New signaling molecules in some gram-positive and gram-negative bacteria.

A new family of putative signaling molecules having a 2(5H)-furanone configuration has been described in this work. They were released during late exponential or stationary phase in different growth media by some gram-positive bacteria, such as Lactobacillus helveticus, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus sanfranciscensis, Enterococcus faecalis, and a gram-negative species, i.e. Salmonella enterica. A pair of 2(5H)-furanones called furanones A and B occurred in all the conditioned media (CMs) of the species considered. These two molecules showed similar retention times and their spectral data shared the key fragments to include them in the 2(5H)-furanones family. However, some differences were observed in the mass fragmentation profiles. In particular the use of PCA analysis of all the mass fragments enabled the grouping of furanone A profiles of S. enterica, L. helveticus, L. plantarum, L. paraplantarum, L. sanfranciscensis and E. faecalis in one unique cluster with only few exceptions. On the other hand, the mass fragmentation profiles of furanone B of the major part of the species and strains could be grouped together and were differentiated from those of L. helveticus. The specific activity of cell-free supernatants of high density cultures of S. enterica confirmed that the release of active molecules, and specifically of furanones A and B, was cell density dependent. Moreover, a preliminary experiment suspending S. enterica cells into cell-free supernatants of L. helveticus previously exposed to an oxidative stress demonstrated that furanones A and B have a strong interspecific activity. In fact cell autolysis and cell envelope damages were observed with Scanning Electron Microscopy (SEM) in S. enterica.

Influence of ethanol on the rennet-induced coagulation of milk.

The influence of ethanol on the rennet-induced coagulation of milk was studied to investigate potential synergistic effects of these two mechanisms of destabilisation on the casein micelles. Addition of 5% (v/v) ethanol reduced the rennet coagulation time (RCT) of milk, whereas higher levels of ethanol (10-20%, v/v) progressively increased RCT. The temperature at which milk was coagulable by rennet decreased with increasing ethanol content of the milk. The primary stage of rennet coagulation, i.e., the enzymatic hydrolysis of kappa-casein, was progressively slowed with increasing ethanol content (5-20%, v/v), possibly due to ethanol-induced conformational changes in the enzyme molecule. The secondary stage of rennet coagulation, i.e., the aggregation of kappa-casein-depleted micelles, was enhanced in the presence of 5-15% ethanol, the effect being largest at 5% ethanol. Enhanced aggregation of micelles is probably due to an ethanol-induced decrease in inter-micellar steric repulsion. These results indicate an interrelationship between the effects of ethanol and chymosin on the casein micelles in milk, which may have interesting implications for properties of dairy products.