Study On Enzymatic Preparation Of Grass Carp Fish Scale Gelatin And ACE Inhibitory Peptides

Only in China, the production of aquiculture was more than the production of fishing,and the production of cultured grass carp in China accounted for more than 90% of global grass carp aquiculture production. Fish scales, the by-products of fish processing, and which are rich in collagens, have not been exploited and are directly discharged into estuaries resulting in environmental pollution and resource waste. If gelatin and angiotensin-I converting enzyme (ACE) inhibitory collagen peptides can be extracted from the fish scales, not only will pollution problem arising from them be solved, but also we can obtain a kind of biological activity product with high appending value, and which also can resolves the gelatin safety problem which arose from bovine spongiform encephalopathy (BSE) and foot-and-mouth disease (FMD).Enzymatic hydrolysis of grass carp fish scales to extract gelatin and ACE inhibitory collagen peptides were investigated as reported in this thesis, the contents and results are as following:HCI solution was used to demineralize the fish scales. The optimum processing technology of the demineralization was as follows: concentration of HCl, 0.4mol/l; ratio of feed liquid, 1:15(w/v); time of demineralization, 90min.The effects of the demineralizing factors and calcium ion on the grass carp fish scale collagen were studied. The content of the solubilized collagen was increasing with decalcification time. The content of the solubilized collagen was decreasing with increasing the HCl concentration from 0.2mol/L to 0.8mol/L. There was no significant effect on shrinkage temperature of the fish scale collagen fibre, when the HCl concentration was increased from 0.2mol/L to 0.6mol/L. But when HCl concentration was increased to 0.8mol/L, the shrinkage temperature of the fish scale collagen fibre decreased to 62℃, and the collagen became unstable. Calcium ion combined -C=O of the collagen through coordination bond resulting in the broken of the hydrogen bond inner and between the collagen molecules, and the helix structures of the collagen were become loose.The properties and structure of fish scale collagens were studied. The grass carp fish scales collagen fibres were rich in praline, hydroproline, glycine and alanine, but none tryptophan, and the content of histidine was lower. The shrinkage temperature of the fish scale collagen fibre was 63.5℃. Fish scales collagen remained tri-helix configuration by X-ray diffraction analysis.Enzyme hydrolysis was used instead of alkali to pre-treat the fish scales. The effects of six kinds of proteases on the hydrolysis of the fish scales were compared quantitatively according to the gelatin yield, viscosity and gel strength and Protease A was chosen to be the optimum protease. The Response Surface Methodology (RSM) was used to optimize the factors affecting Protease hydrolysis. The optimum hydrolytic condition for fish scales was [S] 10.0%(w/v), [E]/[S] 0.24%, pH7.0, time5.5h and temperature 30.4℃,and the enzyme were inactivated at 90℃for 10min.The collagen content of pre-treated fish scales was about 91.04%, which occupied 96.80% of the total protein in the fish scales, and the shrinkage temperature of the pre-treated fish scale collagen fibre was decreased to 55.88℃, which indicated that the stability of the collagen fibre crystal had become lower.The single factor experiment and orthogonal experiment were used to determine the optimum extraction conditions of gelatin from the pre-treated fish scales by hot water. The optimum extracting technology of the fish gelatin was as follows: extracting temperature 55℃, pH value 6, time 5h. The yield of the fish gelatin extracted from the optimum extracting technology by a magnified experiment was 60.23%(w/w), and the viscosity and the bloom strength of fish scales gelatin solution were 8.32 mPa·S and 290 bloom g. X-ray diffraction analysis showed the dried gelatin contained tri-helix structures, while dissolved in hot water, the gelatin lost the tri-helix structures mensurated by circular dichroism spectrum.Physical and chemical properties of gelatin extracted from grass carp fish scales in the optimal conditions were determined. The amino acid composition of grass carp fish scales gelatin was the same as collagen, which were rich in praline, hydroproline, glycine and alanine, but none tryptophan. Compared with the mammal gelatin, the contents of amino acids and hydrophobic acids were lower. Theαandβ-component (α-chain andα-chain dimers) accounted for 88% of total molecular weight of gelatin. The intrinsic viscosity, isoionic points (PI), surface hydrophobic index (S0), gelling point and the melting point were0.73, 7.0, 259.59, 20.8℃and 26.9℃, respectively. Fish scales gelatin almost has no fishy smell.The residues of the fish scales extracted by hot water were hydrolyzed into ACE inhibitory peptides. The effects of six kinds of proteases on the hydrolysis of the fish scales residues were compared quantitatively according to the peptides recovery, the relative molecular weight distribution, the free amino acids content and the ACE inhibitory activity of hydrolysates and A.S.1398 was chosen to be the optimum protease. The optimum hydrolytic condition for fish scales were temperature 50 oC,pH 6.5,[E]/[S] 6%(w/w),[S] 5%(w/v), time 3 h。The percentage of the peptides with molecular weight less than 1500Da was about 90%. After desalting by DA201-C, the ACE inhibitory IC50 of A.S.1398 hydrolysate was 0.45mg/ml.Grass carp fish scales hydrolysate was separated and purificated by DA201-C macroporous adsorption resin, Sephadex G-15, semi-preparative reverse-phase high performance liquid chromatography (RP-HPLC) and analytical RP-HPLC. After these, an active fraction G5-R3-1 was obtained, which was tested to be a purified ACE inhibitory peptide. The amino acids sequence of this peptide was gained using MS/MS analysis, and it was Gly-Pro-Ala-Gly-Pro-Arg. The ACE inhibitory IC50 of the hexapeptide was 52.1μM.The antihypertensive mechanism was discussed through the influence of fish scale collagen ACE inhibitory peptides on the function of human Umbilical vein endothelial cells(HUVEC). The proliferation of the cells can be restrained by the ACE inhibitory peptides which can not cause apoptosis, and the cells were inhibited at S periods. ACE peptides can reduce the amount of ET-1 in the supernatant of the culture media compared with the control group, and the effect of the single peptide(FSCSP) was the same with the mixed peptides(FSCPs) at the same concentration. The results of the real-time fluorescence quantitative RT-PCR showed that in the lower and medium groups of FSCPs the expressions of ACE2mRNA were higher than the control, while in the single peptide group and high content FSCPs group the expressions of ACEmRNA were lower than the control. Both single peptide FSCSP and mixed peptides FSCPs can reduce the expression of ET-1mRNA. The mixed peptides FSCPs also can scavenge the O2-·.