| Collagen is an important part of the organic matrix of bone. Its hydrolysate, collagen peptides, have a lot of physiological activity, such as promote the absorption of amino acids and protein synthesis, and promote mineral absorption, anti-osteoporosis, lowering blood pressure, anti-oxidation, etc. In this paper, we use tilapia skins, which is one of the by-products of tilapia, as raw materials for preparing the collagen peptide to exploring the effect of collagen peptides on promoting osteoblast proliferation. And after preparation and differentiation activity to obtain collagen peptide products with specific efficacy, and to make tilapia by-products more valuable.The tilapia skins’ main compositions were determined in this article. Protein content in dry skin reached90.30%, and66.90%of which are collagen.48h defatted with petroleum ether were used to purify the collagen matrix, the degreasing rate reached62.2%. Hydrolysis ability of four commodities enzymes were compared, where the animal protease enzymatic and flavor enzyme have higher hydrolysis degree on tilapia skin. Orthogonal experiment was used to optimize the two enzymatic hydrolysis conditions, which showed that the optimal conditions of them are the same. Using composite, which consist of animal protease and flavor enzymes, to hydrolysis, both of the dosages are4000U/g protein. Hydrolyzing the tilapia skins under a series of different time (5h,8h,22h) in55℃, pH7.5. Then comparing the effect of the collagen peptide (H5, H8, H22) on the proliferation and differentiation of rat osteoblast activity, and the collagen peptide (Hc) which made by type I collagenase digestion5.5h were used as a positive control. The results show that, with the extension of digestion time, the activity of promoting osteoblast proliferation increased, the growth rate of H8, H22(1mg/mL) were140.35%and144.21%, respectively; the difference was not significant, but it has significant differences with the control group; and it also has an promoting effect on osteoblast differentiation.The degree of hydrolysis, molecular weight distribution and amino acid composition of H5, H8and H22were analysed. The results showed that, the degree of hydrolysis increased followed with digestion time, but the relative molecular weight declined. The components of relative molecular mass which less than1000Da of H5, H8and H22are94.73%,95.56%and97.58%, respectively. And their average molecular weight are51.39Da,324.67Da and253.06D, respectively. Meanwhile, the amino acid composition of H5, H8and H22are closer, which are correspond to the typical amino acid composition of aquatic collagen. But the proline content of them decreased and the hydroxyproline content increased with the digestion time. Nanofiltration membrane with800-1000MWCO(molecular weight cut off) was used to separate H8, and the Permeate1(P1) and Retentatel(R1) were collected to compare the activity. A400-600MWCO Nanofiltration membrane was used to separate the more active one, and the Permeate2(P2) and Retentate2(R2) were collected to compare the activity. The results showed that, comparing to R1, P1has strong activity of promoting the proliferation and differentiation of osteoblast, the cell proliferation rate and differentiation rate reached126.92%and131.77%, respectively. Though both of R2and P2have promoted active on osteoblast proliferation and differentiation, and have reached the level of Hc. P2was stronger than R2, the cell proliferation rate and differentiation rate reached130.48%and120.72%, respectively.The molecular weight distribution and amino acid composition of P2and R2were analysed. The results showed that there was a big difference between them. The component of relative molecular mass less than1000Da of P2and R2accounted for98.65%and96.14%, respectively; average molecular weight are184.62Da and319.59Da, respectively. Furthermore, glycine and alanine of P2accounted for67.2%, and in other amino acids only proline more than5%, reached6.1%, and the ratio of lysine(the typical amino acid of collagen protein) increased by125%. Except proline and hydroxylysine content increased by30%and100%, respectively, the others were closed to H8. Collagen peptide promotes the activity of osteoblast proliferation differentiation may be related to the two kinds of amino acids, or peptides containing these two amino acids. Considering the activity of P2is the highest one, but the yield is extremely low. However, the activity of P1and R2are insignificant, considering the cost, select P1as the final purification peptide. The collagen peptide prepared from this process featured by uniform color and nuance powder, its solubility is excellent, meeting the requirements of SB/T10634-2011<the freshwater fish collagen peptide powder> to the level of the quality of the first-class product. The yield of coarse collagen peptide is18.20%, and the yield of refined peptide is6.67%. |
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