Peptides antimicrobiens

Abstract

The aim of this study is to obtain antimicrobial peptides from bovine and camel casein. The actives peptides are generated by proteolysis of these proteins, inactive in the native state. The hydrolysis is carried out by the action of three gastrointestinal proteases; pepsin, trypsin and chymotrypsin. The hydrolysis is followed by chromatographic (RP-HPLC, C18 column) and electrophoresis (SDS-PAGE, 15 %) analysis. The degree of hydrolysis is measured by the OPhthaldiAldehyde method (OPA). The antimicrobial activity is sought on raw hydrolysates by a plate diffusion assay. The active peptides were identified by mass spectrometry (LC/MS and MALDI-TOF), after split and purification on RP-HPLC. They are characterized by determination of the Minimum inhibitory concentration (MIC) and bactericidal or bacteriostatic properties. A peptide extract enriched antimicrobial peptides is prepared from pepsin hydrolysis of bovine casein by precipitation at 2 M NaCl and neutral pH. The active peptides are derived from αs2-casein and αs1-casein. They correspond to fragments (164-179) (148-166), (180-207) (183-207) and (176-207) of the αs2-casein. New active peptides were identified; (131-174), (153-196) (99-137) (99-163/100-164), (114-176) (115-174), (126-184), (28-90), (138-184) of αs2-casein and (9-68/70-126) of αs1-casein. The peptide extract and a pure peptide αs2 (164-207), showed lower MIC values against Gram-positive bacteria compared to those determined on Gram-negative strains tested. At a concentration equal to the MIC, the tested peptides have a bacteriostatic effect and a bactericidal effect on Gram-positive and on Gram-negative strains, respectively. The action of pepsin on camel caseins also releases antimicrobial peptides. The peptides identified correspond to fragments (66-90), (83-98), (109-126) of αs1 casein; (106-129), (1-19) et (126-142) of αs2 casein; (22-43), (52-69), (50-67) et (77-95) of β casein et (121-141), (116-136), (83-100),(106-124), (12-27) et (61-79) ofκ casein. Under imposed hydrolysis conditions, trypsin and chymotrypsin hydrolysis of casein from the two species, does not release peptides with antimicrobial activity. The study of the action of these proteases in different hydrolysis conditions should be considered. The action of the three proteases on casein follows an intermediate mechanism between the mechanism ""one by one"" and ""zipper"". According to the results, antimicrobial peptides are intermediate hydrolysis products. Thus, the application of hydrolysis conditions favoring the mechanism ""zipper"" deserves to be studied in order to optimize the release of peptides of interest. Bovine caseins were more susceptible to hydrolysis by pepsin and trypsin than camel caseins. The extent of hydrolysis measured over 24H is evaluated at 8.21±0.761% against 5.41±0.057% for pepsin and at 9.91±0.409 % against 8.94±0.106 % for trypsin on bovine and camel caseins respectively. Whereas, when using chymotrypsin, The extent of hydrolysis is similar on both species (8.70±0.413 against % 8.80±0.205 % for bovine and camel caseins, respectively).