The results reported in the present paper and N-terminal sequence homologies established by other authors strongly support the assumption that the gastric protease previously called bovine pepsin I or bovine pepsin B belongs to the aspartate protease group and corresponds to gastricsin (or pepsin C) (EC 3.4.23.3) from other species.Bovine gastricsin was prepared from commercial extracts of adult bovine vells by a procedure involving DEAE-cellulose chromatography, gel filtration on Sephacryl S-200 and a further DEAE-cellulose chromatography. The preparation thus obtained was shown to be free of chymosin and bovine pepsin A by immunodiffusion, selective inactivation in urea and isoelectric focusing. Its molecular weight was estimated by gel filtration to be 32 800.Bovine gastricsin displayed microheterogeneity on isoelectric focusing with pl values of the components ranging from 3.5 to 4.0. Chromatography of bovine gastricsin on hydroxyapatite separated three fractions, none of them being homogeneous by isoelectric focusing. Concanavalin-A-Sepharose 4B bound bovine gastricsin to some extent, but without any significant fractionation. Proteolytic activity could be detected directly on the isoelectric focusing gel for all the components of gastricsin and its fractions from hydroxyapatite and concanavalin-A -Sepharose 4B.Bovine gastricsin and its fractions from hydroxyapatite have similar amino acid compositions, different from those of bovine chymosin and pepsin A but obviously related to those of human, simian and porcine gastricsins.Bovine gastricsin which is inactivated by reaction with diazoacetyl-~~-nor~eucine methyl ester and with 1,2-epoxy-(p-nitrophenoxy)propane in a 1 : I and 1 : 2 stoichiometry, respectively, is able to hydrolyse a synthetic hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, used as reference substrate for aspartate proteases, and exhibits a low activity towards N-acetyl-L-phenylalanyl-L-diiodotyrosine. Its specific clotting activity with x-casein as substrate is only half of that of chymosin and pepsin A.

An inhibition ELISA (enzyme-linked immunosorbent assay) was developed for the determination of caseinomacropeptide (CMP) in order to estimate the proteolysis of/c-casein due to the enzymes of psychrotrophic bacteria in bulk raw milk. The CMP present in milk was quantified specifically by an antibody. The limit of detection was ~ 01 /ig/ml and the CV was < 10%. This method was used to study the proteolytic activity of three strains of psychrotrophic Pseudomonas jiuorescens in raw milk and to analyse different raw milk samples supplied by four dairy plants. The proteolytic activity for different strains of psychrotrophs and for different milk samples varied considerably, but no correlation was established between the level of microbial flora and /c-casein proteolysis. It is thus not possible to determine the extent of proteolysis from the bacterial count alone. However, by CMP determination in bulk raw milk samples after 6 d storage at 4 °C, the mean /e-casein proteolj'sis was ~ 4%. Among the milk samples analysed that contained < 10 7 cfu psychrotrophs/ml, 30% exhibited a proteolysis of /c-casein < 0-5%, i.e. < 5 /tg CMP/ml.

RésuméCette' étude sur l'affinage du Gruyère de Comté, portant sur quatre fromageries, quatre caves d'affinage et six fabrications réparties sur deux saisons, permet de mesurer l'influence de ces facteurs sur la qualité du produit fini. Au total 96 fromages sont analysés à quatre stades successifs de leur affinage.La répétabilité du dosage quantitatif des caséines par électrophorèse est mesurée à partir de 700 résultats. Elle varie linéairement en fonction de la valeur des résultats, de 2,13 % à 5,64 %. Summary Ripening of Gruyère de Comté cheese. IV. Study of proteolysisThe ripening of Gruyère de Comté cheese was studied using four dairies capable of making four identical cheeses from the same vat of milk. Each of these 4 cheeses was ripened in one of four different ripening rooms. Six trials were conducted during six different months on two seasons. As a whole 96 cheeses were analysed at four successive stages of their maturation. Determinations of plasmine contents of milks were performed as weil as quantitative evolutions of cheese protein fractions by polyacrylamide-agarose gel electrophoresis.The repeatability of the electrophoretic determinations was tested from 700 measurements. It va.ied linearely according to the casein levels between 2.13 and 5.64. Seasons significantly affected the products of degradation of J3-casein. Different mechanisms of hydrolysis seemed to exist depending on the season and the tempe rature of the ripening room. The evolution of plasmin levels in milk were compared with the proportions of "1-and deg. J3-fractions at two stages of the ripening of cheeses.

A method for determining chymosin and bovine pepsin A in commercial extracts of bovine veils, based on the use of the synthetic hexapeptide (LeuSer-Phe(NO 2 )-Nle-Ala-Leu-OMe) as reference substrate, is reported. Chymosin and bovine pepsin A were separated chromatographically from extracts and assayed for clotting activity on a reconstituted skim-milk standardized with reference chymosin and bovine pepsin A, themselves standardized in relation to the hexapeptide. The effect of pH on the absorbance difference between the hexapeptide and the LeuSer-Phe(NO 2 ) tripeptide resulting from its hydrolysis was studied. It was found that the ' optimal' pH for determining the activities of the reference enzyme solutions was 4-7.Six chymosin and 3 bovine pepsin A preparations were assayed on the hexapeptide to define the relation between the proteolytic activity and the amount of active enzyme. At pH 4-7 and 30 °C 1 mg chymosin and 1 mg bovine pepsin A hydrolysed 100 and 2700 /ai-peptide/s respectively. The clotting activity of these preparations was assayed on a reconstituted skim-milk to standardize it and thus define the relation between the clotting time and the amount of active enzyme. Chymosin had a specific clotting activity twice that of bovine pepsin A. At equal clotting activities, bovine pepsin A was 55 times more active than chymosin on the hexapeptide at pH 4-7.

Résumé -L'analyse quantitative des diverses caséines du lait de vache a été réalisée par électro-phorèse en gel de polyacrylamide-agarose pH 8,6 et par chromatographie anionique à pH 8,0.L'analyse de 118 échantillons individuels de lait prélevés dans un troupeau de 13 vaches durant une lactation a permis d'estimer les proportions de chaque caséine et de comparer les résultats des 2 méthodes. Une étude sur la capacité de fixation spécifique du colorant bleu de Coomassie R 250 sur chaque caséine a donné des coefficients moyens d'absorption par rapport à la caséine aS1 prise comme référence (1,00) de 0,98 pour la caséine aS2' de 0,95 pour la caséine~et de 0,94 pour la caséine K.. La comparaison des résultats entre les 2 méthodes d'analyse donne des coefficients de corrélation de 0,80 pour la fraction 11' de 0,77 pour la caséine~' de 0,74 pour la fraction 12 et de 0,65 pour les caséines as. Les corrélations sont très faibles pour la caséine K et la fraction 13' L'étude des relations pouvant exister entre les diverses caséines du lait montre que les taux des 3 fractions 1 sont bien corrélés contrairement à d'autres résultats trouvés dans les fromages (Collin et al, 1987