The Impact Of Lactoferrinon Of The Physicochemical Properties And The Proliferation Activity Of Osteoblasts By Heat Treatment

Lactoferrin is one of the four proteins in human milk, accounting for about20%ofthe total protein in it, but it is not milk-specific components, which are widely present inthe milk and body fluids of mammals and is a multifunctional basic protein. But theproduction of infant milk powder, natural milk need to go through multiple heat treatmentprocess, the activity of bovine lactoferrin bound to varying degreesaffected, and this isignored by most people. A large number of data have shown that Lactoferrin is verysensitive to heat and prone to thermal aggregation phenomenon.This study a ims to behardened in the processing aspects of the physical and chemical properties of bovinelactoferrin,the protein thermal aggregation made a preliminary exploration, and investigatethe after heat treatment, the proliferative activity of osteoblasts, so that processing of themilk powder, in particular, functional milk powder processing to provide effectivetemperature control of process parameters.In this study, the crude purified natural bovine lactoferrin re-isolation and purification,purity90%, as to the lactoferrin standard products of Sigma. Molecular weight observedby SDS-PAGE gel electrophoresis analysis of protein bands using Quantity One software,we get a purity of90%Lactoferrin sample, its purity is as tothe90%purity Lactoferrinstandard product purchased from Sigma. Subsequently, the purified Lactoferrinpreparation of iron saturated Lactoferrin (Holo-Lf) and completely iron deficiencyLactoferrin (Apo-Lf), freeze-drying, spare. Preparation of three different iron saturationLactoferrin powder into the same concentration of protein solution, the measuredabsorbance values in the465nm wavelength, the experiment was repeated three times toobtain average to determine the three proteins of iron saturation. Iron saturation of theobtained samples were as follows: the iron saturation of the native Lactoferrin is15.32%,iron saturation of the Holo-Lf is89.10%, iron saturation of Apo-Lf is3.73%, All incompliance with production standards. Three different iron saturation of Lactoferr in ismeasured by Disfenential Seanning Calorimetry (DSC) thermal mutagenic peak, andprovide the basis for the heat treatment temperature.The results are as follows: the Native-Lf has two hot mutagenic peak, respectively are62.66℃and69.83℃，the heat mutagenesis peak of Apo-Lf is62.10℃，and thehot mutagenesis peak of Holo-Lf is70.18℃. According to the test results of DSC,the heating conditions of60℃,65℃,70℃,80℃, and90℃water bath for5min were selected. After heat treatment, by three different microplate determinations of thethree different kinds of Lactoferrin, combined with state lactoferrin iron saturationconditions, compared with the value of the unheated investigated the influence of heattreatment on the saturation of Lactoferrin. The experimental results showed that almost nochange of iron saturation after the heat of the Holo-Lf, under the conditions of heating80℃for5min, Native-Lf shows the phenomenon of loss of iron, under80℃heatingconditions, iron saturation reduction of Apo-Lf is more obvious. Molecular weightchanges of the three heated Lactoferrin were observed by SDS-PAGE gel electrophoresisand software analysis, in order to determin the heat treatment after protein ther--mal aggregation or thermal decomposition.Meanwhile, the three protein samples after heat treatment5000g centrifugation for10min, the protein concentration in the extract supernatant was measured by microplateabsorbance combination with the standard curve to determine the heat treatment. Thisresult and electrophoresis were combined to examine the protein thermal aggregationphenomenon. The study has found that the native Lactoferrin in the heating temperature of70℃arise thermal aggregation phenomenon and protein content starts to decrease.Holo-Lf in the heating temperature of90℃arise slight thermal aggregation phenomenon.Apo-Lf arise thermal aggregation phenomenon when the heating temperature is70℃, andthat is more obvious than native Lactoferrin, moreover, accompanied by the lowest proteincontent.In this study, we successfully separate and determine the skull of osteoblasts bynewborn Wistar rats, and subculture. After heat treatment of three different ironsaturation of Lactoferrin, they were reacted with osteoblasts for48h, and theosteoblast proliferation was determined by MTT assay. When native Lactoferrin heatedto70℃, the activity decreased. When the temperature is80℃, the activity of Holo-Lfbegan to decline. When the heating temperature is70℃, the activity of Apo-Lf issignificantly decreased, but when raise the heating temperature up to90℃, the activityrecovered, which may be related to the proteolysis of Apo-Lf.