Effect of the addition of phytosterols and tocopherols on Streptococcus thermophilus robustness during industrial manufacture and ripening of a functional cheese as evaluated by qPCR and RT-qPCR.

The quality of functional food products designed for the prevention of degenerative diseases can be affected by the incorporation of bioactive compounds. In many types of cheese, the performance of starter microorganisms is critical for optimal elaboration and for providing potential probiotic benefits. Phytosterols are plant lipophilic triterpenes that have been used for the design of functional dairy products because of their ability to lower serum cholesterol levels in humans. However, their effect on the starter culture behavior during cheesemaking has not yet been studied. Here, we followed DNA and RNA kinetics of the bacterium Streptococcus thermophilus, an extensively used dairy starter with probiotic potential, during industrial production of a functional, semi-soft, reduced-fat cheese containing phytosterol esters and alpha-tocopherol as bioactive compounds. For this purpose, real-time quantitative PCR (qPCR) and reverse transcription-qPCR (RT-qPCR) assays were optimized and applied to samples obtained during the manufacture and ripening of functional and control cheeses. An experimental set-up was used to evaluate the detection threshold of free nucleic acids for extraction protocols based on pelleted microorganisms. To our knowledge, this straight-forward approach provides the first experimental evidence indicating that DNA is not a reliable marker of cell integrity, whereas RNA may constitute a more accurate molecular signature to estimate both bacterial viability and metabolic activity. Compositional analysis revealed that the bioactive molecules were effectively incorporated into the cheese matrix, at levels considered optimal to exert their biological action. The starter S. thermophilus was detected by qPCR and RT-qPCR during cheese production at the industrial level, from at least 30min after its inoculation until 81days of ripening, supporting the possible role of this species in shaping organoleptic profiles. We also showed for the first time that the addition of phytosterols at functional concentrations, not only did not affect starter performance but also correlated with a significant increase in target DNA and cDNA levels in most of the time points evaluated throughout cheesemaking. Therefore, these findings suggest that the growth and metabolism of S. thermophilus may be enhanced by the incorporation of these biologically active molecules during cheese production, providing important information for the industrial design of novel fermented foods.

Cell wall modifications during osmotic stress in Lactobacillus casei.

AIMS: To study the modification of the cell wall of Lactobacillus casei ATCC 393 grown in high salt conditions. METHODS AND RESULTS: Differences in the overall structure of cell wall between growth in high salt (MRS + 1 mol l(-1) NaCl; N condition) and control (MRS; C condition) conditions were determined by transmission electronic microscopy and analytical procedures. Lactobacillus casei cells grown in N condition were significantly larger than cells grown under unstressed C condition. Increased sensitivity to mutanolysin and antibiotics with target in the cell wall was observed in N condition. Purified cell wall also showed the increased sensitivity to lysis by mutanolysin. Analysis of peptidoglycan (PG) from stressed cells showed that modification was at the structural level in accordance with a decreased PG cross-link involving penicillin-binding proteins (PBP). Nine PBP were first described in this species and these proteins were expressed in low percentages or presented a modified pattern of saturation with penicillin G (Pen G) during growth in high salt. Three of the essential PBP were fully saturated in N condition at lower Pen G concentrations than in C condition, suggesting differences in functionality in vivo. CONCLUSIONS: The results show that growth in high salt modified the structural properties of the cell wall. SIGNIFICANCE AND IMPACT OF STUDY: Advances in understanding the adaptation to high osmolarity, in particular those involving sensitivity to lysis of lactic acid bacteria.

Adaptation to high salt in Lactobacillus: role of peptides and proteolytic enzymes.

AIMS: To study the influence of peptides and proteolytic enzymes in the osmotic adaptation of Lactobacillus casei. METHODS AND RESULTS: Di- and tri-peptides added individually increased the osmotolerance of Lact. casei when grown in a chemically defined medium (CDM) containing NaCl. Growth stimulation and the re-establishment in their presence of plasmid DNA supercoiling (recovery of the linking number) in hyperosmotic medium indicated that they are used as osmocompatible solutes as carnithine a known osmoprotector does. The investigation of the proteolytic system showed that in high osmolarity medium, the cell envelope-associated proteinase (PrtP), and PepX (X-prolyl-dipeptidyl aminopeptidase) increased activity and lost repression by peptides. PepI, an iminopeptidase was also derepressed. PepQ, a prolidase that specifically liberated proline from dipeptides, was almost unaffected. Derepression in the presence of peptides took place at the transcriptional level. However, the twofold activation of PrtP in CDM hyperosmotic medium was essentially through an increase of the apparent Vmax of the enzyme. CONCLUSIONS: These results strongly suggest a contribution of the proteolytic system peptide supply in the osmotic adaptation. SIGNIFICANCE AND IMPACT OF THE STUDY: Advances in understanding the role of peptides in the adaptation to high osmolarity particularly involved in dairy processes.

Characterization of Lactobacillus carbohydrate fermentation activity using immobilized cell technique.

A microbial bioreactor based on calcium alginate immobilized Lactobacillus cells coupled to a pH electrode was developed for quantitative determination of carbohydrate fermentation activity. A high biomass (10(10) cfu mL(-)(1)) and particular pregrowth conditions were needed. Reduction of catabolite repression by monosaccharides was achieved by pregrowth in lactose. The evolution of acid production in a continuous flow-stopped flow bioreactor was monitored for different sugar solutions in contact with the immobilized bacteria. The resulting slopes (DeltamV/Deltat) were used to quantify the fermentation capability for a defined sugar related to that of glucose, which was taken as 100%. The procedure is simple, being based on pH variation that can give quantitative results compared to other reported techniques for carbohydrate fermentation pattern from which only qualitative results are obtained. In addition, it offers reduction in time and costs and is a suitable tool for the rapid analysis of isolated strains and in studies of modifications of sugar metabolism in mutants.

In Bacillus subtilis DegU-P is a positive regulator of the osmotic response.

The osmosensitivity presented by spo0A and degU null mutant strains of Bacillus subtilis pointed to their protein products as essential regulators for the osmotic response. This was further investigated by analyzing their transcription activity. The results showed that both spo0A-lacZ and degSU-lacZ were induced by the hypertonic medium. The actual phosphorylation state of these proteins was also analyzed by the bias of two reporter gene promoters activity (abrB-lacZ and degQ-lacZ). The absence of repression of abrB in hypertonic conditions suggested that Spo0A was not phosphorylated while the derepression of degQ promoter suggested that DegU-P was formed. These results were in accordance with the observed absence of sporulation in hyperosmotic media and semi-constitutive osmotolerance of degUh mutant strains known to retain phosphorylated DegU. The failure to secrete proteases and to sporulate in hypertonic media suggested that Spo0A acts through abrB regulation in the prevention of alternate responses. The role of DegU-P as positive regulator of the osmotic response seems to be settled and is discussed.

A novel antimicrobial activity of a Paenibacillus polymyxa strain isolated from regional fermented sausages.

A strain isolated from Argentinean regional fermented sausages was found to produce and secrete a compound that inhibited growth of Lactobacillus strains used as indicators. It was characterized as Paenibacillus polymyxa (P13). The antimicrobial activity, named polyxin, was obtained from culture supernatant fluid of late stationary phase and was inhibitory to actively growing cells. It was effective against a wide range of Gram-positive and Gram-negative bacterial species tested including food-borne pathogens. Bacteriocin-like properties such as proteinaceous nature (sensitive to proteases), insensitivity to organic solvents and chelators, stability to heat (up to 10 min at 90 degrees C), and acidic pH but instability in alkaline conditions, were determined. A molecular mass of 10 kDa was estimated by molecular gel filtration.

Osmotic strength blocks sporulation at stage II by impeding activation of early sigma factors in Bacillus subtilis.

In Bacillus subtilis, osmotolerance is a stationary phase-dependent, adaptive response inhibiting sporulation and sharing common regulators with this process. The extent of this inhibition was determined by measuring transcription activity of promoter lacZ fusions to early sigma genes (spoIIG and spoIIA coding for precursors of sigmaE and sigmaF) and to reporters of them (spoIID, spoIIQ and spoIIIG), in the absence and presence of 0.6 M or 1 M NaCl. The transcription activity of these sigma precursors, normally occurring at the onset of the stationary phase, was reduced to 30-50% of their maximal expression in hyperosmotic conditions; expression of genes under their control was, however, more inhibited (<10%). Therefore, sporulation was blocked at the sigma sigmaE and sigmaF activation steps. This assumption was confirmed by electron microscopic examinations of hyperosmotic cultures, which presented asymmetric septa characteristic of stage II mutants. Discussion was focused on the particular composition and/or structure of membranes during hyperosmotic growth and their involvement in the arrest of sporulation.

Variations of the envelope composition of Bacillus subtilis during growth in hyperosmotic medium.

The envelope properties of B. subtilis cultures grown in LB and LBN hyperosmotic media (LB + 1.5 M NaCl) were compared. Since hypertonic cultures showed a Spo-phenotype, a Spo-mutant grown in LB was also analyzed. LBN cultures showed extensive filamentation and presented different sensitivities toward phage infection (phi29 and phi105), or antibiotics whose targets are at wall (lysozyme, penicillin G) or membrane level (polymyxin B, phosphonomycin). Results of the biochemical composition revealed that during hyperosmotic growth, the cell wall increased in thickness, and among the membrane lipids, glycolipid and cardiolipin increased in parallel with a decrease in phosphatidylglycerol. The fatty acid composition was also modified, and an increase in saturated straight chain with a decrease of saturated iso-branched fatty acids was observed. The increase of monounsaturated 18-1 (omega-9) fatty acid was probably related to the absence of sporulation observed in hypertonic media, since its increase has been shown to inhibit the KinA sensor of sporulation. The significance of the other wall and membrane composition variations (and hydrophobic surface properties) in relation to the osmotic adaptation are discussed.

Osmoresistance of spores from Bacillus subtilis and the effect of ssp mutations.

Spores of Bacillus subtilis show similar plating efficiency on media with or without 1.5 M NaCl. In contrast, vegetative cells are osmosensitive unless the stationary phase has been reached. In the present work, loss of heat and osmotic resistance during germination was studied. Their kinetics and sensitivity to protein synthesis inhibition were different: heat resistance was lost first and even in the presence of chloramphenicol, whereas loss of osmotolerance occurred later and was inhibited in the presence of this antibiotic. The influence of spore-associated small acid-soluble proteins (SASPs) on spore osmotolerance was investigated using ssp mutants: all produced spores which germinated poorly and were sensitive to osmotic strength. SASP-E deficiency was particularly significant. Spore osmotolerance was largely restored in complementation assays performed with cloned ssp genes. It is possible that germination-associated degradation of SASP proteins provides osmotically significant levels of amino acids (especially glutamate).

Physiological and genetic characterization of the osmotic stress response in Bacillus subtilis.

Bacillus subtilis cultures submitted to an osmotic upshock (1.5 M NaCl) lysed unless stationary phase had been reached. Several physiological variations were observed, such as delayed growth (adaptation), a filamentous bacterial appearance, RecA-dependent osmoresistance (SOS), and cross-induction by a previous stress (heat shock). Osmoresistance and sporulation seem to share pathways of regulation such as inhibition in the presence of glucose and glutamine and derepression in a catabolite-resistant mutant such as degUh. However, spores were not obtained on hypertonic media. Mutants of later sporulation stages (spoII, spoIII) presented a response similar to that of the wild-type parent, indicating that both processes probably shared early controls. Null mutations in any of the known key modulators of sporulation (spoOA or degU) resulted in similar levels of osmosensitivity. Sensor mutations in kinA and degS also led to strains with altered responses, the kinA mutant being even more osmosensitive than the degS mutant. Several spoOA mutant phenotypes are due to this gene's control of abrB, a regulator of stationary-phase events, and an abrB mutation relieved the osmosensitivity of the spoOA-containing mutant but had no effect on a wild-type strain.