| Sea cucumber(Stichopus Japonicus) viscera has gained growing interest for its health benefits of antitumor, anti-inflammatory, anti-hypertension and so on, which are attributed to the presence of appreciable amounts of bioactive compounds, especially the triterpene glycosides, peptide and polysaccharide. However, large amounts of sea cucumber viscera were discarded during the process of sea cucumber. To rational develop sea cucumber viscera, sea cucumber peptides were extracted by enzymatic hydrolysis, and purified by ultrafiltration and nanofiltration to obtain polypeptide and oligopeptide. Meanwhile, sea cucumber polysaccharides were harvested by alcohol precipitation, and further purified by ion exchange chromatography. Moreover, the purity, monosaccharide composition and proportion of purified polysaccharides were tested. The immunoregulatory activities of sea cucumber oligopeptides were evaluated by delayed type hypersensitivity, serum hemolysin determination, and carbon clearance test. And the antitumor and antioxidant activities of polysaccharide were assessed by cell experiment and antioxidant experiment in vitro. The main accomplishment of this study are listed as follows:.1. Prepatation of sea cucumber peptides by enzymatic hydrolysis of sea cucumber viscera: in this study, hydrolysis of neutral protease, alkaline protease, trypsin, flavor protease and bromelain on sea cucumber viscera were compared by the degree of hydrolysis and polypeptides and oligopeptides content in hydrolyzate. Trypsin was finally choosed for the hydrolysis of sea cucumber viscera. Single factor experiment was used to investigate the effects of substrate concentration, enzyme dosage, hydrolysis time, pH and temperature on hydrolysis degree. Hydrolysis process was optimizated through orthogonal experiment. The result showed that the optimum hydrolysis conditions was as follows: 1.0% of the substrate concentration, enzyme dosage 22,500 U/g, temperature 37℃, pH 8.0, enzymolysis time 5.0 h. The degree of hydrolysis is 48.90% at the optimum hydrolysis conditions. Polypeptide and oligopeptide content in the hydrolyzate were 52.68% and 47.25%.2. Fractionation of polypeptide and oligopeptide: permeate and retentate were secured by ultrafiltration and nanofiltration. The result showed that the content of polypeptides were 25.32% in permeate and 81.40% in retentate. Oligopeptides were 74.08% in permeate and 18.33% in retentate. Polypeptides and oligopeptides were preliminarily isolated.3. Immunoregulation activities of sea cucumber oligopeptides: Mice were divided into low, medium and high dose groups feeding with 85, 170 and 510 mg/(kg.d) sea cucumber oligopeptides, and blank control group was given distilled water. One month later, Ear swelling, phagocytic index and antivolume number of the mice were detected. The results showed that difference of ear weight of low, medium and high dose group mice were 7.79±3.50 mg, 8.64±3.45 mg and 8.91±3.08 mg, respectively, which had a very significant difference(p<0.01) compared with blank control groups(3.79±1.54 mg). The results indicated that sea cucumber oligopeptides could reinforce immune function of experimental mice. The results showed that difference of phagocytic index of low was 3.89±0.58, respectively, which had a significant difference(p<0.05) compared with blank control groups(3.10±0.76 mg), the difference of phagocytic index of medium and high dose group mice were 4.52±1.36 and 4.76±1.15, respectively, which had a very significant difference(p<0.01) compared with blank control groups(3.10±0.76). The results indicated that sea cucumber oligopeptides could reinforce immune function of experimental mice. The results showed that difference of antivolume number of low and medium dose group mice were 110±10.04, 117.67±8.80, respectively, which had no significant difference(p>0.05) compared with blank control groups(108.67±9.18), difference of antivolume number of high dose group mice were 144.33±8.62, respectively, which had a very significant difference(p<0.01) compared with blank control groups(108.67±9.18). The test showed no statistical significace. Therefore,we can come to the conculsion that sea cucumber oligopeptide can enhance immune activity because of the positive of odelayed type hypersensitivity and carbon clearance test.4. Purification of sea cucumber polysaccharide: Sea cucumber polysaccharides were further purified by ion exchange chromatography. Five sea cucumber polysaccharide named HGⅠ、HGⅡ、HGⅢ、HGⅣ and HGⅤwere got by isocratic elution with sodium chloride. The purity and molecular weight of polysaccharides were identified by HPLC with HGⅢ and HGⅣ had high purities. The molecular of HGⅢ and HG Ⅳ were 162.7 kDa and 139.4 kDa, respectively. The monosaccharide composition and proportion of HGⅢ and HGⅣ were detected by PMP derivation. The study showed that HG Ⅲ was mainly composed of D-(+)-Mannose, D-(+)-Glucosamine, D-(+)-Glucose and D-(+)-Galactose. Molar ratio of monosaccharide was 1.00:1.24:0.71:1.16 by setting the D-(+)-Mannose as standard. HG Ⅳ was mainly composed of D-(+)-Mannose, D-(+)-Glucosamine, L-(+)-Rhamnose and D-(+)-Galactose. Molar ratio of monosaccharide was 1.00:1.72:1.52:1.66.5. Antitumor and antioxidant activities of sea cucumber polysaccharides: MTT was used to assess the antitumor activity of HGⅢ and HGⅣ. The results showed that HGⅢ and HGⅣ could inhibit the the proliferation of lung adenocarcinoma cell line A549 with IC50 of 2.07 mg/mL and 1.01 mg/mL, respectively. It suggested that HGⅢ and HGⅣ may proved to be a therapeutic agent for antitumor. DPPH radical, ABTS radical and hydroxyl radical scavenging rate were employed to evaluate the antioxidant activity of HGⅢ and HGⅣ. The results showed that HGⅢ and HGⅣ have excellent antioxidant activity. The IC50 of HGⅢ scavenging DPPH radical, ABTS radical and hydroxyl radical were 1.21, 0.582 and 1.29 mg/mL, respectively, and 3.56, 6.756, and 1.37 mg/mL for HGⅣ.In summary, the preparation and activities of sea cucumber peptides and polysaccharides from sea cucumber viscera were studied in this dissertation, which provided a new idea for the utilization of sea cucumber viscera and theoretical support for comprehensive utilization of sea cucumbers viscera. |
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