| Rana dybowskii is a kind of famous edible and medicinal animal. Female frog fallopian tubes, also known as oviductus ranae, have a very high healthy value. According to the records of Compendium of Materia Medica, oviductus ranae has many functional effects including nourishing yin and moisturizing lung, nourishing the kidney and strengthening the essence and so on, which was very famous all over the world lately. Those byproducts except for oviductus ranae such as skins, bones, cartilages, organs, etc., have a high yield every year, but low values for the byproducts in people’s mind make them often be used easily, for example, being processed into animal feeds or directly discarded, which were not only a waste of resources, but also often caused some environmental problems, therefore, how to use these by-products is one of major problems scientists need to resolve.The topic for this study is to develop and utilize brown frog skins, which is one of the byproducts of Rana dybowskii after being removed oil. Previous studies found that brown frog skins contain large amounts of collagens, which are well known by people and can be used as skin caring products or health foods. Hence, firstly, this study will extract collagen in Rana dybowskii skins according to modern industrial technology, and analyse their physical and chemical characteristics by some modern methods; then enzymolysis approach was used to prepare collagen peptides deprived from Rana dybowskii skins, and next being isolated and some fractions being collected. At last, the bioactivity including antioxidant and ACE inhibitory activity for these fractions after being isolated was studied. This study was aimed at increasing the product scope for Rana dybowskii, and expanding products markets, so that the value of which is not limited to single oviductus ranae, and provide the scientific basis for the development of products industry chain for Rana dybowskii. Specific contents are as follows:1. There were two kinds of collagens that acid-solubilized collagen(ASC) and pepsin-solubilized collagen(PSC) were prepared by using acids and pepsin, respectively, and were compared by measuring the differences of physical and chemical properties and functional characteristics; Results showed that there was a strong absorption peak at 234 nm by analyzing UV spectrums, which demonstrated the two kinds of collagens were in accordance with collagens, characteristics; ASC and PSC had absorption peaks at 3346.0、2952.0、1662.0、1548.0、1242.0 cm-1 and 3322.0、2944.0、1662.0、1551.0、1236.0cm-1 respectively by analyzing IR spectrums, indicating that triple helical conformation of the kinds of collagens were reserved completely; According to analyze amino acids compositions, there were 18 kinds of amino acids were found for ASC and PSC, including 8 kinds of necessary amino acids humans need, compositions of which were a little different between ASC and PSC; The differences including denaturation temperature, hydration and relative solubility for ASC and PSC were not obvious.2. With the degree of hydrolysis(DH) acting as an indicator, choosing a kind of protease from six kinds of proteases(pepsin, papain, alkaline, neutral, trypsin, protamex) as the best protease for this experiment and optimized preparation process of collagen peptides deprived from Rana dybowskii skins using single enzyme, getting the most suitable protease: alkaline protease, whose most suitable process conditions were time 5h, bottom concentration of 6%, an enzyme substrate ratio(E/S) 15%, under these conditions, DH was 12.75%; Molecular weight distribution of collagen peptides deprived from Rana dybowskii skins were measured by gel chromatography, finding that the variation is large, mostly during the 1000 Da ~ 5000 Da.3. Rana dybowskii skins collagen peptides were separated and purified by ultrafiltration and gel permeation chromatography(sephadex G-25), and the antioxidant activity of different fractions were measured by DPPH and ABTS method, in which P2 was measured to be the most potent fraction in scavenging ABTS and DPPH radicals with the IC50 value of 1.882mg/m L, 3.569 mg/m L, respectively; The stability was studied for P2, including digestive enzymes and thermal stability, and the results showed that the DPPH and ABTS radicals scavenging rates of P2 varied a little with the rise of temperature, when digestive time of pepsin and trypsin changing, so did it, which indicated that there were good stability against temperature and digestive enzymes. There were large numbers of amino acids in P2 when assaying its amino acids contents, in which hydrophobic amino acids were higher than others, and about 30%. 4. the ACE inhibitory activity for the three fractions(P1, P2, P3) was assayed by HPLC, in which P2 exhibited highest activity with the IC50 value of 6.325mg/m L; the results in studying P2’s stability showed that its ACE inhibitory activity varied a little with the rise of temperature, no significantly difference among all kinds of groups(P>0.05), but, its activity became stronger than control group after being digested a long time(pepsin 120 min, trypsin 100min)(P<0.05). The correlation study between antioxidant and ACE inhibitory activity found that except for concentration changing, there was a significant correlation for them(P<0.05)), something else(temperature, digestion time of artificial gastric juice, artificial pancreatic juice) changing, there were no significant correlation(P>0.05). |
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