Isolation and characterization of the bioactive fractions from carabao (Bubalus bubalis L.) milk lactoferrin

Lactoferrin (cLF) was isolated from carabao (Bubalus bubalis L.) milk whey by ammonium sulfate precipitation and cation exchange chromatography (CEC) using carboxymethyl cellulose (CMC) as cation exchanger. Bovine lactoferrin ELISA Kit was used for the detection and quantitative of lactoferrin from the samples. Pepsin hydrolysis of the purified cLF was done to determine the fractions of the protein which is bioactive. cLFH fractions were separated and purified by cation exchange chromatography and were characterized. cLF and isolated cLFH fractions were subjected to antibacterial tests in vitro. Bovine LF was likewise tested for antibacterial activity for comparison. Carabao LF was eluted at approximately 0.27-0.30 M NaCl with a 24.97 percent recovery from the whey sample. Average concentration obtained from isolation runs was 22.49 plus or minus 16.52 mg/L. SDS-PAGE of cLF showed RF value corresponding to MW of 77,829. Carbohydrate analysis by phenol-sulfuric acid test showed carbohydrate content of 0.346 percent indicating the glycoprotein nature of the cLF. In vitro antibacterial assay showed that cLF can inhibit growth of enteropathogenic E. coli (EPEC) and S. typhimurium with minimum inhibitory concentrations (MIC) of 800 and 1600 micro g/mL, respectively. Commercial bovine LF on the other hand has twice the activity of cLF with MICs of 400 and 800 micro g/mL for EPEC and S. typhimurium, respectively. Carabao milk LF hydrolysate (cLFH) loaded in CEC-CMC yielded 3 peaks after elution with 0.0-1.0 M NaCl gradient. cLFH 1, 2 and 3 were eluted at salt concentration ranges of 02-0.04 M, 0.08-0.12 M and 0.4-0.55 M, respectively. SDS-PAGE showed fractions with MW of 6,500 and below with cLFH 2 and 3 relatively larger than cLFH 1. cLFH 1 was observed to have the highest antibacterial activity against EPEC and S. typhimurium with minimal inhibitory concentration of 25 micro g/mL compared to 100 to 200 micro g/mL for cLFH 2 and 3. Antibacterial activities of cLF and its cLFH fractions against EPEC and S. typhimurium were stable from pH 3 to 7 and up to 80 deg C.