Impacto de la incorporación de lípidos vegetales y antioxidantes sobre el comportamiento del starter durante la producción industrial de un queso funcional evaluado por técnicas independientes de cultivo

Los fitoesteroles son un conjunto de lípidos vegetales que han sido utilizados para el diseño de productos lácteos funcionales debido a su capacidad para reducir el colesterol sérico. No obstante, su efecto sobre el comportamiento del cultivo starter en la elaboración de quesos no ha sido estudiado. En este trabajo se monitoreó la cinética del DNA y RNA de la bacteria Streptococcus thermophilus, un starter lácteo ampliamente utilizado y con potencial probiótico, durante la producción industrial de un queso funcional conteniendo esteres de ftoesteroles y alfa-tocoferol como compuestos bioactivos. Para este propósito, se optimizaron técnicas de PCR cuantitativa (qPCR) y qPCR con transcriptasa reversa (RT-qPCR). Asimismo, se utilizó un diseño experimental ad hoc para evaluar el umbral de detección de ácidos nucleicos libres. En nuestro conocimiento, este abordaje provee la primera evidencia experimental indicando que el DNA no es un indicador confiable de integridad celular cuando se detecta por qPCR, mientras que el RNA permitiría estimar de modo más preciso integridad bacteriana. El starter S. thermophilus se detectó por qPCR y RT-qPCR durante la producción del queso hasta los 81 días de madurado. Asimismo, se observó por primera vez que la adición de alfa-tocoferol y concentraciones funcionales de fitoesteroles correlacionó con un aumento significativo en los niveles de DNA y cDNA específicos.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. 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 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. An experimental set-up was used to evaluate the detection threshold of free nucleic acids. To our knowledge, this 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 bacterial integrity. The starter S. thermophilus was detected by qPCR and RT-qPCR during cheese production until 81 days of ripening. We also showed for the first time that the addition of alpha-tocopherol and phytosterols at functional concentrations correlated with a significant increase in target DNA and cDNA levels.Fil: Pega, Juan Franco. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Rizzo, Sergio Anibal. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; Argentina.Fil: Rossetti, Luciana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; Argentina.Fil: Perez, Carolina Daiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Diaz, Gabriela Esther. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Ruzal, S.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Descalzo, Adriana Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; Argentina.Fil: Nanni, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; Argentina

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°C), and acidic pH but instability in alkaline conditions, were determined. A molecular mass of 10 kDa was estimated by molecular gel filtration.Fil:Piuri, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Ruzal, S.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Murein hydrolase activity in the surface layer of Lactobacillus acidophilus ATCC 4356

We describe a new enzymatic functionality for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356, namely, an endopeptidase activity against the cell wall of Salmonella enterica serovar Newport, assayed via zymograms and identified by Western blotting. Based on amino acid sequence comparisons, the hydrolase activity was predicted to be located at the C terminus. Subsequent cloning and expression of the C-terminal domain in Bacillus subtilis resulted in the functional verification of the enzymatic activity. Copyright © 2008, American Society for Microbiology. All Rights Reserved.Fil:Acosta, M.P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Palomino, M.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Allievi, M.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Ruzal, S.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Synergistic effects of the lactobacillus acidophilus surface layer and nisin on bacterial growth

We have previously described a murein hydrolase activity for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356. Here we show that, in combination with nisin, this S-layer acts synergistically to inhibit the growth of pathogenic Gram-negative Salmonella enterica and potential pathogenic Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus. In addition, bacteriolytic effects were observed for the Gram-positive species tested. We postulate that the S-layer enhances the access of nisin into the cell membrane by enabling it to cross the cell wall, while nisin provides the sudden ion-nonspecific dissipation of the proton motive force required to enhance the S-layer murein hydrolase activity. Copyright © 2010, American Society for Microbiology. All Rights Reserved.Fil:Prado-Acosta, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Ruzal, S.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Allievi, M.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Palomino, M.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

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 30 min after its inoculation until 81 days 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.Fil: Pega, Juan Franco. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rizzo, Sergio Anibal. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; ArgentinaFil: Perez, Carolina Daiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rossetti, Luciana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; ArgentinaFil: Diaz, Gabriela Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; ArgentinaFil: Ruzal, Sandra Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Nanni, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; ArgentinaFil: Descalzo, Adriana Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto Tecnología de Alimentos; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA).LABINTEX-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD). Département PERSYST; Franci