Purification And Characterization Of Like-Stefins From Silver Carp Egg And Preliminary Effect On Preservation Of Chilled Silver Carp Fillet

In this study, the optimum crude extraction of silver carp cysteine proteinase inhibitors (CPIs) with low molecular weight was selected. Based on this, two low molecular weight CPIs which were named as like-Stefin-Ⅰ and like-Stefin-Ⅱ were purified from silver carp egg by the methods of chromatography and electroeluting. And the molecular weight and purity were characterized by Tricine-SDS-PAGE and HPLC. Moreover, their biochemical properties were characterized, which included inhibitive type, thermal stability, pH stability and Ki. At last, the preliminary effects of silver carp egg like-Stefin-Ⅰ on the preservation of chilled silver carp fillet by determining pH, TVB-N, hearing force and tissue microstructure.The selection of the optimum crude extraction of silver carp CPIs:In the step of homogeneous, the optimal buffer Ⅰ (20mmol/L phosphate buffer, pH 6.0, containing 0.1mmol/L PMSF) was determined by the standard of specific activity.Simultaneously by comparing the specific activtiy of CPIs treated by the different heat and acid conditions, the optimal treatment condition was selected out combined with electrophoresis, ie homogeneous buffer standing at 4℃, with pH3.0 acid treatment, then was ajust to pH 8.0 with alkali. The analysis of SDS-PAGE indicated that the ultrafiltration and interception by 30 kDa-5 kDa ultrafiltration membrane was very effective in separating low molecular weight CPIs.Purification and characterization of silver carp egg low molecular weight CPIs:The ultrafiltrated and concentrated extract of CPIs (30 kDa-5 kDa) from silver carp egg were then used for the purification by chromatography. During this period, the inhibitive activity was assaied using Z-Phe-Arg-MCA. The absorbed fraction with inhibitive activity from Sp Sepharose Fast Flow was collected and applied on Octyl-Sepharose 4 Fast Flow hydrophobic chromatography. And then, six active peaks (Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ) were obtained. The peak Ⅱ and peak Ⅲ with high specific activity were studied futher in this paper.Peak II gave five bands on reduction Tricine-SDS-PAGE, that was coincident with the result from Sephadex G-50 and the CPI of the lowest molecular weight (named like-Stefin-I) from Sephadex G-50 was identified by SDS-PAGE. The 5.921 kDa protein still existed before or after reaction with Papain. Like-stefin-I had only one protein peak at 14.424min (Peak area 89.56%, Tailing factor 1.218) on RP-HPLC.It was found that Like-stefin-I was able to inhibit the activities of silver carp cathepsin B、L and papain, but it had no effect on trypsin and chymotrypsin. And it was stable during a wide range of pH 4.0-pH 9.0 with more than 60% residuary activities; and it could retain the inhibitive activities about 80% between 4-50℃. Like-stefin-I was a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 0.14 nM when tested with the Dixon-plots method.Peak III gave two bands of 17.531 kDa and 6.352 kDa under reducing conditions by Tricine-SDS-PAGE. And both bands were confirmed as inhibitive factor CPIs by the electrophoresis after reaction with papain. The 6.352 kDa Like-stefin-Ⅱ was obtained by electroeluting, and it had two protein peaks, which at 14.198min (Peak area 49.91%, Tailing factor 1.339) and 92.729min (Peak area 30.26%, Tailing factor 1.688) on RP-HPLC.The preliminary research on the effect like-Stefin-I on the preservation of chilled silver carp fillet revealed that adding 1920 units/mg Like-stefin-I, all the biochemical indicators (pH, shear force, TVB-N value and tissue microstructure) of the experimental group were superior to the control group at 4 ± 0.5℃ within 8d.During cold storage, the experimental group fillet reached the lowest pH at 3d, while the control group 2d, and after the storage of 3d, though the pH appeared an increasing trend, the experimental group was significantly (0.01≤p≤0.05) or very significantly lower than the control group (p<0.01).There was no obvious difference (p≥0.05) of the shear stress and TVB-N values between experimental group and control group in the first 2d. After 3d, the shear force was decreased significantly; the shear force of control group was significantly decreased on 3d, while the experimental group did on 4d. During 3d-8d, the shear force and TVB-N values of experimental group were significantly (0.01≤p≤0.05) higher or lower than the control group.The activity of Cathepsin B, L increased gradually at 4 ± 0.5℃ after refrigerated 8d. The activity of Cathepsin L in the control group was significantly (0.01≤p≤0.05) higher than the experimental group on 4d. The activity of Cathepsin B in the control group was significantly (p≤0.01) or very significantky (0.01≤p≤0.05) higher than the experimental group during the whole process. It was indicated that the like-Stefin-I in the experimental group significantly inhibited the activity of endogenesis Cathepsin B or that secreting by the corruption microorganism of the fish fillets.Refrigerated Od, muscle fiber structure of fish fillet was clearly. On 2d, there was no significant difference between the control group and experimental group. On 3d, the muscle fibers of the experimental group had a certain degree of protein degradationin, but the structure remained intact. While, in the control group, the muscle fibrous structure is obvious damaged. On the 6d, in the experimental group, the muscle fibrous structure also had the obvious fracture, while that of the control group was lossed and the surface of fish fillet became blured. On the 8d, two groups of fish fiber structure were disappeared, from the visible extent, the experimental group is better than the control group. Therefore, applying the purification of small molecules Like-stefin-Ⅰ to the silver carp fillet, in a certain extent,could delay the speed of softening and autolysis of frozen fish fillets, maintain the quality of fish fillet.These results suggested that Like-stefin-Ⅰ had the effect on preservation of chilled silver carp fillet.