Establishment Of Simplified Preparation Method For Collagen Fiber And Characterization Of Collagenase In Sea Cucumber(Stichopus Japonicus)

The traditional extraction method of collagen fibers from sea cucumber body wall takes long time. In this study, the simplified extraction method was adopted to extract collagen fiber from body wall of sea cucumber. The extracting condition was optimized by determination of rinsing time or times with deionized water and NaOH solution. The body wall homogenate was mixed with different rinsing solutions at ratio of 1:10 (m/V), and the following steps were performed at 4℃. The results showed that the collagen fibers was obtained by rinsing with deionized water for 30 min and repeating for 3 times, and then rinsing with 0.1 mol/L Tris-HCl (pH 8.0, containing 0.5 mol/L NaCl and 5 mmol/L EDTA) for 8 h, finally treating with 0.1 mol/L NaOH for 24 h. The collagen fibers of sea cucumber body wall were identified by fourier transform infrared spectroscopy. The extracting time is shortened from 7 days to 2 days. A clear band with molecular mass of 116-200 kDa was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The partial sequence of collagen fibers (Gly-Leu-Pro-Gly-Ala-Arg-Gly-Ser-Asn-Gly-Asn-Asp-Gly-Pro-Ala-Gly-Pro-Arg-Gly-Phe-Asp-Gly-Pro-Glu-Gly-Pro-Arg) was detected by liquid chromatography-mass spectrometry (LC-MS/MS), which matched partial alpha collagen type 1 precursor (Paracentrotus lividus). High performance liquid chromatography (HPLC) analysis showed that the component of glycine in collagen fibers was 32.34%, which accounted for about 1/3 of total amino acid, and proline and hydroxyproline were 7.75% and 5.10%, respectively.Sea cucumber intestine collagenase was extracted with 20 mmol/L pH 9 Tris-HCl (10 mmol/L CaCl2). Enzymatic properties were studied by gelatin zymography. The enzyme displayed maximum activity at pH 9 and 40℃. EDTA-Na2 (10 mmol/L) inhibited the sea cucumber intestine collagen degradation enzyme activity significantly. The collagen from sea cucumber body wall and tilapia skin was degraded by intestine collagenase. After treatment, TCA-solube oligopeptide was also increased by using methods of TCA precipitation combining folin-phenol.