Catherine Bertrand-Harb

Proteolysis of β-lactoglobulin and β-casein by pepsin in ethanolic media

Abstract Limited proteolysis of β-lactoglobulin and β-casein by pepsin was performed in the presence of varying concentrations of ethanol. β-Lactoglobulin started to be cleaved by pepsin only in ethanol concentrations greater than 20%, when its secondary structure began to change. In 25% ethanol, the rate of hydrolysis of β-lactoglobulin was slow (40% remained intact after 40 h of hydrolysis) and many short and hydrophilic peptides were observed. The rate of hydrolysis of β-lactoglobulin reached its maximum in 30 and 35% ethanol (80% of β-lactoglobulin was hydrolysed after 10 h), and a mixed population of hydrophilic and hydrophobic peptides of different lengths was observed. Large hydrophobic peptides appeared first, then some shorter products. The rate of hydrolysis of β-lactoglobulin decreased at ethanol concentrations equal to or higher than 40%, when only a few long, hydrophobic peptides were produced. As seen by circular dichroism, the addition of ethanol to β-casein induced α-helix formation and reduced the rate of casein hydrolysis without changing the peptide profile. The only exception was the yield of a single peptide (Pro81 − Met93).

Evolution of β-lactoglobulin and α-lactalbumin content during yoghurt fermentation

The evolution of the concentrations of β-lactoglobulin (BLG) and α-lactalbumin (ALAC) was studied during the early stages of yoghurt fermentation by YC 191, a mixed strain culture from Chr Hansen containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Radial immunodiffusion of samples taken at different times indicated that the concentration of both proteins remained constant during fermentation. Electrophoresis performed on 12% polyacrylamide slab gels confirmed the results obtained with radial immunodiffusion. In model experiments, the strains were incubated either separately or in combination with both whey proteins, one by one or together. BLG proteolysis required a longer time than that used during yoghurt fermentation. ALAC was susceptible to proteolysis, especially by Streptococcus thermophilus. Despite evident possession of adequate proteolytic system, the strains used for yoghurt production did not cleave detectable amounts of the whey proteins during yoghurt fermentation.

Influence of pH on the structural changes of β-lactoglobulin studied by tryptic hydrolysis

The attempt to use trypsin in order to monitor pH (7.5-9.0) induced beta-lactoglobulin conformation changes has revealed differences in the cleavage of specific sites. The tryptic cleavage of two dibasic X-Lys-Lys-Y sites (Lys 69, 70 and 100, 101) shows slighter predominance of symmetrical cut at pH 7.5 and 8.0. Mostly asymmetrical cleavage yielding two C-terminal lysines can be observed at pH 8.5 and 9.0. Atypical cleavage of the Tyr-20-Ser-21 site, which at pH 9.0 is relatively negligible, increases substantially in pH 7.5-8.5. This implies that Tyr-20 probably is the tyrosine reported to be exposed on the surface of the protein during transformation of beta-lactoglobulin molecule occurring in the studied pH range (Tanford et al. (1959) J. Am. Chem. Soc. 81, 4032-4036).