| Lactoferrin is a kind of key basic glycosidoprotein in bovine milk, which hasattracted more and more research attentions. Bovine lactoferrin was a new kind of bonegrowth factor, it played a certain role in bone growth and metabolism and had the abilityto boost growth. Heat treatment which was very common in the dairy industry hadeffects on the proliferation ration of osteoblast This article selected fresh milk as rawmaterial to purity lactoferrin and made the research on variation of molecular structureand properties by Circular dichroism and differential scanning calorimetry afterdifferent heat treatment, meanwhile the method of MTT was used to compare effect ofdifferent heat treatment on the proliferation ration of osteoblast cells.In the present study, lactoferrin was isolated from fresh bovine milk by defattingand casein-removing treatments, and subsequently purified by SP Sepharose Big Beadion exchange chromatography, Superdex200gel chromatography, ultrafiltration andvacuum freeze drying. The purified form of lactoferrin was obtained with purity of94.71%. The denaturation temperature of lactoferrin was determined to be73.1℃bydifferential scanning calorimetry. Furthermore, the effects of heat treatment on themolecular properties of lactoferrin have been determined. The heat-processed parameterhas been set:62～65℃30min，72℃10s，85℃10min，95℃10min Combined withthe heat treatment parameters of the actual dairy processing. Electron microscopy andlaser diffraction particle size analyzer study on the influence of heat treatmentlactoferrin showed that with increase of the treatment temperature the size of proteinaggregates increased, that is to say, the degree of Lactoferrin thermal aggregationenhanced. Circular dichroism studies on the influence of heat treatment on thesecondary structure of lactoferrin showed that with increase of the treatmenttemperature the α-helix conformation of lactoferrin was gradually transformed toβ-sheet conformation, indicating that the protein molecules became better ordered andmore compact with significantly increased trends of protein denaturation. Fluorescencespectrophotometer tudies on the influence of heat treatment on the secondary structureof lactoferrin showed that with increase of the treatment temperature protein surfacehydrophobicity decreased at first and then increased, indicating that protein surfacehydrophobicity reached the minimum when the heat temperature was close to thedenaturation temperature.Osteoblast cells were extracted from calvaria in newborn SD rats by enzymedigestion, the proliferation ration of osteoblast cells was determined by MTT assay. Thedifferent effects of temperature-time on the bioactive activity of lactoferrin were compared. The extracted cells were osteoblast cells by HE staining, alkalinephosphatase staining and mineralized nodules staining. The most of osteoblast cellswere fusiform or triangular with two or three projections by morphological observation.Results showed that unheated lactoferrin (native-LF) can significantly promote theproliferation of osteoblasts in a dose-dependent manner at the concentration range of50-500μg/mL. Moreover, lactoferrin has different effects on the proliferation ofosteoblast at different incubation time of24h,48h and72h. Compared with thenative-LF, the treatments of65℃/30min and72℃/10s didn’t have significant differenceon the activity. However,85℃/10min and95℃/10min have shown significant effectson the proliferation of osteoblast, especially on the high concentration group. Underhigh intensity heat treatment conditions of85℃/10min and95℃/10min, the activity oflactoferrin will decrease with the increasing concentration of lactoferrin.This study described heat treatment had effects on the structure， molecularproperties and osteoblast proliferation activity of lactoferrin This article laid theoreticalbasis on development of lactoferrin relevant product. |
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