| Lactoferrin is a non-heme iron-binding glycoprotein with natural activity in milk.It has a wide range of biological activities such as anti-oxidation,anti-inflammatory,broad-spectrum antibacterial,inhibition of cancer cell growth and immune regulation,which gives it considerable market potential.Therefore,the research on the separation and preparation of lactoferrin is of great significance for the flourishing development of the lactoferrin industry.In recent years,the raw materials for the isolation and preparation of lactoferrin in China are mainly bovine colostrum and there are few studies on the separation and preparation of lactoferrin from fresh milk.In this paper,fresh milk is used as raw material and enzymatic,membrane and chromatographic techniques are continuously used.The optimum process parameters are determined by using lactoferrin purity,recovery rate and membrane efficiency as indicators thus providing the industrilazation of the separation and preparation of lactoferrin with a certain theoretical basis.Via the optimization research of crude lactoferrin extraction using enzyme curd method,the following conclusions are drawn:The curd temperature is 38?—46?,the curd pH is 5.86.6,the curd time is40 min60min,the chymosin addition is 0.005%0.007%,the lactoferrin purity is the highest,whey turbidity the best.The optimal process parameters:curd temperature 42?,curd pH 6.2,curd time40 min,curd enzyme addition amount 0.005%.The lactoferrin content was?2.24±0.25?%,and the whey turbidity A500 was 0.25±0.05.Via the optimization research of lactoferrin separation using membrane technol-ogy,the following conclusions are drawn:?1?The protein content of the 0.45?m ceramic membrane microfiltration perme-ate is higher than the protein content of the 0.10?m ceramic membrane microfiltration permeate,and the permeate protein content of the two pore size ceramic membranes is microfiltration with 0 to 3 hours.The prolongation of time was significantly reduced?P<0.05?.There was no significant change in protein content after 3 hours?P>0.05?.The microfiltration membrane could not continue to function normally,so choose to wash back once every 2 hours.?2?Quadratic multiple regression equations for the relationship between mem-brane efficiency?Y1?,lactoferrin purity?Y2?,feed liquid temperature?X1?,operating pressure?X2?and feed liquid pH?X3?are as follows:Y1=0.28+9.653×10-3X1+2.093×10-3X2-0.026X3+8.235×10-3X1X2-2.430×10-3X1X3-1.080×10-3X2X3-0.038X12-0.022X22-0.085X32Y2=20.76+0.73X1+0.094X2-1.63X3-0.1X1X2+0.31X1X3+0.18X2X3-1.81X12-0.095×X22-4.51X32?3?The optim?m process parameters for ultrafiltration of 100kDa ultrafiltration membrane are:feed liquid temperature 35.65?,operating pressure 0.202 MPa and feed liquid pH 6.85.Under this condition,membrane efficiency is 0.287g/?m2·min?;?4?The optimum process parameters for ultrafiltration of 50kDa ultrafiltration membrane are:feed liquid temperature 35.90?,operating pressure 0.267 MPa and feed liquid pH 6.83.Under this condition,the purity of lactoferrin is 20.97%.?5?The verification test results showed that the membrane efficiency was?0.283±0.021?g/?m2·min?when ultrafiltration was performed using a 100 kDa ultrafiltra-tion membrane.Using 50 kDa ultrafiltration membrane ultrafiltration?20.92±0.09?%,close to the theoretical value.The optimization research of lactoferrin purification using chromatographic technology leads to the following conclusions:?1?Among the three gels of CM-sephadex C-25,SP-Sepharose FF and CM-sepharose FF,CM-sepharose FF had the largest adsorption capacity of 17.73mg/mL.?2?According to the single factor results,the optimal process parameters were obtained by orthogonal experiment:the eluent ionic strength was 0.9mol/L,the load-ing rate was 1.0mL/min,and the elution rate was 0.8mL/min.The verification test was carried out according to the above conditions,and the results showed that the purity of lactoferrin under the conditions was?91.24±0.43?%,and the recovery was?86.33±0.31?%. |
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