Listeria monocytogenes inhibition by Carnobacterium strains and crude bacteriocins on sterile and commercial vacuum-packed cold-smoked salmon stored at 4 degrees C and 8 degrees C was investigated. Carnobacterium piscicola V1 was bactericidal against L. monocytogenes at the two temperatures, whereas Carnobacterium divergens V41 presented a bacteriostatic effect. C. piscicola SF668 delayed L. monocytogenes growth at 8 degrees C and had a bacteriostatic effect at 4 degrees C. Listeria growth was not affected by a non-bacteriocin-producing C. piscicola. Crude extracts of piscicocins were bactericidal at 4 degrees C and 8 degrees C. Listeria growth was delayed by divercin V41 at 8 degrees C and was inhibited at 4 degrees C. Nisin delayed Listeria growth at 8 degrees C and was bacteriostatic at 4 degrees C. The present study demonstrates that L. monocytogenes growth could be prevented on vacuum-packed cold-smoked salmon by Carnobacterium and associated bacteriocins at chilled temperatures. Moreover, no product spoilage could be observed with the use of such bacteriocin-producing strains as demonstrated by good sensorial analyses and low biogenic amine production.

Lactobacillus helveticus can possess one or two cell envelope proteinases (CEPs), called PrtH2 and PrtH. The aim of this work was to explore the diversity of 15 strains of L. helveticus, isolated from various origins, in terms of their proteolytic activities and specificities on pure caseins or on milk casein micelles. CEP activity differed 14-fold when the strains were assayed on a synthetic substrate, but no significant differences were detected between strains possessing one or two CEPs. No correlation was observed between the proteolytic activities of the strains and their rates of acidification in milk. The kinetics of hydrolysis of purified ␣ s1 -and ␤-casein by L. helveticus whole cells was monitored using Tris-Tricine sodium dodecyl sulfate (SDS) electrophoresis, and for four strains, the peptides released were identified using mass spectrometry. While rapid hydrolysis of pure ␤-casein was observed for all strains, the hydrolysis kinetics of ␣ s1 -casein was the only criterion capable of distinguishing between the strains based on the number of CEPs. Fifty-four to 74 peptides were identified for each strain. When only PrtH2 was present, 22 to 30% of the peptides originated from ␣ s1 -casein. The percentage increased to 41 to 49% for strains in which both CEPs were expressed. The peptide size ranged from 6 to 33 amino acids, revealing a broad range of cleavage specificities, involving all classes of amino acids (Leu, Val, Ala, Ile, Glu, Gln, Lys, Arg, Met, and Pro). Regions resistant to proteolysis were identified in both caseins. When strains were grown in milk, a drastic reduction in the number of peptides was observed, reflecting changes in accessibility and/or peptide assimilation during growth.

Summary -The seasonal variations in milk fat composition, especially du ring the grazing period, often lead to poor eye formation in Swiss-type cheese. The influence of free fatty acids on the grow1h and metabolism of the dairy propionibacteria has been studied in this work. Linoleic (C 1B : 2 ), laurie (C 12 : 0 ), myristic (C14:0) and oleic acids (C1B:1) inhibited the growth and acid production of P treudenreichii subsp shermanii in the reference medium. The antibacterial activity of Iinoleic acid can be overcome by additions of cholesterol and soya lecithin to the medium. The four species and !WO subspecies of dairy Propionibacterium could be divided into !WO groups according to their high susceptibility to unsaturated fatty acids (P freudenreichii subsp shermanii and subsp freudenreichit) or low susceptibility (P acidipropionici, P jensenii and P thoeniJ). Nevertheless, this inhibitory action of free fatty acids was not observed in milk, retentate media or lactic curd. A total extraction of the milk lipids led to the recovery of this inhibitory effect on Propionibacterium. The possible modes of action of these molecules are discussed on the basis of the observed potassium effluxes and disturbances in the cell membranes.

(Reçu le 17 janvier 1995 ; accepté le 22 février 1995)Résumé -Grâce à la mise au point d'un équipement prototype de pressage, une amélioration de la méthodologie d'extraction de la phase aqueuse des fromages proposée initialement par Barthel et al (1928a) a été réalisée. Les phases aqueuses d'emmental à 4 stades d'affinage: sortie saumure, entrée et sortie cave chaude, commercialisation, ont été extraites et caractérisées sur les plans bactériologi-que, physico-chimique et biochimique. La teneur en substance sèche initialement de 90 g/kg de phase aqueuse atteint 170 g/kg dans le fromage affiné. Cet accroissement découle de l'élévation (multiplication par 5) de la teneur en azote solubilisé du fait de la protéolyse, notamment lors de l'affinage en cave chaude; 56% de l'azote total déterminé dans le fromage affiné sont sous la forme d'acides aminés libres. L'évolution des teneurs en sucre et en acides organiques est conforme à leur métabolisation par les bactéries lactiques et propioniques (consommation de 25% du lactate majoritairement de la forme L). L'analyse des formes minérales montre une évolution considérable des sels phosphocalciques par rapport au lait: rapport Ca/P variant entre 8,2 et 5,4 au lieu de 1,3. Les teneurs en Na et CI ne sont pas en accord avec leurs proportions dans la molécule de NaCI, ce qui conduit à penser qu'une partie de l'ion CI est mobilisé dans d'autres formes salines du type chlorhydrate. La caractérisation de la phase aqueuse des fromages apparaît au travers de cette première étude comme une méthodologie privilégiée de suivi de la fabrication et de l'affinage des fromages. emmental 1 phase aqueuse / affinage / composition / presse hydraulique Summary -Extraction and composition of the aqueous phase of Emmental during ripening. A new pressing unit was designed to improve the extraction methodology of the cheese aqueous phase, originally proposed by Barthel et al (1928a). Emmental aqueous phases extracted from cheeses at

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Lactobacillus helveticus exhibits a great biodiversity in terms of protease gene content, with 1 to 4 cell envelope proteinases. Among them, proteinases PrtH and PrtH2 were shown to have different cleavage specificity on pure α(s1)-casein. The aim of this work was to investigate the proteolytic activity of 2L. helveticus strains in cheese matrix: ITGLH77 (PrtH2 only) and ITGLH1 (at least 2 proteinases, PrtH and PrtH2). Cell viability, proteolysis, autolysis, and stretchability of experimental Emmental cheeses were measured during ripening. The peptides identified by mass spectrometry showed very different profiles in the 2 cheeses. Regardless of the casein origin, the number of different peptides containing more than 20 amino acids was greater in cheeses manufactured with strain ITGLH77. This accumulation of large peptides, including those from α(s1)- and α(s2)-caseins, was in agreement with the lower overall extent of proteolysis obtained in ITGLH77 cheeses, which can be attributed to the presence of one cell envelope proteinase of the lactobacilli strains or lesser release of intracellular peptidases into the cheese aqueous phase. In parallel, stretchability was measured throughout ripening time. Emmental strands observed by confocal laser scanning microscopy showed microstructure similar to that of mozzarella strands. Stretchability was correlated with a specific type of peptide (hydrophobic), as shown by principal component analysis, and with a lower degree of proteolysis.