Preparation And Isolation Of Lactoferrin Antimicrobial Peptide

Lactoferrin is a bioactive protein in the bovine colostrum, with a variety of biological functions, such as broad-spectrum antibacterial activity, reinforced iron transfer and absorption, antivirus functions. But its poor thermal stability seriously effect its applications. After hydrolysed by pepsin, it not only can improve the inhibitory activity, but also can strengthen the thermal stability. This lead it broad application prospects in food, medicine, feed industry and so on. This study is very meaningful for the improvement of nature antibacterial agent.This paper studies the preparation, the processing of desalination and isolation and purification of lactoferrin antimicrobial peptide. At the end, antibacterial activity was studied.First of all, preparing lactoferrin antimicrobial peptide from the enzymatic hydrolysis of lactoferrin was studied. Through single factor experiment and response surface regression analysis of the hydrolysis process, the optimal parameters were: enzyme of pepsin, lactoferrin concentrations of 30 mg/mL, the ratio of pepsin and lactoferrin 1.5%, temperature 44℃, pH 2.4, hydrolysis time 2 h. Under optimal conditions of the enzyme hydrolysis, the degree of hydrolysis was 10.76% and inhibitory activity was 97.97%. Amino acid composition analysis showed the ratio of hydrophobic and basic amino acid of lactoferrin antimicrobial peptides were 32.90% and 19.25%, respectively. Studying on the enzymatic hydrolysis product of the molecular weight distribution, found that molecular mass of lactoferrin antimicrobial peptide distributed mainly in 200~6000.Secondary, desalination for lactoferrin antimicrobial peptides in static and dynamic states of macroporous adsorption resin DA201-C was investigated. Based on physical performance of resins, the static dynamics curve and equation of adsorption quantity, adsorption isotherms, and the best detergent were obtained by static adsorption experiments. While the concentration of lactoferrin antimicrobial peptides was 10 mg/mL, equation of adsorption quantity was Y=0.5035X-0.3971(R~2=0.9045). while for 30 mg/mL, the equation was Y= 0.4887X+1.0815(R~2=0.9412). The equation of adsorption quantity was Qe=1.7461C+0.5703 (R~2=0.9993). The best detergent was 85% ethanol. The dynamic adsorption experiments showed that the resin to desalt lactoferrin antimicrobial peptides with 85% ethanol as desorption agent, desalination rate was 97.40%, and reclaim rate was 92.54%. The results of stability studying showed lactoferrin antimicrobial peptide had strong resistence digestion stability and thermal stability and storage stability at 4℃at acid condition.Finally, the separation and purification procedure was studied. Lactoferrin antimicrobial peptide-b was isolated by Sephadex G-50. Its inhibitory activity was 99.17% at the concentration of 1 mg/mL. Then purier lactoferrin antimicrobial peptide-b1 was isolated by semi-preparative RP-HPLC, its inhibitory activity to E.coli was 99.05% at the concentration of 0.1 mg/mL. Finally, lactoferrin antimicrobial peptide-b1-1 separated by RP-HPLC, when it was in the 0.1 mg / mL, inhibitory activity was 99.12% and its molecular weight was around 2500. The results of the antimicrobial activity showed that the lactoferrin antimicrobial peptide can inhibit common food spoilage and pathogen organisms including Gram-negative and Gram-positive bacteria. It had a broad spectrum antimicrobial activity.