Study On Establishment Of The Testing Method Of Lactoferrin In Milk

Lactoferrin(LF)is a glycoprotein existed in external secretions such as milk, especially colostrum. LF has various bioactive functions such as broad-spectrum antimicrobial activity,promotion of iron transfer and absorption, immunomodulatory effects, regulation of in flammatory reactions, antiviral activity, and inhibition of lipid peroxidation. Therefore, it has great potential to be widely used in foods, pHarmaceuticals,and cosmetics.The experiment established a quick and accurate colloid gold method to determine LF. By purifying LF , preparing polyclonal antibody of lactoferrin , building and optimizing colloid gold method. colloid gold method of determining LF has been built.The ion exchange chromatograpHy of purification LF in laboratory condition was studied The experiment used the CM-SepHarose Fast Flow to dynamically absorb the LF, applied the 1.6% and 5% NaCl to step elute, and optimized the process of laminar analysis and the method of sample reception. The result shows that the purity of LF reaches 96.7% and the concentration of 3.2mg/ml by elution and the optimization of the method of sample reception.Preparation of LF antibody: The experiment took big white rabbit of New Zealand and Roman hens as immunological object , immunological dosage of New Zealand white rabbit was 1 mg/kg , the immunological period was three weeks, immune times was four. The immunological dosage of Roman hen for egg laying was 0.5 mg/kg , the immune period was two weeks. The treatment of antiserum and egg adopted the method of saturated ammonium sulpHate, saturation were 50% and 33% by two deposits repeatedly. Indirect ELISA was used to measure antibody liter, the antiserum was 1:48000.Establishment and optimization of the method of Colloidal gold LF detection. By checking wavelength , the best gold probe, solution pH, temperature and BSA thickness , we reached the best detecting condition that is, the detecting wavelength is 630 nm; The best Gold probe thickness is 1:2; The best PH value is 7.4; In diluting solution, the BSA thickness is 15 ug/ml; The best detecting temperature is 0-20min for 250C as effect time section. Taking LF in checking milk as example , we build up a new and quick method of detecting LF.Appraisal and application of the method. The experiment took LF in checking milk as example , by repetitive test , reclaiming test and comparative test to appraise the method. The method was used in milk and colostrums to test the application and utility. The result shows that the method is sensitive, which can test 10-2500ng/ml, the recovery rate is 95.89%, the coefficient of differentiation of the same batch is 3.57%, while the different batch is 4.74%.