Fractionation of β-Lactoglobulin Tryptic Peptides by Ampholyte-Free Isoelectric Focusing

Abstract

Solutions of tryptic hydrolysate of bovine beta-lactoglobulin were fractionated by liquid-phase IEF in a preparative Rotofor cell at constant power for 2 h without ampholytes in order to identify interactions between peptides. The 20 peptide fractions collected were analyzed by capillary electrophoresis and SDS-PAGE under native, denaturing, and reducing conditions. The hydrolysate was shown to be composed mainly of acidic peptides (pI 2-5, 62%) of molecular mass below 6 kDa, and numerous disulfide bonds were detected. Purified peptides (beta-LG 15-20, 71-75, 76-82, and 84-91) were also focused individually and in mixtures and matched to components of the IEF fractions obtained from the tryptic hydrolysate of beta-LG. The separation of acidic (beta-LG 84-91) and basic (beta-LG 76-82) peptides was achieved by IEF, whereas uncharged peptides (beta-LG 15-20 and 71-75) were poorly separated due to their low electrophoretic mobility. Because no peptide-peptide interaction could be identified by IEF fractionation, it is suggested that electrical fields may decrease electrostatic interactions between charged peptides.

Stimulation of Interleukin-10 Production by Acidic β-Lactoglobulin-Derived Peptides Hydrolyzed withLactobacillus paracaseiNCC2461 Peptidases

“…Acidic and basic peptides were separated from the tryptic-chymotryptic hydrolysate of BLG by ampholytefree isoelectric focusing by the protocol described by Groleau et al (9). Briefly, hydrolysate (0.5 g) was rehydrated in 40 ml of deionized water and fractionated for 2 h at 4°C by liquid-phase isoelectric focusing in a preparative Rotofor cell (Bio-Rad Laboratories, Hercules, Calif.) at constant power (15 W).…”

Effects of bifidobacterial cytoplasm peptide and protein fractions on mouse lymphocyte proliferation and cytokine production

“…Cytoplasmic contents were separated by ampholyte-free liquid-phase isoelectric focusing, using the method described by Groleau et al (2002). Briefly, extracts (0.1 g) were re-suspended in 40 ml of deionized water and fractionated for 2 h at 48C in a preparative Rotofor cell (Bio-Rad Laboratories, Hercules, CA) at constant power (15 W).…”

“…)/150 mm long was used with a Waters HPLC system (model) under the following conditions: flow rate, 1 ml min (1 ; column temperature, 398C; solvent A, trifluoroacetic acid (TFA) 0.11% (v/v) in HPLC-grade water; solvent B, acetonitrile/ water/TFA 60%/40%/0.1% (v/v). Elution was done with a linear gradient of solvent B from 0Á/60% over 45 min (Groleau et al 2002). Major peaks corresponding to peptide or protein from B. lactis Bb12 were collected individually at different elution times, thoroughly dried using a Speed Vac concentrator (Savant Instrument Inc., Farmingdale, NY) and re-suspended in water for immunoactivity assays.…”

“…Peaks collected from RP-HPLC were analysed using a MSD QUAD Agilent 1100 Series mass spectrometer (Agilent Technologies, Palo Alto, CA) following the method of Groleau et al (2002). The effect of TFA was reduced by re-suspending dried fractions in 20% (v/v) propionic acid prior to infusion in the MS interface.…”

Recent advances in the application of capillary electrophoresis for food analysis

“…In other studies related to the breakdown of milk proteins, CE has been applied in a study of the tryptic hydrolysates of bovine b-lactoglobulin [13]. Certain bioactive peptides are formed in such hydrosylates that are of interest as potential nutraceuticals or functional food ingredients, but peptide-peptide interactions have been identified as a possible obstacle to the successful isolation of these products.…”