花鲈冰藏过程中肌原纤维蛋白生化特性与新鲜度变化的相关性

Correlation between changes in freshness and protein biochemical properties of Lateolabrax japonicas during iced storage

摘要:

为研究冰藏期间花鲈鱼片的品质及蛋白质生化特性的变化，通过感官评分、pH、质构、色差、硫代巴比妥酸值(TBA值)、挥发性盐基氮(TVB-N)值、菌落总数(TVC)和肌原纤维蛋白含量、巯基、羰基含量的测定及十二烷基硫酸钠—聚丙烯酰胺凝胶电泳(SDS-PAGE)确定了冰藏花鲈鱼片理化指标变化，并对肌原纤维蛋白生化特性与主要的新鲜度指标进行相关性分析。结果显示，花鲈鱼片冰藏期间亮度值、红度值、硬度值分别由57.92±0.66、−(4.34±0.39)、174.92±16.80降低至46.62±1.54、−(5.21±0.08)、122.25±15.73；pH呈先降低后增加的趋势；TBA值、TVB-N值随贮藏时间的延长分别由(0.35±0.01) mg/kg与(9.41±1.03) mg/100 g增加至(0.97±0.04) mg/kg与(33.18±0.83) mg/100 g；贮藏至第16天时菌落总数达(6.35±0.41) lg(CFU/g)，已超过二级鲜度标准，且感官分值不可接受。贮藏期间肌原纤维蛋白含量、巯基含量分别由(104.21±3.42) mg/g与(145.81±1.02) μmol/g prot下降至(72.03±5.25) mg/g与(137.28±1.29) μmol/g prot，羰基含量由(0.63±0.03) nmol/mg增加至(1.45±0.06) nmol/mg。SDS-PAGE电泳图谱显示，在30 ku附近产生新的蛋白条带。相关性分析表明，TBA值、菌落总数、蛋白含量与羰基含量之间具有良好的相关性。研究表明，花鲈鱼片冰藏至第16天时达到不可食用程度，贮藏后期微生物的大量繁殖、脂质氧化与蛋白质氧化间的相互作用可能是品质劣变的主要原因。

关键词:花鲈;冰藏;新鲜度;蛋白质;生化特性;相关性

Abstract:

Sea bass (Lateolabrax japonicus) is an economically important marine species widely cultured in Asia. Due to the high content of water and protein in fish, it is susceptible to the influence of microorganisms, fat oxidation and endogenous enzymes during circulation, resulting in quality deterioration. Freezing was a common storage method, but the increase of ice crystals can easily cause quality loss such as low water holding capacity, protein denaturation, and microstructure damage. Iced storage, which can effectively inhibit the growth of microorganisms, has gradually become one of the primary methods for prolonging the shelf life of L. japonicus. In recent years, there have been many studies on quality change in fish fillets during frozen storage, while there were few studies on the correlation between changes in freshness and protein biochemical properties of L. japonicus. Therefore, the quality change mechanism of L. japonicus during iced storage needs to be further explored. In this study, the effects of storage time on the freshness and protein biochemical properties of L. japonicus fillets during iced storage were investigated. The value of sensory characteristics, pH, texture, color, thiobarbituric acid (TBA), total volatile based nitrogen (TVB-N), total viable count (TVC) and protein content, SDS-PAGE, sulfhydryl and carbonyl contents of L. japonicus fillets were measured periodically to analyze changes of L. japonicus fillets during iced storage. And correlation analysis between the main freshness indicators and biochemical characteristics of myofibrillar protein were conducted. During the storage, the brightness of L. japonicus fillets values decreased from 57.92±0.66 to 46.62±1.54, the redness values decreased from −(4.34±0.39) to −(5.21±0.08), the hardness values decreased (174.92±16.80 to 122.25±15.73) significantly, the pH increased in the early stage and then decreased. Moreover, the value of TBA and TVB-N value increased from (0.35±0.01) mg/kg and (9.41±1.03) mg/100 g to (0.97±0.04) mg/kg and (33.18±0.83) mg/100 g, respectively. After 16 d of storage, the TVC was increased to (6.35±0.41) lg (CFU/g), which exceeded the secondary freshness standard, and the sensory score was unacceptable. The content of myofibrillar protein and sulfhydryl decreased from (104.21±3.42) mg/g and (145.81±1.02) mmol/g prot to (72.03±5.25) mg/g and (137.28±1.29) mmol/g prot, respectively. While the carbonyl content increased from (0.63±0.03) nmol/mg to (1.45±0.06) nmol/mg. The SDS-PAGE pattern of myofibrillar protein showed a new protein band around 30 ku, which may be the degradation product of troponin T. Troponin T, which was involved in the contraction of striated muscle bound by myosin, may be phosphorylated, improving the activity of endogenous protease and promoting the degradation of troponin in the late storage period. Moreover, the 90 ku protein band I almost completely disappeared, and the protein may be completely degraded by endogenous proteases. Correlation analysis showed that the correlation coefficients remain high between TBA value, TVC, protein content and carbonyl content. TBA were significantly negatively correlated with protein content, which may be related to the large amount of non-heme iron, which was released after the denaturation of myoglobin and hemoglobin, promoted fat oxidation. The content of myofibrillar protein was significantly decreased on the 16^th day, and the protein appeared significantly degraded in the pattern of SDS-PAGE. This result was consistent with the significant negative correlation between the TVB-N value and the protein content. The structure of myofibrillar protein of fish was easily destroyed during the long-time storage, and the hydrophobic residues were cross-linked to form insoluble aggregates, which reduced the solubility of the protein. In addition, the content of myofibrillar protein was significantly negatively correlated with the content of carbonyl groups. Active oxygen could cause the oxidation of amino acid side chains and protein backbones, leading to protein breaks or protein cross-linking aggregation. These modifications would also reduce the solubility of protein, which was not conducive for the freshness of fish. The results of this study showed that the L. japonicus fillets reached the inedible level after 16 days of iced storage. The freshness indexes of L. japonicus fillets all had a good correlation with storage time apart from pH value. These indexes can be used to characterize the freshness quality of L. japonicus fillets during iced storage. Mass reproductive microorganisms and the interaction between lipids oxidation and protein oxidation in the late ice storage may be the main reason for the quality deterioration. Studies also found that there was a good correlation between the freshness index, protein degradation, protein oxidation and fat oxidation of fillets during iced storage. In the future, metabolomics, proteomics and other omics can be used to further identify different characteristic biomarkers related to metabolites and oxidized proteins. Mass spectrometry and bioinformatics could also be used to further analyze the new bands appearing in the electrophoresis pattern, and the protein of fish muscle myofibril during storage can be further identified. In future, the molecular mechanism of proteins degradation and oxidation could be investigated in depth. And the mechanism of fish softening still needs further exploring, including the interaction relationship between activity of proteases and microorganisms. In addition, it can be seen that the seabass fillets exceeded the acceptable range when they were frozen to 16 days from the sensory score and some other indexes of freshness, while the TVB-N value was still within the edible limit. Accordingly, although the traditional quality detection methods are rigorous and scientific, there is a significant lag in the detection of aquatic products, and a single indicator could not be a reliable indicator for the freshness identifying of fish samples. Therefore, electronic noses and hyperspectral imaging, nuclear magnetic resonance and other new technologies, combined with different modeling methods, are promising ways to be used to evaluate and predict the shelf life of different aquatic products.

Key words:Lateolabrax japonicas;iced storage;freshness;protein;biochemical properties;correlation