The speed of absorption of dietary amino acids by the gut varies according to the type of ingested dietary protein. This could affect postprandial protein synthesis, breakdown, and deposition. To test this hypothesis, two intrinsically 13 C-leucine-labeled milk proteins, casein (CAS) and whey protein (WP), of different physicochemical properties were ingested as one single meal by healthy adults. Postprandial whole body leucine kinetics were assessed by using a dual tracer methodology. WP induced a dramatic but short increase of plasma amino acids. CAS induced a prolonged plateau of moderate hyperaminoacidemia, probably because of a slow gastric emptying. Whole body protein breakdown was inhibited by 34% after CAS ingestion but not after WP ingestion. Postprandial protein synthesis was stimulated by 68% with the WP meal and to a lesser extent (؉31%) with the CAS meal. Postprandial whole body leucine oxidation over 7 h was lower with CAS (272 ؎ 91 mol⅐kg ؊1 ) than with WP (373 ؎ 56 mol⅐kg ؊1 ). Leucine intake was identical in both meals (380 mol⅐kg ؊1 ). Therefore, net leucine balance over the 7 h after the meal was more positive with CAS than with WP (P < 0.05, WP vs. CAS). In conclusion, the speed of protein digestion and amino acid absorption from the gut has a major effect on whole body protein anabolism after one single meal. By analogy with carbohydrate metabolism, slow and fast proteins modulate the postprandial metabolic response, a concept to be applied to wasting situations.

Mechanisms of protein gain during protein feeding have been investigated using a combination of oral and intravenous labeled leucine in healthy young men. The oral labeled leucine was administered as a free oral tracer ([13C]- or [2H3]leucine) added to unlabeled whey protein or as whey protein intrinsically labeled with L-[1-13C]leucine. When the oral tracer was free leucine, it appeared in the plasma more rapidly than the unlabeled leucine derived from the whey protein, and this resulted in an artifactual 88% decrease of protein breakdown. When the oral tracer was protein bound, protein breakdown did not change significantly after the meal. In contrast, nonoxidative leucine disposal (i.e., protein synthesis) was stimulated by 63% by the meal. In conclusion, 1) an intrinsically labeled protein is more appropriate than an oral free tracer to study postprandial leucine kinetics under non-steady-state conditions and 2) protein gain after a single whey protein meal results solely from an increased protein synthesis with no modification of protein breakdown.

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.

-Le phosphocaséinate natif (PPCN) était séparé du lait cru par microfiltration tangentielle sur membrane (diamètre des pores: 0,2 l1m) suivie d'une purification par diafiltration avec de l'eau. Le suivi analytique des liquides obtenus de part et d'autre de la membrane montrait une perméation conforme aux lois de la filtration des protéines majeures du lactosérum (j3-lactoglobuline et a-lactalbumine), du lactose et des sels minéraux solubles et au contraire, une rétention élevée des composants solubles à haut poids moléculaire ainsi que des protéoses-peptones. Le rétentat final était séché par atomisation. Ses teneurs en matière azotée (N x 6,38) et en cendres étaient respectivement de 89% et de 9% par rapport à la substance sèche. L'aptitude à la coagulation par la présure, de la solution reconstituée a été comparée à celle du lait cru. Le temps de coagulation est réduit de 53%; la cinétique d'organisation du. gel (appréciée par le K2Q) et la fermeté finale (appréciée à 30 min) sont également accrues de plus de 50%. La propriété de former un gel sous l'action de la présure après un traitement thermique de 100 oC-5 min est conservée, contrairement au lait qui, soumis au même traitement, ne coagule plus. microfiltration 1 phosphocaséinate 1 coagulation présure 1 traitement thermique Summary-Native micellar casein separation through cross flow membrane microfiltration. Native phosphocaseinate (PPCN) was separated from raw milk by tangential membrane microfiltration (pore diameter: 0.2 um) followed by purification through water diafiltration. Analytical survey of both tiquids issued by membrane treatment showed permeation rates matching mathematical laws for the main whey proteins (fJ-lactoglobulin and a-Iactalbumin), lactose and soluble mineraI salts. On the contrary, a high retention of high molecular weight soluble components and proteose-peptone fraction was observed. Finaldiafiltratedretentate was spray-dried. Its protein (N x 6.38) and ash contents in total solids were respectively 89% and 9%. Rennet coagulating ability of PPCN reconstituted solution was compared to that of raw milk. Coagulation time was reduced by 53%; gel development kinetics (estimated by Krol and final firmness (estimated at 30 min) were increased by more than 50%. When a severe heat treatment (100 oC-5 min) was applied, milk lost its ability to coagulate alter rennet action; PPCN solution did not, but ifs coagulation time was increased and the curd was slightly weaker.

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(Reçu le 2 août 1990 ; accepté le 26 novembre 1990) Résumé -La rétention de 7 espèces bactériennes représentatives de la flore contaminante des laits crus par les membranes de microfiltration mises en oeuvre dans le procédé Bactocatch a été ét-udiée. Ces espèces étaient ensemencées dans 4 fluides laitiers: perméat d'ultrafiltration; microfiltrat de lait obtenu sur membrane 0,2 urn: solution de phosphocaséinate; lait écrémé pasteurisé. Les résultats obtenus montrent que le nombre de réductions décimales observé est en moyenne de 2,6 et est indépendant du niveau initial de contamination, ce qui amène à la conclusion que les membranes utilisées dans le procédé fonctionnent comme des filtres en profondeur et non pas comme des filtres écrans. Le taux de rétention des bactéries par la membrane 1,4 um varie avec le volume cellulaire moyen entre 99,93% et 99,99%. La comparaison des rétentions observées en fonction de la composition des fluides mis en oeuvre laisse supposer un effet positif non négligeable des composants solubles du lait, qui interréagissent avec les grains d'alumine composant la membrane avant de favoriser l'adsorption interne des microorganismes.

Summary -Proteins from skim milk, paracaseinate and whey have been successfully analyzed by reverse-phase high-pertormance liquid chromatography (RP-HPLC) cou pied with electrospray ionization mass spectrometry (ESI-MS). The major milk proteins were identified by comparison of molecular masses determined by ESI-MS to molecular masses calculated from amino acid composition deduced from primary structures and cDNA sequences for some proteins. This method has permilted the simultaneous identification of caseins and whey protein variants. Observed molecular masses of major milk proteins were found to be 19038.0 ± 2.2 Da forK-CN A-l P (number of experiments n = 6); 19007.0 ± 1.1 Da for K-CN B-1P (n = 4); 25230.0 ± 2.1 Da for as2-CN A (n = 9); 23617.2 ± 1.3 Da for as1-CN B-8P (n = 14); 24 092.0 ± 1.7 Da for~-CN B-5P (n = 6); 24 025.3 ± 1.0 Da for~-CN A 1-5P (n = 14); 23 984.8 ± 0.7 Da for~-CN A2_5P (n = 14); 18278.3 ± 2.2 Da for the monomeric form of~-LG B (n = 5); 18 364.8 ± 1.6 Da for the monomeric form of~-LG A (n = 5); 14 179.21 ± 3.14 Da for a-LA (n = 5). Hence an accuracy of 0.01 % was obtained by ESI-MS analysis. It was also shown that the on-line coupling of HPLC with ESI-MS offers a very promising alternative for studying the proteolysis and determining the specificity of used enzymes in some technological treatments as shown for milk-clotting enzymes.

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