Study On The Structure And Physical Properties Of Collagen From Walleye Pollock (Theragra Chalcogramma) Skin

To make more effective use of underutilized resources, the distribution and morphology of collagen fiber was studied in the leftover in aquatic product processing-walleye pollock (Theragra chalcogramma) skin, from which, acid-soluble collagen was isolated to investigate the molecular structure and physical properties.1. Histological observation clarified the presence of collagen fibre in walleye pollock skin. The results of picric acid-sirius red stain indicated that typeⅠcollagen was found in pollock skin. And electron microscopic observation revealed that skin of pollock consisted of bundles of collagen fibrils with a repeating period composed of one dark segment plus one light segment, but collagen fibrils differed in diameter.2. Acid-soluble collagen (ASC) was successfully extracted from the skin of pollock. It was identified as typeⅠcollagen with the existence of helical arrangements of collagen. The denaturation temperature (Td) and shrinkage temperature (Ts) were 24.6℃and 47℃, respectively, both lower than those of mammalian collagens.3. Acid-soluble collagen from pollock skin was further purified using a combination of DEAE-52 cellulose and Sephacryl S-300 HR gel filtration chromatography. The purified collagen showed a structure of filamentary fibril with helical arrangements that the distance between the molecular chains was 1.18nm, and the unit height, typical of the triple helical structure, was 0.27nm. Peptide mapping revealed that the collagen exhibited the homology with the hypothetical protein sce1263 of sporangium cellulosum'So ce 56'with the sequence coverage of 31%.4. Dynamic viscoelastic properties of collagen solutions with concentrations of 0.1~1.0% (w/w) were characterized by means of oscillatory rheometry at temperatures ranging from 20 to 50℃. Results showed that both storage modulus (G') and loss modulus (G'') increased with the increase of frequency. According to a three-zone model, dynamic modulus of collagen solutions in the studied concentrations and temperatures showed terminal-zone and plateau-zone behavior. Entanglements could be viewed when the concentration of collagen solution was higher, as well as the temperature reaching 40 and 50℃. But there were no significant entanglements in collagen solution at the temperature of 30℃. On the other hand, the functional properties of collagen from pollock skin were evaluated. Results showed that pollock skin collagen showed the better solubility and worse emulsifying activity, but its emulsion was stable.5. Aggregation kinetics of collagen from pollock skin was studied. The main factors including mass concentration of collagen, pH value and ionic strength, which affect the aggregation property, were discussed. The results proved that the aggregation characterization of collagen was closely related to the factors mentioned above. When mass concentration of collagen was increased to 0.6g/L and above, evident aggregation behavior was found in solution. And collagen showed higher aggregation rate at pH7.2. Ionic strength slowed down the collagen aggregation, but when the NaCl concentration reaching 30 or 60mmol/L, it was easier to form large aggregates. So these factors could be considered when the aggregation property of collagen from pollock skin was studied and applied.The aggregation behavior of collagen from pollock skin was investigated by the fluorescence probe pyrene. Results showed that the aggregation property of collagen showed mass concentration dependence, and the threshold mass concentration was found, namely the critical aggregation mass concentration (CAMC). Using plots of pyrene I1/I3 ratio versus logarithm of different mass concentration of collagen, the CAMC was found to be at 0.48g/L. The subsequent dynamic fluorescence decaying study revealed that the aggregation number of collagen was not a constant, varying with different mass concentrations. The structure of aggregates tended to be integrated, when collagen mass concentration reaching 1.2g/L and above.The fluorescence probe pyrene was also used to monitor the dynamic gelatin formation process. The concentration of pyrene was chosen as 40μmol/L in 6.67% (w/w) gelatin from pollock skin. It was shown that the changes of I1/I3 ratio in pyrene fluorescence spectra were well related to the cross-linking of gelatin. 6. Gelatin from pollock skin was cross-linked, respectively, by the natural plant polyphenols-gallic acid and rutin. Results showed that the maximal gel strength of gelatin could be obtained when the content of gallic acid was 20mg/g gelatin, or when the content of rutin was 8mg/g gelatin, but the gelling and melting point of gelatin showed no significant increase after gallic acid or rutin added. The shrinkage temperature of gelatin was about 10℃higher with the introduction of 6mg/g gelatin and 8mg/g gelatin rutin, while there were no obvious effects on the thermal stability of gelatin with addition of 20mg/g gelatin and 30mg/g gelatin gallic acid. Scanning electron microscopy (SEM) revealed that greater cross-linking of gelatin was found after gallic acid or rutin added, but it was evident in rutin-gelatin. There were two peaks in the X-ray diffraction diagrams of gelatin with and without ingredients (gallic acid or rutin). But the distance between the molecular chains of gelatin was smaller after the addition of gallic acid or rutin, revealing that they could cross-linked with the polypeptide chains of gelatin. FTIR investigations suggested that gallic acid and rutin mainly interacted with C-N-C group and carboxyl group.The effect of gallic acid and rutin on swelling behavior of gelatin was investigated. It was shown that the equilibrium swelling ratio decreased with the introduction of 20mg/g gelatin and 30mg/g gelatin gallic acid, as well as 6mg/g gelatin and 8mg/g gelatin rutin, but it was evident in rutin-gelatin. In the primary swelling process, the swelling behavior of gelatin with and without cross-linking agent (20mg/g gelatin gallic acid, 6mg/g gelatin and 8mg/g gelatin rutin) followed the Fick's law of diffusion, while that of gelatin containing 30mg/g gelatin gallic acid obeyed the non-Fick's law of diffusion. Besides, the relaxation of polypeptide chains was the rate-determining step in the whole swelling process of gelatin with and without cross-linking agents.Differential scanning calorimetry (DSC) was employed to probe the state of water in the hydrogel, and the effect of gallic acid and rutin on the state of water was discussed. The results indicated that there were freezing free, freezing bound and nonfreezing bound water in gelatin, when the swelling equilibrium was reached. Addition of gallic acid and rutin did not show obvious effect on equilibrium water content of gelatin, but produced much effect on the distribution of frozen and non-frozen water. The non-frozen water was lower after addition of 20mg/g gelatin and 30mg/g gelatin gallic acid, while it was higher after addition of 6mg/g gelatin and 8mg/g gelatin rutin.