Reduction of immunoreactivity of bovine beta-lactoglobulin upon combined physical and proteolytic treatment.
Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin before, during and after treatment at 600 MPa and pH 6.8 for 10 min at 30, 37 and 44 degrees C. The extent of beta-lactoglobulin hydrolysis under pressure was noticeably higher than at atmospheric pressure, particularly when chymotrypsin was used. Addition of proteases at ambient pressure to previously pressure-treated beta-lactoglobulin gave only a modest increase in proteolysis with respect to the untreated protein. Products of enzyme hydrolysis under pressure were separated by reverse-phase HPLC, and were found to be different from those obtained at atmospheric pressure when chymotrypsin was used. The residual immunochemical reactivity of the products of combined pressure-enzyme treatment was assessed on the unresolved hydrolysates by ELISA tests using polyclonal and monoclonal antibodies, and on individual hydrolytic fractions by Western Blotting using sera of paediatric patients allergic to whey proteins in cow milk. The immunoreactivity of the whole hydrolysates was related to their content of residual intact beta-lactoglobulin, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolysis under pressure. The results indicate that chymotrypsin effectively hydrolysed hydrophobic regions of beta-lactoglobulin that were transiently exposed during the pressure treatments and that were not accessible in the native protein or in the protein that had been previously pressure treated.
Bonomi
Francesco
F
Dipartimento di Scienze Molecolari Agroalimentari, University of Milan, Italy.
Fiocchi
Alessandro
A
Frøkiaer
Hanne
H
Gaiaschi
Antonella
A
Iametti
Stefania
S
Poiesi
Claudio
C
Rasmussen
Patrizia
P
Restani
Patrizia
P
Rovere
Pierpaolo
P
D001274:Atmospheric Pressure; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007782:Lactoglobulins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D014357:Trypsin / Q000378:metabolism*
D007782:Lactoglobulins; D002918:Chymotrypsin; D014357:Trypsin