Separation And Purification Of Antimicrobial Peptides In Lactoferrin Hydrolysates

Antimicrobial peptides not only have broad-spectrum antibacterial effect, but also can restrain or kill the viruses, parasites and cancer cells, etc. Antimicrobial peptides have been found in lactoferrin hydrolysates(LHs) in previous study. They are natural substances, safe, harmless and easy to be absorbed by human body. They have broad application prospects in food additives, especially beverage additive. The main content of this experiment was to separate and purify the antimicrobial peptides which we previously obtained from LHs.The LHs were prepared by pepsin hydrolysation in this study. The conditions of hydrolysating lactoferrin were substrate concentration 5%, reaction temperature 37℃, reaction time 4 h, enzyme/substrate 1000 U·g-1, p H adjusted to 2.5. The LHs were purified by salting-out, gel filtration chromatography and semi-preparation reversed-phase high performance liquid chromatography(RP-HPLC). The antimicrobial activity was determined using Escherichia coli as the sensitive bacteria. Specific research results were as follows:(1) Antimicrobial peptides was separated from LHs after lactoferrin was hydrolysised into different fragments of peptides. Sephadex G-25 was used as separating gel. The separation condition were as followed: 0.1 mol/L phosphate buffer solution(PBS) as the eluent, the elution rate 0.5 m L/min, the loading quality of sample 1 mL, sample concentration 25 mg/m L, and ultraviolet detector at 280 nm in room temperature. There was one tailing peak in the gel filtration chromatograms, that suggested further seperation. The separated products were collected and freezed dried, the antimicrobial activity was detected, and the bacteriostasis rate was 19.0 %.(2) Salting-out classification was carried out before gel filtration. Ammonium sulfate solutions of different saturation levels(20 %-60 %) were select to salt out LHs, salting products were separated by Sephadex G-25 gel filtration and antibacterial activities of LHs were analyzed. The experimental results showed that the bacteriostasis rate of LHs was the strongest at 35 % saturation of ammonium sulfate. There were two peaks(peakⅠand peakⅡ) in gel chromatograms that also suggested further seperation. The products were collected, freezed drying, and antimicrobial activity was analyzed. The bacteriostatic rate of peakⅠ(88.0 %) was higher than the others. Thus the target antibacterial peptide was in the peakⅠseparation products. At the same time, desalination was performed.(3) The collection of peakⅠwas purified again by RP-HPLC. The purification conditions was the sample loading quality 100 μL, the sample concentration 40 mg/mL, the column temperatureadjusted to 35 ℃, mobile phase solution A: 0.1 % trifluoroacetic acid aqueous solution, mobile phase solution B: chromatographic grade of 98 % acetonitrile and 0.1 % trifluoroacetic acid solution, the elution rate 3.0 m L/min, ultraviolet detector wavelength 254 nm. There were a sharp peak a and a few small miscellaneous peaks in the chromatogram. All the seperated products from different peaks were analyzed after collection and freezed dring, and the bacteriostatic rate of peak a was 92.0 % which was obviously higher than that of the former two levels of purification products. It was suggested that the target antimicrobial peptides were purified from LHs.