Studies On Extraction, Characterisation And Effect Of Temperature On The Secondary Structure Of Pepsin-solubilized Collagen From Fish Scale

Pepsin-solubilized collagen (PSC) was extracted from fish scale of grass carp by pepsin, the effects of extraction media, pre-treating, stirring, solid/solvent ratio, extraction times and time on the yield of collagen were studied to ascertain the optimal technology. Purified collagen type was identified by SDS-PAGE, FT-IR and amino acid composition, meanwhile solubility and rheological characteristics (apparent viscosity and gel strength) of fish scale PSC were studied systemically. Effect of temperature on the secondary structure of PSC was studied by FT-IR, Raman and CD. The aggregation behaviors of fish scale PSC at different concentration, temperature and pH were studied by AFM. The main results were shown as follows:1. The optimum extraction conditions were 0.50mol/L malic acid with a solid/solvent ratio of 1:10 (w/v) as extraction media, stirring using a magnetic stirrer during the whole process, extracting three times and 48 hours every time after immersing in distilled water for 5 hours, removing impurity by microwave and decalcifing with 4% malic acid. SDS-PAGE, FT-IR and amino acid composition analysis indicated that the extracted collagens were typical type I collagen and reached electrophoretic purity.2. Fish scale PSC was found to be eutectic in 0.50mol/L acetic acid solution and the highest soluble concentration reached 4.0mg/mL. PSC had the highest relative solubility at pH3. NaCl had little effect on the solubility of PSC at concentration below 3% (w/v), however, a sharp decrease in solubility was observed as the concentration of NaCl was above 3% (w/v).3. Intrinsic viscosity of fish scale PSC was 1.07L/g. Apparent viscosity of PSC went up prominently with the increase of PSC concentration as well as presence of glycerol, ethanol or polyglycol. Glucose and sucrose could also increase slightly apparent viscosity of PSC solution, yet NaCl and CaCl₂ decreased evidently apparent viscosity of PSC solution. PSC had the highest apparent viscosity at pH3. Apparent viscosity decreased with the rising of shear rate, but few changes were found during heating preservation.4. The gel strength of fish scale PSC went up with the increase of PSC concentration and heating temperature, but declined evidently when heating time prolonged. Gel strength increased quickly at the first four hours of maturation time, and reached steady after 18 hours. NaCl and CaCl₂ could decrease gel strength of PSC, while glucose and sucrose could increase gel strength of PSC slightly. Effects of glycerol, ethanol and polyglycol on gel Strength of PSC were accordant, all of them decreased gel strength at the low concentration, then increased with the increase of additive concentration.5. Denaturation temperature (Td) of the lyophilized PSC was 37.1℃by DSC, yet PSC in acetic acid denatured at 32.0℃by Capillary Viscometer, which demonstrated that the lyophilized PSC was more stable than PSC solution. Rotary Rheometer indicated that Td of PSC aquatic solution was 31.5℃, which was higher than that of those using malic acid (25.4℃) or acetic acid (28.0℃) as solvents. This suggested that aquatic solution of PSC was more stable than acidic solution of PSC. Td of PSC went up slightly with the presence of 4% glycerol, 4% ethanol, 4% polyglycol, 5% glucose and 5% sucrose respectively, yet the presence of 1.5% NaCl or 2%CaCl₂ could led Td fall to 22.0℃, Which indicated that different solvents and additives had effects on Td of PSC solution to some extent.6. FT-IR revealed that fish scale PSC had typically characteristic absorptions of collagen, peaks at 1658cm^-1, 1552cm^-1 and 1238cm^-1 were assigned to be amideâ… , amideâ…¡and amideâ…¢respectively. When the temperature increased, amide A and amide B shifted to lower frequency, the absorption of 1658cm^-1 split into several absorption peaks, the absorption at 1552cm^-1 had a slight red-shift followed by a distinct blue-shift, and the frequency of 1238cm^-1 declined. Raman spectra showed that the absorptions of amideâ… , amideâ…¡and amideâ…¢appeared at 1669cm^-1, 1557cm^-1 and 1245cm^-1 respectively, which were higher than those in FT-IR spectra. Furthermore, the characteristic absorptions of proline at 921cm^-1 and 855cm^-1 only appeared in Raman spectra. CD spectra demonstrated a rotatory maximum at 221.6nm and a negative peak at 204.4nm of PSC solution, which were typical spectral characteristics of the collagen triple helix structure. The structure changes of the lyophilized PSC appeared mainly between 35℃and 60℃in FT-IR and Raman spectra, yet CD spectra demonstrated that the configurational changes of PSC in acidic solution appeared in the range of 20℃to 35℃, which indicated that the lyophilized PSC was more stable than the acidic solution of PSC.7. AFM showed that the aggregation behaviors of fish scale PSC at different concentration, temperature and pH were evidently different. In acidic solution of pH2.7, fish scale PSC existed as fibrillar monomers and linear aggregates were simultaneously observed in solution at low concentration of 10μg/mL. Fish scale PSC self-assembled to network with the increase of concentration, and random coils structure was formed at the concentration of 4.0mg/mL. In pH7.2 Tris-HC1 solution, fish scale PSC could self-assemble to network structure even at low concentration of 10μg/mL, and the fibril diameter and height increased evidently with the increase of concentration. The aggregation behaviors changed obviously from linear fibrils to globular aggregates after treating for one hour at 40℃, and the higher-order structure of PSC was almost destroyed by heating for one hour at 80℃, which induced that just a few anomalistic aggregates could be seen in AFM images.