Broad‐spectrum inhibition of proteolytic enzymes by allicin and application in mitigating textural deterioration of ice‐stored grass carp (Ctenopharyngodon idella) fillets

The inhibitory effect of allicin on proteolytic enzymes and textural deterioration of ice‐stored grass carp (Ctenopharyngodon idella) fillets was investigated. The results of in vitro study showed that allicin inhibited the activity of cathepsin B, L and D, calpain and collagenase in crude extract of grass carp muscle. Among endogenous enzymes, cathepsin B, L and collagenase were the most susceptible to allicin. Proteolysis of myofibrillar proteins by either crude enzyme or cathepsin B and L was almost prevented by allicin when employed at a concentration higher than 100 mm . After storage of 21 days, shear force of fillets treated with allicin at 10–100 mm was 39–51% higher than that of control. Myofibrillar proteins of fillets during storage were well protected against degradation when allicin concentration increased to 100 mm , as evidenced by SDS‐PAGE. Therefore, allicin could be a potential broad‐spectrum inhibitor to retard softening of fish fillets via mitigating myofibrillar proteolysis by endogenous enzymes especially cathepsin B and L during ice‐storage.     

Effects of Chilling and Partial Freezing on Rigor Mortis Changes of Bighead Carp (Aristichthys nobilis) Fillets: Cathepsin Activity,Protein Degradation and Microstructure of Myofibrils

To investigate the effects of chilling and partial freezing on rigor mortis changes in bighead carp (Aristichthys nobilis), pH, cathepsin B, cathepsin B+L activities, SDS‐PAGE of sarcoplasmic and myofibrillar proteins, texture, and changes in microstructure of fillets at 4 °C and –3 °C were determined at 0, 2, 4, 8, 12, 24, 48, and 72 h after slaughter. The results indicated that pH of fillets (6.50 to 6.80) was appropriate for cathepsin function during the rigor mortis. For fillets that were chilled and partially frozen, the cathepsin activity in lysosome increased consistently during the first 12 h, followed by a decrease from the 12 to 24 h, which paralleled an increase in activity in heavy mitochondria, myofibrils and sarcoplasm. There was no significant difference in cathepsin activity in lysosomes between fillets at 4 °C and –3 °C (P > 0.05). Partially frozen fillets had greater cathepsin activity in heavy mitochondria than chilled samples from the 48 to 72 h. In addition, partially frozen fillets showed higher cathepsin activity in sarcoplasm and lower cathepsin activity in myofibrils compared with chilled fillets. Correspondingly, we observed degradation of α‐actinin (105 kDa) by cathepsin L in chilled fillets and degradation of creatine kinase (41 kDa) by cathepsin B in partially frozen fillets during the rigor mortis. The decline of hardness for both fillets might be attributed to the accumulation of cathepsin in myofibrils from the 8 to 24 h. The lower cathepsin activity in myofibrils for fillets that were partially frozen might induce a more intact cytoskeletal structure than fillets that were chilled.     

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.     

Proteolytic degradation of myofibrillar components by carp cathepsin L

Carp cathepsin L, which is the best candidate to produce textural change in the arai-treated carp fillet, exhibited maximum hydrolytic activity for Z-Phe-Arg-MCA and soluble casein at pH 5·0–5·5. The proteolytic action of the enzyme was evaluated by complete degradation of various carp myofibrils at pH 5·0 over 30 min and by potent degradation of the same proteins at pH 5·5–6·0 over 20 h. All myofibrillar components were partially degraded by the enzyme at pH 6·5–7·0, but varying amounts of them remained undegraded after 20 h. These findings indicate that carp cathepsin L degrades not only carp myofibrillar components but also their resultant products between pH 5·0 and 7·0 and that it markedly acts on myosin heavy chain, α-actinin and troponin-T and -I. Carp cathepsin L likely contributes to postmortem muscle tenderisation of carp fillet over an extensive pH range during storage. © 1998 SCI.     

The impact of collagen on softening of grass carp (Ctenopharyngodon idella) fillets stored under superchilled and ice storage

The objective of this work was to study the impact of collagen on softening of grass carp (Ctenopharyngodon idella) fillets during chilled storage. The fillets were stored under superchilling (?1.5 ± 0.2 °C) and with ice (0.2 ± 0.1 °C) for 21 days, and texture properties, collagen and the related enzyme activities were measured. Results showed that firmness and collagen content were strongly influenced by storage temperature and time. Fillet firmness decreased by 32.3% (superchilling) and 49.6% (ice stored) of the initial values after 3 days of storage, respectively. Total collagen content decreased with time, but different collagen fractions showed variations. Collagen degraded to different extents depending on storage conditions as indicated by SDS‐PAGE and amino acid analysis. In addition, collagenase activity declined significantly during the first 3 days, followed by a slow increase. This study demonstrated that collagen degradation was involved in grass carp fillet softening and provided useful information for fillet quality improvement.     

Inhibition of endogenous protease activity and protection of histomorphical integrity during refrigerated storage of grass carp fillets by treatment with natural edible di- and tri-carboxylic acids

In this study, by dipping grass carp fillets with 10 mM di- and tri-carboxylic acid solutions individually before storage at 4 °C for 10 days, the application potential of these acids in mitigating softening of fish was investigated through analysing shear force, pH, endogenous cathepsin activities, myofibrillar proteolysis and histomorphological characteristics. The results suggested that maleic acid, fumaric acid and tartaric acid were significantly effective against softening of grass carp muscle, as shown by shear force 26%–31% higher than that of control after storage. Acid treatments led to a decline in the pH of muscle. Besides, the inhibitory effect of these acids on cathepsins was demonstrated by activity reduction by 20%–90%. Further, sodium dodecyl sulphate–polyacrylamide gel electrophoresis and heat shock protein (HSP) analysis supported the protection of these acids against degradation and dissociation of myosin and actin through suppressing HSP loss. Moreover, histomorphological images with higher intensity of acridine orange fluorescence in muscle revealed the improvement of tissue integrity by acid treatments. The inhibitory potential of these acids in suppressing softening of fish muscle is particularly relevant to their capacity of mitigating cathepsin-induced proteolysis and improving lysosome stability. Therefore, di- and tri-carboxylic acids could play pleiotropic functions in the quality control of fish fillets.     

Myosin heavy chain degradation during post mortem storage of Atlantic cod (Gadus morhua L.)

Post mortem proteolytic degradation of fish fillets leads to textural changes like muscle softening and gaping. In this study proteolytic degradation of myosin heavy chain (MHC) was monitored during storage of muscle and of isolated myofibrils at different temperatures and pH-values by the use of MHC-specific antibodies. The ability of cathepsin D to associate to myofibrillar proteins was also studied. Muscle stored at 6 °C and isolated myofibrils stored at 0 °C, 6 °C and 20 °C were degraded at pH 6.3 or lower. Cathepsin D could be found associated with extensively washed myofibrils. Inhibition of cathepsin D during storage affected the observed MHC-degradation at pH 5.5, but not at pH 6.3. This indicates that cathepsin D to a less extend than formerly believed, is responsible post mortem degradation of MHC.     

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.     

Cathepsin Degradation of Pacific Whiting Surimi Proteins

Cathepsin B was the most active cysteine protease in Pacific whiting fish fillets; cathepsin L was predominant in surimi. Cathepsin L showed highest activity at 55°C in both fish fillets and surimi, indicating its function in myosin degradation during conventional heating of fillets and surimi, gels. Washing during surimi processing removed cathepsin B and H but not cathepsin L. Myosin heavy chain was the primary substrate during autolysis of surimi paste and actin and myosin light chain showed limited hydrolysis during 2 hr incubation. Purified Pacific whiting cathepsin L hydrolyzed myofibrils, myosin and native and heat-denatured collagen. The degradation pattern of myofibrils by the protease was the same as the autolytic pattern of surimi.     

Textural and ultrastructural changes during processing and storage of lightly preserved salmon (Salmo salar) products

The effect of a controlled lactic fermentation on textural and ultrastructural properties of salmon fillets was evaluated. When compared with a non‐inoculated cured sample, fermented salmon had unique textural properties since it exhibited significantly higher force (p < 0.05) and work (p < 0.001) values in a penetration test on two sampling days but a consistently lower hardness (p < 0.05) in a Texture Profile Analysis (TPA) rheological test. Study of the myofibrillar ultrastructure showed that basic structures were drastically damaged during processing and storage of fermented and cured salmon. Nevertheless, Z‐lines were better preserved in fermented samples. It is suggested that the slight protective effect of lactic fermentation with the starter L sake may be the consequence of a lower pH, by putatively inhibiting neutral or alkaline proteolytic enzymes. © 2000 Society of Chemical Industry     

Relationship between proteolytic changes and tenderness in prerigor lactic acid marinated beef

A meat tenderising procedure involving injection of a lactic acid solution into prerigor muscle was investigated using beef M pectoralis profundus. The distribution of lysosomal enzymes in subcellular fractions, densities of myofibrillar protein bands after SDS‐PAGE and shear force were measured in non‐injected, 0.5 M and 1.0 M lactic‐acid‐injected samples during a 21 days ageing period. The activities of cathepsin B + L and β‐glucuronidase in the soluble fraction increased with level of lactic acid and with time post‐mortem (P < 0.001). Lactic acid and storage decreased densities of SDS‐PAGE bands migrating at the position of myosin heavy chain (MHC) and α‐actinin and increased densities of a 150 kDa band (P < 0.01). SDS‐PAGE of isolated perimysium cleaved with CNBr showed proteolytic cleavage of collagen after prolonged storage. Lactic acid injection significantly reduced shear force (P < 0.001). The cathepsin B + L activity in the soluble fraction correlated to shear force (r = −0.8), the degradation of MHC and α‐actinin (r = −0.88 and −0.90) and the generation of the 150 kDa fragment (r = 0.90) but not to the generation of a 31 kDa fragment (r = 0.05). A major part of the tenderness improvement after lactic acid injection was complete at 24 h post‐mortem, and was therefore due to a rapid process, eg pH‐induced swelling of the muscle structure. The data on enzyme activities and protein degradation, however, suggested that the action of lysosomal cathepsins also contributed to textural changes. © 1999 Society of Chemical Industry     

Relevance of collagen solubility and gelatinolytic proteinase activity for texture softening in chilled grass carp (Ctenopharyngodon idellus) fillets

The aim of the present study was to investigate the underlying mechanisms of softening texture in chilled grass carp filletsunderpinning collagen solubility, gelatinolytic proteinase activity and physicochemical parameters. Acid-soluble collagen (ASC) and heat soluble collagen (HSC) increased markedly, while a significant decline was detected in total collagen and insoluble collagen (ISC) during the first 3 days of storage, coinciding with a loss of shear force and water-holding capacity (WHC) (P < 0.05). Moreover, the activity of gelatinolytic proteinases was gradually activated and reached the peak at day 3 (P < 0.05). Pearson coefficient analysis showed that gelatinolytic proteinase activity revealed a significant correlation with collagen solubility. Total collagen, ASC, ISC and HSC were significantly correlated with shear force and WHC. Our study clarified that the increase of collagen solubility by gelatinolytic proteinases played an important role for texture softening in the early stage of chilled grass carp fillets.     

Changes on enzymatic activity and on sarcoplasmic and myofibrillar proteins of frozen-stored hake (Merluccius merluccius) pre-treated by high pressure

High-pressure (HP) pre-treatments were applied on European hake (Merluccius merluccius) followed by a frozen accelerated experiment (−10 °C). A central composite design ranging pressure levels (150–450 MPa) and frozen storage time (0–150 days) was used, being evaluated the enzymatic activities and muscle proteins. Acid phosphatase and calpain activities decreased after 150 days of frozen storage (58%/56% and 38%/56% for non-/HP-treated samples, respectively). Cathepsin B showed higher reductions (98%) for longer storage times. Furthermore, HP and frozen storage did not affect significantly cathepsin D activity, only slightly decreasing at 169 MPa. Furthermore, HP seemed to not affect myofibrillar proteins, while sarcoplasmic proteins were clearly affected by HP and frozen storage time, resulting in a reduction of about 53% or 23% for 431 or 450 MPa, respectively. Thus, HP could be used to lowering the deleterious effect of proteases on frozen European hake.     

Quality changes and microbiological spoilage analysis of air‐packed and vacuum‐packed silver carp (Hypophthalmichthys molitrix) fillets during chilled storage

The effects of air‐packed (AP) and vacuum‐packed (VP) on quality and microbial characteristics of silver carp (Hypophthalmichthys molitrix) fillets during chilled storage (4 ± 1 °C) were investigated. The fillets were analyzed for sensory scores, total volatile basic nitrogen (TVB‐N), ATP‐related compounds (ATP, IMP, HxR, and Hx), K value, and biogenic amines (BAs). The results proved that VP inhibited the increase of microorganisms, TVB‐N, Hx, and putrescine in silver carp, and slowed the reduction in sensory score. Therefore, VP can be applied for preservation of the silver carp fillets to improve its quality. For identification, 16S rRNA genes of the isolated pure strains were sequenced and analyzed. On the initial day of storage, Chryseobacterium was the dominant bacterial genus. At the end of shelf life, Pseudomonas was the most common group in AP fillets and Aeromonas followed by Yersinia were found mainly in VP samples.

Practical applications

Silver carp (Hypophthalmichthys molitrix) are distributed widely in fresh water systems. The world aquaculture production of silver carp was 4,354,638 tons, and it ranked second highest among freshwater fish species in 2015, but they are perishable during storage because of microbial spoilage and biochemical reactions. Vacuum packaging (VP) has proved to be effective for extending the shelf‐life of aquatic products by excluding oxygen that prevents the growth of spoilage bacterial. However, little information is available on the microbial succession of VP silver carp. Therefore, this work was to determine the differences of microbiological succession on chilled silver carp fillets under air‐packed (AP) and VP conditions using a combination of culture‐based and 16S rRNA gene analysis methods. Furthermore, this study will give valuable information about development and spoilage of VP silver carp fillets.     

Effects of chitosan coating combined with essential oils on quality and antioxidant enzyme activities of grass carp (Ctenopharyngodon idellus) fillets stored at 4 °C

Effects of chitosan coating combined with essential oils from clove, cinnamon and lemon grass on quality and antioxidant enzyme activities of grass carp fillets stored at 4 ± 0.5 °C were evaluated. The quality parameters (including pH, total volatile basic nitrogen (TVB‐N), K value, thiobarbituric acid (TBA) value, shear force and total viable count (TVC)) and antioxidant enzyme activities were analysed periodically. The results indicated that composite chitosan coatings presented better preservation effects than chitosan coating alone. In addition, chitosan–clove essential oil coating had the best quality enhancement effects among treatments by inhibiting deterioration of physicochemical quality and microbial growth and maintaining antioxidant enzyme activities of fillets during refrigerated storage. The negative correlation was observed between the changes in TBA values and antioxidant enzyme activities in fillets. Based on these findings, chitosan–essential oil coatings effectively mitigated oxidative stress and extended shelf life of refrigerated grass carp fillets.     

Application of Artificial Neural Network to Predict K-Value,Inosine Mono-Phosphate,and Hypoxanthine Concentrations of Grass Carp (Ctenopharyngodon idellus) Fillets During Storage

K-value, inosine mono-phosphate, and hypoxanthine concentrations of grass carp (Ctenopharyngodon idellus) fillets were determined during storage at 273, 277, 281, 288, and 293 K. Simultaneously, a feed-forward artificial neural network was developed to predict these changes in grass carp fillets during storage, and a comparative study on K-value prediction between the artificial neural network and Arrhenius model was also performed. The results showed that the K-value and hypoxanthine concentrations increased with storage time, while inosine mono-phosphate reached a peak and then decreased with time. The artificial neural network was successful in predicting changes in the K-value, inosine mono-phosphate, and hypoxanthine concentrations throughout storage, and it was even more effective in predicting K-value with lower relative errors than the Arrhenius model. The high regression coefficient (R²) and low mean squared error indicated that the artificial neural network could be a potential tool in modeling changes in K-value, inosine mono-phosphate, and hypoxanthine concentrations of grass carp fillets within 273–293 K.     

Effects of konjac glucomannan on physicochemical properties of myofibrillar protein and surimi gels from grass carp (Ctenopharyngodon idella)

The cryoprotective effect of konjac glucomannan (KGM) on myofibrillar protein from grass carp (Ctenopharyngodon idella) during frozen storage at −18 °C and the influence of five levels of KGM (0%, 0.5%, 1%, 1.5%, and 2%) on texture properties, water-holding capacity, and whiteness of grass carp surimi gels were investigated. KGM as a novel cryoprotectant could significantly mitigate the decrease in salt extractable protein (SEP), Ca²⁺-ATPase activity, and total sulphydryl and active sulphydryl contents of myofibrillar protein during frozen storage. KGM at the level of 1% showed the same good cryoprotective effect as a conventional cryoprotectant (10% sucrose–sorbitol, 1:1, w/w). As the levels of KGM increased, breaking force and deformation of grass carp surimi gels increased significantly. Water-holding properties of the surimi gels are improved with the increasing addition of KGM, but the whiteness decreased and the colour became darker. The optimum addition level of KGM was suggested to be 1%.     

Modelling quality changes in Songpu mirror carp (Cyprinus carpio) fillets stored at chilled temperatures: comparison between Arrhenius model and log‐logistic model

To understand the quality changes in Songpu mirror carp (Cyprinus carpio) fillets during chilled storage, sensory score, total aerobic counts (TAC), total volatile basic nitrogen (TVB‐N) and electrical conductivity (EC) of the fillets stored at 270, 273, 276, 282 and 288 K were investigated. Furthermore, both Arrhenius model and log‐logistic model were established to predict the quality changes. The high regression coefficients (R² > 0.90) represented the acceptability of both Arrhenius model and log‐logistic model. Relative errors between predicted values and observed values were all within ±5% for models based on EC, within ± 25% for models based on TAC and TVB‐N (except the TVB‐N value on 9th and 15th day). The relative errors of sensory score were all within ±35% in the first 9 days. It is concluded that both Arrhenius model and log‐logistic model based on EC, TAC or TVB‐N can be applied in modelling quality changes in Songpu mirror carp fillets during storage within 270–288 K.     

The impact of desmin on texture and water‐holding capacity of ice‐stored grass carp (Ctenopharyngodon idella) fillet

The objective of this work was to analyse the role of desmin in texture softening and water‐holding capacity (WHC) of ice‐stored grass carp fillet. Results indicated that shear force of the fillet decreased sharply within 3 days, while drip loss increased during the whole ice storage. Water mobility and distribution were measured by low‐field ¹H NMR T₂ relaxation that revealed a positive correlation (P < 0.01) between WHC and the mobility of the immobilised water (T₂₁ relaxation time). Meanwhile, negative correlation could be established between intact desmin and drip loss (P < 0.01). Intact desmin was extremely correlated with shear force (P < 0.01) and positively correlated with calpains activity (P < 0.05). This study demonstrated that the degradation of desmin was involved in the mobility of the myofibrillar water and softening of grass carp fillet during ice storage. Furthermore, calpains autolysis seemed to result in desmin degradation.     

Myofibrillar protein degradation of carp (Labeo rohita (Hamilton)) muscle after post‐mortem unfrozen and frozen storage

The degradation of myofibrillar proteins of rohu carp (Labeo rohita (Hamilton)) muscle was analysed after post‐mortem storage. Muscle fillets were kept either unfrozen at 2 °C for up to 15 days or frozen at ?8 °C or ?20 °C for up to 6 months. A co‐ordinated histochemical, biochemical and electrophoretic study showed a differential response of the carp muscle, revealing clear degenerative/degradative changes specific to the post‐mortem storage temperatures. The myofibrillar protein fractions, namely myosin light chains and α‐actinin, showed degradative changes during the above storage conditions, whereas other protein fractions in the high‐molecular‐weight range fragmented to give lower‐molecular‐weight proteins. The importance of the post‐mortem storage temperature for controlling the degradation of the myofibrillar proteins was emphasised. This is the first report on this popular fish species, known for its culinary importance, showing that specific protein fractions of the myofibrils degrade during post‐mortem storage. © 2001 Society of Chemical Industry