Freshness indicator protein, endogenous protease related to quality traits of mandarin fish during low-temperature storage process and application

Abstract

The present application relates to a freshness indicator protein related to quality traits of low-temperature stored Siniperca chuatsi, endogenous proteases and applications. The freshness indicator endogenous proteases are related to postmortem proteolysis and meat softening of Siniperca chuatsi, including cathepsin B, cathepsin L and calpain. The freshness indicator proteins are related to quality changes occurring during postmortem cold storage of Siniperca chuatsi, including myoactin 1, myosin binding protein, LDB protein, actin-related protein, etc. The present application determines the change rule of endogenous protease activity of Siniperca chuatsi at different storage stages, and the dynamic influence on muscle proteolysis and muscle degradation of Siniperca chuatsi, and reveals the internal relationship with fish meat sensory quality and processing characteristics, and clearly defines the "critical point" of quality deterioration of Siniperca chuatsi. It provides clear guidance for the control of endogenous enzyme-mediated quality deterioration in the processing and storage process of Siniperca chuatsi.

Description

Technical Field

[0001] This invention relates to the field of fish product technology, and in particular to a freshness indicator protein, an endogenous protease, and their applications related to the quality traits of mandarin fish during low-temperature storage. Background Technology

[0002] Mandarin fish (Siniperca chuatsi) is a valuable edible fish widely distributed in major rivers and lakes of my country. It has tender flesh, high nutritional value, and significant economic value, with market demand showing a steady upward trend. The related processing industry for mandarin fish has significant economic benefits. However, due to its high stress tolerance, poor tolerance to low oxygen levels, and high mortality rate after being caught, mandarin fish requires stringent processing, transportation, and storage conditions. Furthermore, the sale of live mandarin fish is seasonally limited by market supply. Therefore, developing timely and effective preservation technologies is crucial for the development of the mandarin fish industry, driven by the need to expand the distribution range of mandarin fish products and extend their shelf life.

[0003] Low-temperature preservation technology is currently the most commonly used method for preserving aquatic products due to its low damage to raw materials and low cost. However, the quality deterioration of fish meat during low-temperature preservation has become a bottleneck problem that the freshwater fish processing industry urgently needs to solve. Shark muscle is tender and rich in endogenous enzymes and psychrophilic microorganisms, participating in its muscle protein metabolism and post-mortem biochemical reactions, maintaining high activity even under refrigeration conditions. After fish death, the apoptosis program is initiated, and changes in the biochemical environment lead to the activation of endogenous proteases. Cathepsins and calpains, present in lysosomes, are released from the damage sites in the fish muscle, causing muscle decomposition and softening. Many studies have shown that cathepsins and calpains are closely related to protein degradation and quality deterioration in aquatic products, with the degradation of cytoskeletal proteins being the fundamental cause of softening, decreased freshness, and reduced taste. The cathepsins that cause textural deterioration are mainly B, L, and D; environmental temperature, pH value, and muscle ionic strength may have a synergistic effect with endogenous muscle protein hydrolases. Therefore, by studying the changes in enzyme activity related to protein degradation in mandarin fish, the effects of endogenous enzymes on the main muscle protein—myofibrillar protein, and the activation and synergistic effects of endogenous enzymes in fish meat, it is of great significance to explore the biochemical mechanisms of post-mortem quality changes in mandarin fish and to control and identify the quality of mandarin fish.

[0004] Because fish possess a diverse range of endogenous proteases with varying functions, participating in different major biochemical reactions and playing different roles at different stages of fish life, the detailed mechanisms underlying the synergistic effects of multiple endogenous proteases on texture and quality degradation during low-temperature preservation of fish meat remain unclear. Therefore, proteomics technology is needed to elucidate these complex biochemical processes. Previous reports have utilized proteomics to study changes in muscle proteins in different fish species during storage. Related studies used 2-DE proteomics to assess the freshness of turbot under 4°C refrigeration conditions, finding that changes in the abundance of phosphoglucose mutase 1, pyruvate kinase, myosin heavy chain 1, troponin T, desmin, and actin were significantly correlated with freshness indicators QI, K value, TVB-N, and TVC. These key proteases and differentially expressed proteins can serve as freshness markers related to turbot quality. Cao used iTRAQ quantitative proteomics to reveal the impact of thawing methods on the quality of largemouth bass fillets. 47 proteins were identified as differentially expressed proteins, of which 14 were significantly associated with pH, ​​color, cooking loss, and centrifugation loss. This study elucidated the biomolecular signaling mechanism of quality changes in largemouth bass fillets from a proteomics perspective.

[0005] Currently, there are no reported studies on the proteomics of mandarin fish. The impact of endogenous proteases on myofibrillar protein degradation during fish storage remains unclear, and there is a lack of evidence regarding key proteases causing fish quality deterioration and protein indicators of freshness changes. This project aims to study the quality changes, protein degradation, and endogenous enzyme mechanisms of mandarin fish during low-temperature preservation. Using proteomics, we will explore the protein metabolic pathways leading to quality deterioration in low-temperature preserved mandarin fish and the impact of endogenous proteases on muscle protein degradation. The goal is to obtain freshness indicator proteins and key proteases related to the quality traits of mandarin fish during low-temperature storage, revealing at the molecular level the mechanism by which endogenous enzymes regulate the quality changes of low-temperature preserved mandarin fish. This will provide theoretical and practical guidance for controlling the quality deterioration of low-temperature preserved mandarin fish and developing green biological preservation technologies for high-quality freshwater fish processed products. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides a freshness indicator protein, an endogenous protease and their application related to the quality traits of mandarin fish during low-temperature storage.

[0007] To achieve the objectives of this invention, the technical solution is as follows:

[0008] An endogenous freshness indicator protease related to the quality traits of mandarin fish during low-temperature storage, which is associated with post-mortem protein hydrolysis and meat softening of mandarin fish, wherein the endogenous freshness indicator protease includes cathepsin B, cathepsin L and calcium-activated protease.

[0009] A freshness indicator protein related to the quality traits of mandarin fish during low-temperature storage is disclosed, which is associated with quality changes that occur during post-slaughter cold storage of mandarin fish. The freshness indicator protein includes myogenin-1b, myosin-binding protein H, LDB domain-binding protein, nebulin, troponin I (TnI), myosin heavy chain 9 (Myosin-9), adenylate cyclase-associated protein 1 (CAP1), phosphoglycerate kinase 1 (PGK1), phosphoglycerate mutase (PGAM2), triose phosphoryl isomerase (TPI1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycogen phosphorylase L (PYGL), aldolase A (aldolase a), fructose diphosphate (ALDOA), heat shock protein B1 (HSPB1), and hemoglobin.

[0010] The endogenous protease or freshness indicator protein related to the quality traits of mandarin fish during the low-temperature storage process is used for the preservation control of mandarin fish.

[0011] The beneficial effects of this invention are as follows:

[0012] 1. To determine the changing patterns of endogenous protease activity in mandarin fish at different storage stages, and its dynamic impact on muscle protein hydrolysis and degradation, and to reveal its intrinsic relationship with the sensory quality and processing characteristics of the fish, clearly defining the "critical point" of quality deterioration in mandarin fish. This will provide clear guidance for controlling endogenous enzyme-mediated quality deterioration during the processing and storage of mandarin fish.

[0013] 2. To elucidate the correlation between endogenous protease activity at the molecular level and macroscopic quality changes in mandarin fish, and to identify key endogenous proteases and differentially expressed proteins related to quality characteristics using proteomics technology; and to screen protein biomarkers that indicate the freshness of mandarin fish through bioinformatics analysis. This will provide theoretical and technical support for the low-temperature preservation of specialty freshwater fish from the biochemical perspective of enzyme activity inhibition. Attached Figure Description

[0014] Figure 1 pH changes in mandarin fish stored at 4℃. Note: Different letters indicate significant differences between samples (P < 0.05).

[0015] Figure 2 Changes in water-holding capacity of mandarin fish stored at 4℃. Note: Different letters indicate significant differences between samples (P < 0.05). Figure 3Changes in myofibril fragmentation index of mandarin fish stored at 4℃. Note: Different letters indicate significant differences between samples (P < 0.05).

[0016] Figure 4 Color changes of mandarin fish stored at 4℃. Note: Different letters indicate significant differences between samples (P < 0.05).

[0017] Figure 5 SDS-PAGE of mandarin fish meat stored at 4℃;

[0018] Figure 6 Changes in cathepsin B activity in mandarin fish stored at 4℃; Note: Different letters indicate significant differences between samples (P < 0.05), * indicates significant differences between two groups of samples (P < 0.05).

[0019] Figure 7 Changes in cathepsin L activity in mandarin fish stored at 4℃; Note: Different letters indicate significant differences between samples (P < 0.05), * indicates significant differences between two groups of samples (P < 0.05).

[0020] Figure 8 Changes in the activity of calcium-activated protease in mandarin fish stored at 4℃; Note: Different letters indicate significant differences between samples (P < 0.05), * indicates significant differences between two groups of samples (P < 0.05).

[0021] Figure 9 Correlation analysis between quality indicators and endogenous proteases of mandarin fish under 4℃ refrigeration conditions; Note: * Significant differences exist between different representative samples (P < 0.05); A: Blank group; B: Treatment group

[0022] Figure 10 Volcano plot of differentially expressed proteins (DEPs) in mandarin fish on day 0 and day 5 (A). Red dots represent upregulated DEPs (FC>1.2, P<0.05), and green dots represent downregulated DEPs (FC<0.83, P<0.05). Hierarchical cluster analysis of differentially expressed proteins in mandarin fish on day 0 vs. day 5 (B) (blue represents lower intensity, red represents higher intensity, and white represents undetectable changes). Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited to the scope described in the embodiments.

[0024] Example 1

[0025] 1. Quality changes of mandarin fish during storage

[0026] 1.1 pH value change

[0027] like Figure 1 As shown, the pH values ​​of the mandarin fish muscle in the control group and the treatment group were 7.2 and 7.12 on day 0, respectively. The pH value fluctuated during storage. On day 10, the pH value of the mandarin fish muscle in the control group decreased compared to day 0, while the pH value of the treatment group increased compared to day 0. The initial pH decrease was mainly due to the production of lactic acid from anaerobic respiration and the accumulation of phosphate from ATP degradation. The later decrease in pH value in the control group may be due to the metabolism of nutrients in the fish meat by microorganisms, producing acidic metabolites. The later increase in pH value in the treatment group may be due to the degradation of proteins by endogenous proteases, producing basic amines.

[0028] 1.2 Changes in water holding capacity (WHC)

[0029] Figure 2 The changes in water-holding capacity of mandarin fish meat stored at 4℃ for 10 days were observed: the water-holding capacity of fresh mandarin fish muscle was 75% (control group) and 78% (treatment group), respectively. The water-holding capacity of both groups generally showed a trend of first increasing and then decreasing. Except for day 7, the water-holding capacity of the treated group was higher than that of the control group. This may be because the protein reactions (hydrolysis, oxidation, denaturation) in the control group were more intense, leading to a weakened ability of the muscle to retain water, thus causing water loss from the fish meat tissue.

[0030] 1.3MFI Changes

[0031] Changes in MFI of mandarin fish meat stored at 4℃ are as follows: Figure 3 As shown: Both groups of MFI values ​​showed an increasing trend, indicating that the myofibril structure of the fish meat was damaged and broken as the storage time increased. The MFI value of the control group increased by 2.3 times on day 10, while the MFI value of the treatment group increased by 1.9 times, indicating that microbial proliferation has a certain promoting effect on the increase of MFI value, but the degradation of myofibril protein is still mainly dominated by the action of endogenous proteases. 1.4 Color changes of mandarin fish during storage

[0032] Meat color is a decisive factor influencing consumer purchasing behavior and also a significant factor affecting the shelf life of fresh meat. It can be directly used as an important sensory indicator of meat quality. During storage, changes in fish meat color occur due to fat oxidation, protein denaturation, and pigment degradation.

[0033] L*, Whiteness: With prolonged storage, the brightness and whiteness values ​​of both groups of mandarin fish continuously decreased, with significant differences (p < 0.05). The continuous decrease in brightness value indicates that the luster of the mandarin fish muscle decreased during storage, and the muscle color gradually darkened. The color was relatively bright in the early stage of storage, but became slightly duller in the later stage, and the acceptability of the mandarin fish meat gradually decreased. This is because the accumulation of methemoglobin and methemoglobin in the muscle tissue on the surface of the mandarin fish meat led to a browning reaction.

[0034] a*: After the mandarin fish is slaughtered, the purplish-red reduced myoglobin combines with oxygen under high oxygen partial pressure to form bright red oxymyoglobin, giving the fish meat its bright red color. Then, during freezing, under low oxygen partial pressure, the ferrous ions in the myoglobin hemoglobin are oxidized to ferric ions, forming a brownish-red Fe-containing... 3+ The high concentration of methemoglobin in the fish meat causes it to lose its original luster, gradually darkening and turning reddish-brown, thus continuously decreasing the redness value. As shown in the figure, the redness value of both groups of mandarin fish meat decreased with prolonged storage time, but the overall change was not significant. With prolonged storage time, microorganisms proliferated in the fish meat, fat was oxidized to produce lipid peroxides, and the accumulation of methemoglobin gradually increased, leading to a decrease in the redness value of the fish meat. The ProClean 300 treatment group inhibited the growth of microorganisms in the fish meat, so the change in redness value was not significant (P < 0.05).

[0035] b*: As the storage period lengthens, the fish fat is gradually oxidized under the influence of light, temperature, enzymes, and other initiators, causing the flesh to gradually turn yellow. During storage at 4℃, the yellowness values ​​of the fish meat in both groups showed a slow upward trend, indicating that the mandarin fish meat tends to turn yellow with the extension of the storage period.

[0036] 1.5 Changes in myofibrillar protein during mandarin fish storage

[0037] SDS-PAGE images of mandarin fish muscle ( Figure 5 The results showed that the intensity of myosin heavy chain (MHC) and myosin light chain (MLC) degradation fragments significantly increased with prolonged storage time. Myosin heavy chain degradation is a major reason for the softening of fish meat texture. Myosin heavy chain (MHC) is easily oxidized and cross-linked through disulfide and non-disulfide covalent bonds, which facilitates the formation of high molecular weight polymers and aggregates. Protein oxidative damage also promotes protein degradation; MHC oxidation and the formation of actin cross-links and aggregates may be the causes of degradation. The similar changes in the intensity of the two groups of MHC degradation fragments suggest that the role of endogenous proteases is the main cause of myosin heavy chain degradation. Myosin heavy chain produces 150-170 kDa fragments; in the first 5 days of storage, the band color change was not significant, indicating that refrigeration at 4°C helps inhibit degradation and maintain the structure of the fish meat.

[0038] Cytoskeletal proteins—actinin (105 kDa), actin (42 kDa), and tropomyosin (34 kDa)—lighten in color and degrade during storage. Even minor degradation of cytoskeletal proteins can lead to structural changes in muscle, thus affecting fish quality. Tropomyosin, a regulatory protein in myofibrillar proteins, is tightly wrapped around actin filaments and exhibits water solubility and thermal stability. The intensity of tropomyosin molecular bands in fish meat increases during the first 5 days of storage, possibly because actin in the myosin complex is partially replaced by myosin; this replaced portion of tropomyosin is less prone to degradation during storage. When storage exceeds 5 days, tropomyosin begins to degrade, possibly related to the combined action of cathepsins B, D, and L.

[0039] Desmin (53 kDa) is an important member of the intermediate fibrous filament family, playing a crucial role in connecting myofibrils to the cell membrane at the Z-disc level. SDS-PAGE analysis showed that desmin degradation was not significant during storage.

[0040] 2. Changes in endogenous protease activity

[0041] like Figure 6 As shown, the cathepsin B activities in the control group and the treatment group increased by 1.3-fold and 1.2-fold, respectively, during the first 3 days of refrigeration. The activities decreased in the later stages and then increased again, with the enzyme activity being higher than that of fresh mandarin fish. This indicates that cathepsin activity in the muscle of mandarin fish remained high during storage at 4℃. The increased cathepsin activity may be due to the sustained expression of genes related to cathepsin synthesis and apoptosis.

[0042] Figure 7 This study describes the changes in cathepsin L activity in mandarin fish muscle during storage. Unlike cathepsin B, cathepsin L activity consistently increased during the first 7 days of storage, but remained lower than that of cathepsin B. On day 7, cathepsin L activity increased 2.0-fold (control group) and 2.2-fold (treatment group), respectively. During the first 3 days of storage, the enzyme activity in the control group significantly increased and remained relatively stable in the later stages; the enzyme activity in the treatment group maintained an increasing trend during the first 7 days of storage.

[0043] Changes in the activity of calcium-activated protease in mandarin fish meat stored at 4℃ are as follows: Figure 8 As shown: During storage, the activity of both groups of calcium-activated proteases increased with fluctuations, but their activity was relatively low compared to cathepsins.

[0044] During refrigeration, there was no significant difference in enzyme activity between the two groups of fish, indicating that ProClean 300 treatment did not have a significant effect on the activity of endogenous proteases.

[0045] 3. Correlation Analysis of Quality Data

[0046] Figure 9 The correlation analysis between endogenous proteases and fish meat quality indicators is shown. Figure A represents the control group, and Figure B represents the treatment group. As shown in the figure, texture indicators are negatively correlated with the myofibril fragmentation index, indicating that myofibril breakage leads to changes in fish meat structure. When the pH value of the fish meat increases, the values ​​of texture indicators such as meat firmness decrease, indicating that proteins are broken down to generate basic amines. Simultaneously, pH value shows a high correlation with cathepsin B and calcium-activated protease, suggesting that pH value may affect the activity of endogenous proteases.

[0047] 4. Identification of Differentially Expressed Proteins (DEPs)

[0048] Based on the identification results of differentially expressed proteins, 66 proteins showed significant changes in the day 5 group compared to the day 0 group (Table 1). Figure 10 As shown in Figure A, the identified differentially expressed proteins included 26 upregulated proteins and 40 downregulated proteins. This confirms that physiological and biochemical reactions induced significant changes in the muscle proteins of mandarin fish under cryogenic storage. Compared to the Day 0 group samples, the 40 differentially expressed proteins showed lower abundance in the Day 5 group samples, mainly including troponin I, F-actin, myosin, tranexamic acid, and keratin. The reduced expression of these structural proteins is closely related to muscle contraction and the integrity of skeletal muscle structure, indicating that protein denaturation, oxidation, and degradation were induced during cryogenic storage due to the growth of psychrophilic microorganisms, the action of endogenous enzymes, and biochemical processes. Compared to the Day 0 group samples, the abundance of 26 differentially expressed proteins increased in the Day 5 group samples, including phosphoglycerate kinase 1 (PGK1), adenosine triphosphate isomerase B (TPI1), mannose-6-phosphate isomerase (MPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glycogen phosphatase (PYGL). Increased expression of these metabolic enzymes is closely related to carbohydrate transport and metabolism, leading to accelerated protein and lipid degradation, as well as oxidation in mandarin fish muscle during cold storage.

[0049] Cluster analysis was performed on differentially expressed proteins to better reveal the differences in protein abundance between fresh and frozen mandarin fish. Figure 10B). The clustering algorithm used in the clustering analysis was based on data similarity, classifying samples and variables along two dimensions: vertically representing sample clusters and horizontally representing protein clusters. The results showed that the protein content of mandarin fish varied significantly during refrigerated storage. The slight differences in color observed in the three replicates of each group are likely due to individual differences between individual mandarin fish.

[0050] 5. Correlation analysis between quality changes and differential proteins in mandarin fish

[0051] Correlation analysis was performed on 66 differentially expressed proteins (DEPs) identified during refrigeration (0d vs 5d) and the quality indices of mandarin fish meat (firmness, chewiness, cohesiveness, elasticity, pH, WHC, MFI, L*, a*, b*, whiteness) and the activities of cathepsin B, L, and calcium-activated protease. The results were expressed using Pearson correlation coefficients. The results showed that all 66 identified DEPs were correlated with changes in mandarin fish meat quality and enzyme activity. 27 DEPs were highly significantly correlated with both quality indicators and enzyme activity changes (P < 0.01), including metabolic enzymes, structural proteins, and transport proteins.

[0052] Structural proteins play a crucial role in maintaining the integrity and stability of muscle tissue. Differentially expressed proteins related to their function are influenced by the activity of endogenous proteases in mandarin fish muscle, thus affecting the texture properties of mandarin fish muscle. Table 2 shows that the interactions of proteins related to carbohydrate transport metabolism, such as phosphoglycerate kinase 1 (PGK1), phosphoglycerate mutase 2 (PGAM2), triose phosphoisomerase 1 (TPI1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glycogen phosphorylase L (PYGL), may affect energy metabolism and homeostasis in muscle tissue. These interactions are negatively correlated with the stability of mandarin fish muscle proteins.

[0053] Table 1. Bioinformatics analysis of differentially expressed proteins (DEPs) in the muscle of mandarin fish stored for 0 and 5 days.

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[0061] The above embodiments are merely preferred technical solutions of the present invention and should not be considered as limitations on the present invention. The embodiments and features described in this application can be arbitrarily combined with each other without conflict. The scope of protection of the present invention should be limited to the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. That is, equivalent substitutions and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. A freshness indicator protein composition related to the quality traits of mandarin fish during low-temperature storage, characterized in that, The freshness indicator protein composition comprises myoproliferator-1, myosin-binding protein, LDB protein, con-actin, troponin I, myosin heavy chain 9, adenylate cyclase-associated protein 1, phosphoglycerate kinase 1, phosphoglycerate mutase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, glycogen phosphorylase L, acetalase A, fructose diphosphate, heat shock protein B1, and hemoglobin.

2. The use of the freshness indicator protein composition related to the quality traits of mandarin fish during low-temperature storage as described in claim 1 in the preparation of products for the preservation and control of mandarin fish.

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