Characterization by ionization mass spectrometry of lactosyl beta-lactoglobulin conjugates formed during heat treatment of milk and whey and identification of one lactose-binding site.
The extent of the early stage of the Maillard-type reaction that impaired functional properties of whey proteins was evaluated by electrospray ionization mass spectrometry. Under conditions of mild heat treatment (63 degrees C for 20 s) applied to milk before whey separation at room temperature 23 degrees C), a modification of the relative molecular mass of beta-lactoglobulin (beta-LG) was observed that differed from that of the native form by 324. This specific modification of beta-LG occurred in acidified whey as well as in sweet whey and increased with the extent of the heat treatment. Incubation of purified beta-LG dissolved in milk ultrafiltration permeate or in lactose solution at 50 to 80 degrees C demonstrated the presence of a lactosyl residue that was covalently bound to beta-LG; beta-casein, used as a control, showed no mass modification. Studies of kinetics showed that a maximum of 35% of the beta-LG was lactosyl-beta-LG conjugate after heat treatment at 70 degrees C for 1 h. This study provides the first direct evidence of specific lactosylation of beta-LG during the initial stage of the Maillard reaction. One of the first lactose-binding sites was identified as a Lys47 by protease mapping and analysis by means of on-line liquid chromatography combined with mass spectrometry. In addition, collision-activated dissociation performed on the lactosylated peptide beta-LG (f 46-51) showed the rearrangement reactions occurring during the fragmentation process by electrospray. A mechanism is proposed.
Leonil
J
J
Institut National de la Recherche Agronomique, Laboratoire de Recherches de Technologie Laltière, Rennes, France. jleonil@labtechno.roazhon.inra.fr
Molle
D
D
Fauquant
J
J
Maubois
J L
JL
Pearce
R J
RJ
Bouhallab
S
S
D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D006358:Hot Temperature; D006868:Hydrolysis; D007782:Lactoglobulins / Q000737:chemistry / Q000378:metabolism*; D007785:Lactose / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D015416:Maillard Reaction; D013058:Mass Spectrometry; D008892:Milk / Q000737:chemistry*; D008894:Milk Proteins / Q000737:chemistry*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D014357:Trypsin; D000067816:Whey Proteins
D007782:Lactoglobulins; D008894:Milk Proteins; D010446:Peptide Fragments; D000067816:Whey Proteins; D014357:Trypsin; D007785:Lactose; D008239:Lysine