| The use of lactic acid fermentation could not only preserve the fish, but also modify their functional and sensory properties, which is receiving considerable attention. Our previous studies have determined a method to produce a tasty, elastic fermented fish surimi, clarified the mechanism of acid-induced gelation during fermentation of fish surimi and investigated influence of Pediococcus pentosaceus proteases on gel properties. However, effect of endogenous fish muscle enzymes on gelation and antioxidant properties of fermented product has not been studied systematically. Fish enzymes are rich in vivo and their impact on the quality of products cannot be neglected. Therefore, in this study, based on the established fish fermentation techniques, the properties of endogenous enzymes influencing the quality of fermented fish surimi were investigated, and their influence on gelation and antioxidant properties were also studied. This study is of significant importance for further understanding gelation and modori phenomenon of fermented fish surimi, and understanding the mechanism of changes in antioxidant properties during fish fermentation. In addition, this study provides an evident theoretical foundation for controlling key techniques of fish fermentation, as well as to produce a high-quality fish surimi product, thus making contribution to increasing the level of fish processing industry in China.Firstly, enzymes influencing changes of surimi protein during fermentation were determined. Behaviors of endogenous proteases in the range of fermentation parameters were determined by measurement of proteolytic activities under different pH and temperatures, by response surface methodology, and by calculation of activation energy; hydrolysis patterns of fish protein under fermentation conditions by endogenous proteases were analyzed by using myofibrillar protein as substrate; the effects of Lactobacillus plantarum proteases and endogenous proteases on degradation of protein were compared by proteolytic activities,SDS-PAGE of sarcoplasmic and myofibrillar protein, and amino acid nitrogen(AAN); the effects of different endogenous enzymes on crucial protein of fish were determined by specific enzymatic activities, SDS-PAGE or production of specific bonds. The results showed that endogenous proteases had the highest activities at pH 4.0 and the lowest at pH 7.0;influence of pH and temperature changes on endogenous proteolytic activities were more evident under fermentation range(25oC – 37oC) than refrigerated temperature(near 4oC),indicating that control of pH and temperature was more important in the fermentation stage than storage at refrigerated temperature; at lower pH(4.5 and 5.0), myofibrillar proteins tended to aggregate and then they were hydrolyzed, while at higher pH(5.5 – 7.0),myofibrillar protein did not aggregate and was only slightly hydrolyzed in the final stage;endogenous proteases had both exopeptidases and endopeptidases; during fermentation,lysosomal proteases, such as cathepsin B, L and D increased significantly while collagenases,serine proteases and calpains decreased dramatically; in addition, acid proteases, such as cathepsin B, L and D, could hydrolyze more MHC and actin than other proteases; endogenous TGase activity declined fast during fermentation and the ability to produce ε-(γ-Gln)-Lys bonds was low under acidic conditions, therefore, it had little impact on protein crosslinkingduring fermentation.Then, properties of endogenous proteases and some kinetics indicators were investigated.Subsequently, effect of endogenous proteases on gel properties and the reason were investigated. The most important proteases on gel strength were determined by specific enzyme inhibitors; effect of different hydrolysis degrees on gel strength were studied by controlling hydrolysis time; main factors influencing endogenous hydrolysis was determined by TCA-soluble peptides at different pH and in different protein structures; fluorescence spectroscopy, UV spectroscopy and Raman spectroscopy were used to analyze the reason of modori caused by endogenous proteases. The results showed that endogenous cysteine proteases such as cathepsin B, L, and H and aspartic proteases such as cathepsin D had the highest influence on modori and the impact was attributed mainly in the final stage.Metalloproteinases and serine proteases such as calpains and collagenases had less influence and it was attributed mainly at the beginning. Endogenous TG also influenced gel properties at the beginning and the impact was small in the whole stage. Endogenous proteases had negative effect on the gel strength from the beginning to the end. Moderate hydrolysis could not enhance the gel strength. Protein aggregation hardly affected the endogenous hydrolysis activities but pH could change these activities significantly. The decline of pH could transform zymogens into activated enzymes. Endogenous proteases could cleave hydrophobic amino residues, resulting in decrease of hydrophobic interactions and gel strength.Endogenous proteases did not change content of disulfide bonds significantly, but could makeα-helix resolved, β-sheet declined and random coils formed, explaining the decrease of gel strength from view of structure..At last, effect of endogenous proteases on antioxidant properties of fermented fish surimi was studied and the reason of occurrence of antioxidant properties was found. The change of antioxidant activities during fermentation was determined by ABTS and DPPH scavenging activities, and Fe2+ chelating activities; the correlation analysis was performed between antioxidant properties and degree of hydrolysis(DH), free amino acids and molecular weight distribution; the influence of peptide sequences and structure on change of antioxidant properties was determined by separation with Sephadex-15 gel filtration chromatography and identification with LC-MS. The results showed that ABTS scavenging activities increased with fermentation time while DPPH scavenging activities increased and followed by a decrease. The two values at the end of fermentation were both near 80%. Fe2+ chelating activities were relatively low during fermentation, which was lower than 20%. The increase trend of DH was not completely in accordance with the change of antioxidant properties, but they had some correlation. Most hydrophobic free amino acids(FAA) and the total content of FFA increased significantly during 48 h of fermentation. Meanwhile, peptides >10000 Da increased and then decreased; peptides <5000 Da decreased and then increased;low-molecular-weight peptides(2000 Da – 500 Da) went up fast at the last stage of fermentation. The change in antioxidant properties had more correlation with the change in peptides content than that in FAA content. Endogenous proteolysis had the highest correlation while microbial proteolysis had the lowest. LC-MS showed that acid and endogenous hydrolysis could induce peptides with Phe, Tyr, N-terminal hydrophobic amino acids andC-terminal hydrophilic amino acids, leading to the increase of antioxidant activities. Each group was lack of Glu, Asp and His, which might result in lower Fe2+ chelating activities.In this study, endogenous cathepsins were determined as the most important proteases for gel properties of fermented fish surimi. They had high activities, heat- and acid- stability, and high abilities to hydrolyze fish proteins during fermentation. Cleavage of hydrophobic groups was the reason for modori caused by endogenous proteases. Production of peptides with hydrophobic amino acids by acid and endogenous proteases was the reason for increase of scavenging activities. |
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