The interactions of fatty acids with porcine and bovine beta-lactoglobulins were measured using tryptophan fluorescence enhancement. In the case of bovine beta-lactoglobulin, the apparent binding constants for most of the saturated and unsaturated fatty acids were in the range of 10(-7) M at neutral pH. Bovine beta-lactoglobulin displays only one high affinity binding site for palmitate with an apparent dissociation constant of 1 x 10(-7) M. The strength of the binding was decreasing in the following way: palmitate > stearate > myristate > arachidonate > laurate. Caprylic and capric acids are not bound at all. The affinity of beta-lactoglobulin for palmitate decreased as the pH of the incubation medium was lowered and BLG/palmitate complex was not observed at pH's lower than 4.5. Surprisingly, chemically modified bovine beta-lactoglobulin and porcine beta-lactoglobulin did not bind fatty acids in the applied conditions.

The cathepsin L propeptide (phcl-2) was expressed in Saccharomyces cerevisiae using a human procathepsin L/alpha-factor fusion construct containing a stop codon at position -1 (the C-terminal amino acid of the proregion). Since the yield after purification was very low, the cathepsin L propeptide was also obtained by an alternate procedure through controlled processing of an inactive mutant of procathepsin L (Cys25Ser/Thrl10Ala) expressed in Pichia pastoris, by small amounts of cathepsin L. The peptide resulting from the cleavage of the proenzyme (phcl-1) was then purified by HPLC. The purified propeptides were characterized by N-terminal sequencing and mass spectrometry and correspond to incomplete forms of the proregion (87 and 81 aa for phcl-1 and phcl-2 respectively, compared to 96 aa for the complete cathepsin L propeptide). The two peptides were found to be potent and selective inhibitors of cathepsin L at pH 5.5, with Ki values of 0.088 nM for phcl-1 and 0.66 nM for phcl-2. The Ki for inhibition of cathepsin S was much higher (44.6 nM with phcl-1), and no inhibition of cathepsin B or papain could be detected at up to 1 microM of the propeptide. The inhibitory activity was also found to be strongly pH-dependent. Two synthetic peptides of 75 and 44 aa corresponding to N-terminal truncated versions of the propeptide were also prepared by solid phase synthesis and displayed Ki values of 11 nM and 2900 nM, respectively, against cathepsin L. The data obtained for the 4 propeptide derivatives of various lengths indicate that the first 20 residues in the N-terminal region of the propeptide are more important for inhibition than the C-terminal region which contributes little to the overall inhibitory activity.

Summary -The fluorescence spectra of raw (NHa), heated (NHP), homogenised (HOM) and homogenised + heated (HOP) milks were recorded using a variable angle front-surface accessory. The emission fluorescence spectra of tryptophans in proteins, vitamin A and anilino-naphthalene sulfonie acid and the excitation fluorescence spectra of vitamin A and anilino-naphtha1ene sulfonic acid were collected. The spectra showed that the treatments applied to milk induced changes in the fluorescence characteristics of the probes. Principal component analysis was applied to the normalised fluorescence spectral data in order to distinguish between milk samples. It was shown that the map defined by principal components 1 and 2 discriminated NHa, NHP, HOM and HOP samples as a function of homogenisation and heating, respectively. The potential of fluorescence spectroscopy in combination with a chemometric method to discriminate between heated and homogenised milk sampi es was demonstrated. milk / protein / vitamin A / front face fluorescence / multivariate analysis Résumé -Intérêts des méthodes spectroscopiques pour la caractérisation des produits laitiers. I.Utilisation de la fluorescence frontale pour la caractérisation de laits natif, chauffé et homogénéisé. Les spectres de fluorescence de lait natif (NHa), chauffé (NHP), homogénéisé (HOM) et homogénéisé + chauffé (HOP) ont été enregistrés au moyen d'un accessoire de fluorescence frontale. Les spectres d'émission de fluorescence des tryptophanes, de la vitamine A et de l'acide anilinonaphtalène sulfonique, ainsi que les spectres d'excitation de la vitamine A et de l'acide anilinonaphtalène sulfonique, ont été enregistrés. Les traitements appliqués au lait induisent des modifications dans les spectres de fluorescence. L'analyse en composante principale a été appliquée sur les spectres de fluorescence normés. La carte factorielle 1-2 permet de séparer les échantillons en fonction du traitement appliqué au lait. lait / protéine / vitamine A / fluorescence frontale / analyse multivariée 658 E Dufour, A Riaublanc

Soft cheeses exhibit a wide range of textures and, as a consequence, of structures. The objective of the present study was to investigate intrinsic fluorophores of cheese in order to discriminate between eight different soft cheeses. Protein tryptophan emission spectra and vitamin A excitation spectra were recorded directly on cheese samples using front face fluorescence spectroscopy. The eight soft cheeses were discriminated using their spectra by applying multivariate statistical methods such as principal component analysis and factorial discriminant analysis. From the tryptophan fluorescence data set, a good classification was observed for 95% and 92% of the principal and test samples, respectively. A better classification (96% and 93% for principal and test samples) was obtained from the vitamin A spectra. The spectral pattern associated with the principal components provides characteristic wavelengths which are the most suitable for separating the spectra. They allow information on the protein structure at the molecular level to be derived, in relation with cheese texture. cheese / identification / structure / fluorescence / protein Résumé-La spectroscopie de fluorescence frontale pour identifier et caractériser la structure de fromages à pâte molle. Les fromages à pâte molle présentent une large gamme de texture et, en conséquence, de structure. L'objectif de cette étude était d'évaluer le potentiel des fluorophores intrinsèques du fromage dans la discrimination de 8 types de fromages à pâte molle. Les spectres d'émission des tryptophanes de protéines et les spectres d'excitation de la vitamine A ont été enregistrés directement sur des fromages au moyen de la fluorescence frontale. L'analyse des collections de spectres par des méthodes statistiques multidimensionnelles telles l'analyse en composantes

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