlspot, milk fish and tilapia. Significant linear correlations were observed between decrease in enzyme activities and other biochemical indices and sensory scores. The results indicated that loss of activities of ATPase and LDH in fish muscle was significantly related to early changes in quality of frozen stored fish.     

Effect of Washing Treatment on Quality of Minced Mullet Flesh

Washing treatments were evaluated for quality improvement of minced mullet. Thiobarbituric acid (TBA) number generally decreased with increased washing temperature from 5–35°C and increased at higher temperatures. At 35°C, a wash water pH of 6.0 or above resulted in decreased TBA number. Response surface analysis indicated maximum whiteness index, minimum TBA number, and minimum cooking loss with washing at 29–33°C for up to 10 min with a water to flesh ratio from 23–26. Highest springiness was indicated with washing ratio of 25 to 30 at 28–31°C The simultaneously optimized washing conditions for highest minced mullet quality were 31.4°C for 7.5 min at a ratio of 24.     

Instant infusion pasteurisation of bovine milk. II. Effects on indigenous milk enzymes activity and whey protein denaturation

Direct heat treatment of two milk types, skimmed and nonstandardised full‐fat, was performed by instant steam infusion and compared with indirect heating. Infusion conditions were temperatures of 72–120°C combined with holding times of 100–700 ms, and indirect heat conditions were 72°C/15 s and 85°C/30 s. The activity of indigenous enzymes such as alkaline phosphatase, lactoperoxidase, xanthine oxidase and γ‐glutamyl transpeptidase was evaluated. Infusion temperature was the main determinant of inactivation. Whey protein denaturation represented by β‐lactoglobulin increased significantly with infusion temperature. The nonstandardised milk had a higher denaturation rate than skimmed milk. The effect of instant infusion on pH and milk fat globule size in relation to whey protein denaturation and association is discussed.     

Rheological and differential scanning calorimetry studies on structural and textural changes in frozen Atlantic mackerel (Scomber scombrus)

The viscoelastic behaviour and thermal stability of Atlantic mackerel fillets stored at ?20 and ?30 °C for up to 2 years were investigated. An increase in elastic (G′) and viscous (G″) modulus values, reflecting protein aggregation, was observed in samples stored at ?20 °C compared with those stored at ?30 °C, as well as with storage time. The results indicate that toughening on frozen storage is not just limited to lean gadoid fish but also occurs in fatty fish, leading to texture deterioration. Differential scanning calorimetry of fillets stored at ?20 °C showed a shift to a lower transition temperature (T_m) and a decrease in enthalpy (ΔH) compared with control fillets stored at ?30 °C; this change was enhanced when fillets were stored for a longer period of time, confirming protein denaturation and the formation of aggregates reported previously by the authors (J Sci Food Agric 82: 579–586 (2002)). The contribution of lipid oxidation to protein aggregation was shown by storing minced mackerel with or without the antioxidant vitamin E at ?10 °C. The G′ and G″ values were higher in samples stored without vitamin E than in samples stored with vitamin E; thus antioxidants may be used to minimise protein aggregation in fatty fish. The role of lipid oxidation in promoting protein aggregation and deterioration in the texture of fatty fish has not been reported hitherto. Antioxidants such as vitamin E may be used not only to prevent lipid oxidation but also to minimise protein damage in order to prolong the shelf‐life of fatty fish. Copyright © 2004 Society of Chemical Industry     

Implication of Muscle Alkaline Proteinase in the Textural Degradation of Fish Meat Gel

Unlike the actomyosin paste, white croaker meat paste showed poor elastic gel when heated around 60°C. The hydrolysis of muscle protein by muscle proteinase was observed in the poor gel. There are at least four kinds of proteinases in white croaker muscle: cathepsin D, neutral proteinase, calpain and alkaline proteinase. Among them only alkaline proteinase could act at pH of meat paste and around 60°C. Above facts confirmed the previous work that the heat-stable alkaline proteinase is probably implicated in the textural degradation of fish meat gel around 60°C.     

Autolysis and the endogenous proteinases characterised in beardless barb (Anematichthys apogon) muscle

Beardless barb is a common fish species used in fermentation of fish paste Ka-pi-plaa. Autolytic profile of beardless barb muscle showed the maximum autolysis was at 50 °C, at both acidic and alkaline pH values. With augmentation concentration of NaCl, autolytic activity slightly decreased. Endogenous proteinases isolated from fish muscle in crude extract forms were also characterised. The acidic proteinases had optimum activity at pH 3.0 and 50°C, and they showed higher proteolytic activity than the alkaline proteinases which were optimally active at pH 9.0 and 50 °C. Proteinases in peak at pH 3.0 were inhibited by pepstatin A, but those in peak at pH 9.0 were highly inhibited by PMSF, TLCK and soybean trypsin inhibitor, suggesting that both aspartic and serine proteinases were existed in beardless barb muscle. The proteinases were stable in pH range of 2.0-5.0 but unstable at the temperatures higher than 40 °C. NaCl suppressed the proteolytic activity, ATP activated the proteinase activity, while CaCl₂, MgCl₂ and CoCl₂ exhibited no influence on the activity. The results implied that cathepsin D is the predominant proteinase responsible for autolysis in beardless barb. The findings were useful to improve the processing and qualities of Ka-pi-plaa product using beardless barb as raw material.     

Evaluation of pH‐treated fish sarcoplasmic proteins on rheological properties of fish myofibrillar protein mediated by microbial transglutaminase

Fish sarcoplasmic protein (SP) could be exploited in the water‐holding agent for fish protein gels, except that the gel strength is reduced. The adjustment of pH could modify protein interactions to overcome the inferior effect. Fish SP solutions were adjusted to pH 3 or 12, neutralised to pH 7 and lyophilised to be pH‐treated SPs. These SPs along with lyophilised untreated SP (Normal SP) were incorporated into fish myofibrillar protein (MP) with microbial transglutaminase (MTG). The denaturation temperature (T_d) of MP mixed with normal SP was 66 °C with the lowest shear stress value. The denaturation of MP mixed with pH‐treated SP reduced to be 57 °C, resulting in increased shear stress. The cooking loss of MP gel was reduced by adding pH‐treated SPs, while the breaking forces were similar to control. The result indicated that pH‐treated SPs could be used to reduce cooking loss of MTG‐mediated MP gels without affecting the gelling properties.     

Characterization and Cheese-Making Properties of Rennet-Like Enzyme Produced by a Local Algerian Isolate of Aspergillus niger

An ochratoxin free extracellular acid protease was produced by solid state cultivation of Aspergillus niger FFB1. The purified enzyme (48.7 kDa) showed an optimal milk clotting activity at pH 5.5 and 45°C in the presence of 0.01 M CaCl₂. The enzyme was stable at least 24 h at 35°C in the pH range of 5.5–7.0. Thermal denaturation started above 45°C. Fresh cheese manufactured with reconstituted cow milk and the purified enzyme showed similar basic characteristics (pH 4.5, acid taste, white color) as marketed cheeses obtained with calf rennet. This emphasizes the value of exploiting local biological resources for value added food processing in developing countries.     

Thermal Transitions of Salt-soluble Proteins from Pre- and Postrigor Chicken Muscles

Salt-soluble protein (SSP) was extracted from pre- and postrigor chicken muscles at various pH values, and protein thermal denaturation was studied using several techniques. Heating at 1°C/min from 20 to 70°C induced a three- to fourfold increase in breast and leg hydrophobicity. Differential scanning calorimetry of breast and leg SSP showed a major transition occurring within the range 55 to 64°C, with the value dependent on rigor state and pH. Protein-protein association, as measured by turbidity change upon heating, underwent two transitions for leg SSP and two or three for breast SSP. The specific transition temperature and rate were dependent on pH, muscle type and rigor state. However, muscle type and pH had a greater effect than muscle rigor state on SSP denaturation.     

Denaturation of Cod Myosin during Freezing after Modification with Formaldehyde

Formaldehyde at concentrations occurring in frozen fish increased the extent of denaturation as measured by loss in solubility, decrease in ATPase activity and increase in surface hydrophobicity, of partially purified cod myosin stored at -25°C or -80°C. Most denaturation occurred during the freezing/thawing process. Formaldehyde caused some crosslinking of myosin, but crosslinking and insolubilization appeared to be relatively independent. The presence of diamines led to some increase in crosslinking but had little effect on protein solubility.     

Comparison of the Proteolytic Activities of New Commercially Available Bacterial and Fungal Proteases toward Meat Proteins

The hydrolytic activity of 3 commercially available protease preparations (bacterial protease G, fungal 31000, and fungal 60000) were examined using fluorescent‐labeled casein, azo dye‐impregnated collagen, and meat protein extracts from bovine M. semimembranosus and Achilles tendon, and compared to that of papain. Assays showed that all proteases exhibited little activity at low temperature (5 °C), and maximal activity at 45 °C. The pH, at which optimal activity was observed for each of the protease preparations, differed and ranged from pH 5.0 to 8.0. Kinetic parameters (K_M and V_max) were also different between protease preparations, with the bacterial protease G and papain exhibiting significantly higher V_max values (P < 0.001) and lower K_M values (P < 0.01) for the casein substrate than the 2 fungal protease preparations. Meat protein hydrolysis was displayed on SDS‐PAGE and proteins analyzed with mass spectrometry. The protease preparations were shown to have varying affinity toward different meat proteins. The bacterial protease G preparation was efficient at hydrolyzing most myofibril and collagen proteins, and appeared to be more efficient than papain at hydrolyzing collagen proteins. On the other hand the 2 fungal protease preparations showed a selective specificity toward meat myofibrillar proteins, and the fungal 60000 protease preparation exhibited high affinity toward collagen γ and collagen type I chain B proteins. The results generated in this study demonstrated that these commercial proteases have good potential for use in meat tenderization applications due to their mild and complementary effects on different meat proteins. Practical Application : Bacterial and fungal protease preparations exhibited varying affinities for hydrolyzing meat proteins. This selective moderate capability of microbial proteases compared to papain is potentially an advantage in avoiding over‐tenderization in meat. On the other hand, the bacterial protease G preparation, which appeared to be more efficient at hydrolyzing connective tissue proteins than papain, could be beneficial in tenderizing meat with high connective tissue content. The synergistic effect of these protease preparations could be incorporated into a meat tenderizing formula to give the tenderizer a broad activity spectrum, thus able to target different cuts of meat.     

Thermal stability of fish myofibrils: a differential scanning calorimetric study

The variation in myofibrillar protein thermostability was compared for various fish species, using differential scanning calorimetry. The tropical fish, catfish ( Clarius gariepinus ), carp ( Cyprinus carpio ), Nile perch ( Lates niloticus ), red snapper ( Lutianus sebae ), red mullet ( Parpeneus barberinus ), sea bream ( Gymnocranius rivalatus ), and cold-water reared trout ( Salmo gairdneri ) and cod ( Gadus morhua ) were analysed. Onset temperature of myofibrillar protein denaturation occurred at up to 11°C higher for tropical species (43.5°C, catfish), than cod (32.6°C) at pH 7 and low ionic strength (I). As pH (6.0-8.0) and I (0.05-1.00) were increased, thermal denaturation temperatures of myosins from tropical, but not cold-water, species decreased. Enthalpies of myofibrillar denaturation decreased for all species with increasing pH and I. Only one thermal transition was detected for myosin at pH 6 and low I, increasing to three as pH and I were increased. Changes in thermal characteristics of myosin subunits over iced and frozen storage suggest more rapid deterioration in cold-water than in tropical fish. The differences in myofibrillar stability of fish from different habitat temperatures have implications for the processing and storage of tropical fish.     

HEAT TREATMENT EFFECT ON TEXTURE CHANGES and THERMAL DENATURATION of PROTEINS IN BEEF MUSCLE

Meat tenderness is one of the most important quality criteria when evaluating results of cooking conditions. Changes in tenderness and weight losses of heat-treated meat (semitendinosus muscle) for different time-temperature combinations were analyzed; the relationship between protein denaturation and textural changes was studied.
Heat treatments of meat samples (1.5 cm in diameter, 2 cm long) were performed in a thermostatic bath in the 60–90C range. Maximum heating times were 180 min. Meat hardness was determined by Warner-Bratzler measurements using an Instron testing machine. Protein denaturation was followed by Differential Scanning Calorimetry (DSC) analyzing peaks for myosin (I and II), sarcoplasmatic proteins and collagen (II) and actin (III).
Between 60 and 64C, hardness decreased with cooking time until reaching the lowest asymptotic values. This was related to protein denaturation of peak I and II. Between 66 and 68C, hardness decreased at first but increased later due to actin denaturation; at the temperatures 81 and 90C no modifications were observed and hardness remained at its higher values.
The kinetic model proposed fit the experimental results satisfactorily. Activation energies of tenderizing and toughening processes are similar to those of protein denaturation of peak II and III. Weight losses due to cooking were also modelled increasing through the entire temperature range.     

Effects of Kudoa spores,endogenous protease activity and frozen storage on cooked texture of minced Pacific hake (Merluccius productus)

The effects of Kudoa infection of Pacific hake (Merluccius productus) on endogenous protease activity and on cooked mince texture were investigated. Texture was significantly (p < 0.05) negatively correlated with spore counts as well as protease activity. Soft texture (maximum force <150 g) was observed in fish with 10⁴–10⁶Kudoa thyrsites spores g⁻¹ mince, compared to 10⁵–10⁸K. paniformis spores g⁻¹ mince, suggesting that especially for fish having lower infection levels, K. thyrsites may have a greater impact than K. paniformis on Pacific hake quality. Pre-incubation for 15 min at 52 °C prior to cooking resulted in softer texture in some samples due to endogenous proteolytic action. This pre-incubation effect was not consistently observed in fish held 6 months or longer at −25 °C or after freeze-thaw cycling, which may be explained by an opposing toughening effect attributed to protein denaturation and aggregation during prolonged or abusive frozen storage.