| Lactoferrin(LF) is a basic glycoprotein mainly in the whey protein. As a novel bone growth factor, it can fulfill its function by regulating bone growth and metabolic processes, its related basic and applied research has attracted wide attention.After ingested by the human body, LF must be absorbed through the gastrointestinal tract. It has not reported that LF plays a role of osteogenesis in a form of intact protein,molecular fragments after gastric digestion or both.This study used simulated gastric fluid to hydrolysis LF in different time and made it break down into polypeptide fragments with different molecular weight. LF peptides were purified by SP Sepharose Fast Flow ion exchange chromatography, Superdex Peptide 10/300 GL gel chromatography and vacuum freeze drying, meanwhile the method of MTT was used to compare the effect of different LF peptide on the proliferation of osteoblast cells.The use of mass spectrometry techniques to identify amino acid sequence. The amino acid sequences of LF and most of other reported proteins with osteogenesis activity would be analyzed and contrasted by bioinformatics,then clarified the relationship between the structure of LF hydrolysis polypeptide peptide and its effect on the proliferation of osteoblast, and we can speculate the active mechanism of LF that digested in the gastric environment.The principal contents and results were concluded as followed:In this study, LF was hydrolysised with simulated gastric fluid in 5 min, 10 min, 30 min, 60 min, 90 min, 120 min, 180 min, 240 min.Using gel electrophoresis to analysis the molecular weight of LF hydrolysis peptides.With the extension of hydrolysis time,LF was gradually hydrolyzed into low molecular weight peptides, and the concentration of small molecular weight peptides increased gradually.After 90 min of hydrolysis,the tend of hydrolyzate was unchanged, and the minimum molecular weight was about 5k Da.With the the method of MTT was used to compare the effect of different LF peptide on the proliferation of osteoblast cells, we found that the hydrolyzate produced at 5 min(H5) can significantly promote the proliferation of osteoblasts compared with other time points.Using SP Sepharose Fast Flow cation exchange chromatography to purify H5,five peaks were got, and the fifth peak(P5)can significantly promote the proliferation of osteoblasts.Then using Superdex Peptide 10/300 GL gel chromatography to further purify P5, two peaks were got and the first peak(P5-1) can significantly promote the proliferation of osteoblasts.P5-1 contained two components analysised through gel electrophoresis, and the molecular weight of each component was 9.35 k Da and 6.95 k Da.The study speculated that these two lactoferrin peptides played an important role inpromoting the proliferation of osteoblasts.Compare the amino acid sequence of LF with bone morphogenetic protein(BMP2/4/6/7/9) through bioinformatics. It shows that the similarity between LF and BMP2/4/6/7/9 was 28.56% and the mainly similar parts was in the position from the522 to 597 amino acid of LF. This sequence had 76 amino acids and its molecular weight was about 9 k Da, it was close to one peptide contained in P5-1. Speculating the peptide contened in P5-1 was from this similar sequence. Compared the hydrophobic region, phosphorylation sites, and predicted the secondary structure of the LF hydrolysis peptide then compare the second structure with BMPs,to analyze the similarity of them.This article was to study the osteogenic proliferation activity of LF peptides hydrolysised in the simulated gastric environment, combined with bioinformatics to infer the relationship between the structure and activity of lactoferrin and further elaborate the effect of structure on its osteogenic proliferation activity.These work will sure to establish some basic theoretical foundaments for the application of b LF when it is used as a functional component in the research and development of dairy products. |
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