Behavior of Brevibacterium linens and Debaryomyces hansenii as ripening flora in controlled production of smear soft cheese from reconstituted milk: growth and substrate consumption dairy foods.

Experimental cheeses inoculated with Debaryomyces hansenii and Brevibacterium linens were ripened for 76 d under aseptic conditions. Triplicate cheese-making trials were similar as a result of efficient control of the atmosphere. In all trials, D. hansenii grew rapidly during the first 2 d and then slowed, but growth remained exponential until d 10 (generation time around 70 h). Total cell counts were higher than the number of viable cells, and after 10 d they remained around 3 x 10(9) yeast/g of DM. This difference resulted from the nonviability of a fraction of D. hansenii. After d 15, the pH of the rind was close to 7, and B. linens grew exponentially until d 25 (generation time around 70 h). The growth rate subsequently decreased but remained exponential (generation time around 21 d). Cell counts of D. hansenii and B. linens were correlated with the environmental technical conditions. Total D. hansenii counts were also correlated with total B. linens counts. Viable B. linens counts were related to rind lactate, and total counts depended on rind pH, internal lactate, and D. hansenii viable counts. The internal pH of the cheese depended on lactate concentrations, whereas surface pH was related to internal lactose, temperature, and relative humidity. These results suggest a determining role of the diffusion of the carbon sources in the ripening of smear soft cheese.

Behavior of Brevibacterium linens and Debaryomyces hansenii as ripening flora in controlled production of soft smear cheese from reconstituted milk: protein degradation.

Model smear soft cheeses, prepared with Debaryomyces hansenii and Brevibacterium linens as ripening starters, were ripened under aseptic conditions. Results of the cheese-making trials, in triplicate, were similar and showed similar patterns of protein degradation. In all of the trials, the acid-soluble nitrogen and nonprotein nitrogen (NPN) indexes and NH3 concentrations of the rind were low until d 10. The acid-soluble nitrogen and NPN of the rind then increased to 100 and 18% of total nitrogen, respectively, at d 76. The NH3 concentrations remained low until d 24 and increased until d 70, reaching about 1.8 g of NH3/kg of DM, and then remained constant. The acid-soluble nitrogen and NPN indexes and NH3 concentrations in the inner cheese mass were lower than in the rind. They showed the same evolution, reaching about 18% for acid-soluble nitrogen, 10% for NPN, and 1.5 g of NH3/kg of DM. It was shown that the inner cheese pH and populations of D. hansenii and B. linens have an effect on proteolysis. Viable cell counts of D. hansenii and B. linens were correlated with the environmental conditions and with proteolytic products. The determining role of carbon source and NH3 diffusions on the cheese ripening process were confirmed.

Major protein components in the cell envelope of Clostridium tyrobutyricum.

The overall composition of the Clostridium tyrobutyricum cell envelope did not vary significantly during cell growth and was characterized by a high protein content (about 40% dry weight). Teichoic and teichuronic acids were absent and the neutral sugar content low. Insoluble peptidoglycan represented only 10-12% of the cell envelope (dry weight basis); it contained glucosamine, muramic acid, alanine, diaminopimelic acid and glutamic acid (molecular ratio 1/1/2/1/1). SDS-PAGE revealed the presence of about 50 proteins in this cell envelope; however, one high molecular weight protein was largely predominant. They were not covalently bound to the peptidoglycan and their relative amounts were practically constant through cell growth and with various extraction treatments. A brief heat treatment of whole cells in PBS caused selective release of the major cell envelope proteins together with flagellin; this method was used to characterize these proteins in 37 strains of C. tyrobutyricum and some other clostridia. The major envelope proteins had molecular weights ranging from 96 to 145 Kd and the flagellins from 32 to 72 Kd.

[Demonstration of 2 main serological groups in Clostridium tyrobutyricum]. / Mise en évidence de deux groupes sérologiques principaux chez Clostridium tyrobutyricum.

Antisera prepared against heated cells of 10 strains of Clostridium tyrobutyricum gave cross-reactions with several other clostridial species. Such reactions could be completely eliminated by absorption with C. beijerinckii, C. fallax and C. tetanomorphum. Both agglutination and immunofluorescence tests with these antisera showed that 85 strains of C. tyrobutyricum were divided into two main serological groups, A and B. The 56 strains of group A possessed the same thermostable species-specific antigen, whereas those of group B were lacking in it. Antisera prepared against either formol- or ethanol-treated cells or low-heated cells of C. tyrobutyricum showed only low titre cross-reactions with other clostridial species. When these were removed by absorption, only the homologous strains and a few other strains of C. tyrobutyricum group B were still agglutinated at a full titre by these antisera. Therefore, only strains of group A of C. tyrobutyricum can be easily identified by a single type-specific antiserum.

A high-molecular-mass cell wall protein released from Clostridium tyrobutyricum by heat treatment.

Analysis of the cell wall of 4 strains of Clostridium tyrobutyricum reveals an unusually high protein content (35-40% dry weight). Brief heat treatment of whole cells of these stains causes release of two proteins, flagellin and a cell wall component of high molecular mass (110-125 kDa in the different strains). This component represents approx. 5% of the dry cell weight.

Antagonistic activity of Debaryomyces hansenii towards Clostridium tyrobutyricum and Cl. butyricum.

A study of the inhibitory action of Debaryomyces hansenii (31 strains) on Clostridium tyrobutyricum (5 strains) and Cl. butyricum (2 strains) on laboratory media showed that Deb. hansenii inhibited the growth of these organisms, and that this effect was due not only to competition for nutrients but also to the production of both extra- and intracellular antimicrobial metabolites. The inhibitory effect varied with strain and occurred whether the yeasts were grown aerobically or under reduced O2 tension.

Peptide hydrolases of Lactobacillus casei: isolation and general properties of various peptidase activities.

Discovery of an endopeptidase by gel chromatography and separation of 3 exopeptidases (a dipeptidase, an aminopeptidase and a specific carboxypeptidase) from Lactobacillus casei NCDO 151 by affinity chromatography is described. The 3 exopeptidases were strongly inhibited by the metal chelators EDTA and 1,10-phenanthroline but were reactivated with Co2+ and Mn2+. The pH optima for aminopeptidase, dipeptidase and carboxypeptidase activities were 6.5, 7.6 and 7.2, respectively. Maximum activity was obtained at 45 degrees C for the aminopeptidase, at 30 degrees C for the dipeptidase and at 40 degrees C for the carboxypeptidase. The substrate specificities of the 3 enzymes were also studied. The properties of these 3 enzymes are compared with those of other bacteria.

[Taxonomic characters of "Clostridium tyrobutyricum" (author's transl)]. / Caractères taxonomiques de Clostridium tyrobutyricum.

The taxonomic characters of 77 strains of Clostridium tyrobutyricum have been studied and compared to those of known strains. This work shows the insufficiency of the Bergey's Manual (8th edition) nine characters distinguishing Clostridium groupe I to identify C. tyrobutyricum. To be more representative of the species, addition of one or two characters to this key of determination and some modifications to the complementary description of C. tyrobutyricum in this manual are proposed.

[The spore germination of "Clostridium tyrobutyricum" III.--An hypothesis on the mechanism of initiation (author's transl)]. / La germination de la spore de Clostridium tyrobutyricum III.--Hypothèse sur le mécanisme de la phase initiale

Spores of C. tyrobutyricum do not contain 3-phosphoglyceric acid (PGA) but a polysaccharide which could replace PGA as an energy source during germination. The absence of PGA, which is an inhibitor of phosphotransacetylase, confirms the role of the acetyl-CoA synthesizing system in the germination initiated by acetate. Spore extracts of C. tyrobutyricum, as extracts of vegetative cells, were found to contain a ferredoxin and exhibited a NADH-ferredoxin oxydase activity which required the presence of an acetyl-CoA regenerating system, suggesting that this enzyme is also involved in germination. From this results, an hypothesis on the role of initiators (acetate and NH4+) in the mechanism of initiation of spore germination in C. tyrobutyricum is proposed. Acetate would have an effect on the utilisation of the endogenous polysaccharide and on glucose catabolism, and therefore, would be an effector for the production of the energy required particularly to transport cations into the spore.

[An intracellular polysaccharide involved in sporulation of "Clostridium butyricum" I. Cytology, production and preliminary enzymic analysis (author's transl)]. / Polyoside intracellulaire impliqué dans la sporulation de Clostridium Buryricum. I. Cytologie, production et analyse enzymatique préliminaire

Free glucose concentration and polysaccharide production in Clostridium butyricum cells have been studied with an enzymatic method. Results indicated a substantial decrease in intracellular glucose content simultaneously with a production of polysaccharide prior to the end of exponential growth. Then the polysaccharide accumulated rapidly to reach a maximum just before the first refractile spores appeared, and it decreased by 50% during the last stages (V and VI) of sproulation. Electron micrographs of ultrathin sections have demonstrated that most of the polysaccharide is located inside the mother cell cytoplasm as large granules when the remaining is dispersed within the spore cytoplasm beginning during stage III of the sporulation. Overall results showed that production and use of C. butyricum polysaccharide were closely related to sporulation. The isolated polysaccharide exhibited poor water solubility, iodine spectrum with a lambda max at 545 nm and 72% beta-amylolysis. Total hydrolysis occurred with amyloglucosidase indicating an alpha-glucan containing alpha(1 leads to 4) and alpha(1 leads to 6) glucose linkages. The debranching from its beta-dextrin limit by pullulanase revealed the presence of a glycogen like-type structure with some external chains which are longer than those of a normal glycogen. This glycogen arrangement appeared to be of clusters linked by linear chains at least as long as the longest external chains.