Estrategias para mejorar la fagorresistencia de bacterias lácticas (BAL)

Las infecciones fágicas de bacterias lácticas usadas como cultivos iniciadores en la industria láctea fermentativa, son una de las causas principales de fallas en su capacidad acidificante. Una prioridad de la investigación en esta área durante los últimos 25 años ha sido el desarrollo de cultivos fagorresistentes con buenas propiedades tecnológicas para procesos industriales. Diferentes estrategias han sido empleadas para el mejoramiento de cepas: 1) Técnicas basadas en ADN recombinante (Per, ARN antisentido y Sistema suicida), que han sido desarrolladas principalmente para lactococos, resultando altamente efectivas pero muy específicas hacia los fagos. Por otro lado, estas cepas ?ingenierizadas? no pueden todavía aplicarse en alimentos. 2) Transferencia conjugativa de plásmidos de fagorresistencia , muy empleada para obtener cepas industriales en varios países, y 3) Aislamiento de mutantes espontáneos fagorresistentes (a partir de cultivos secundarios o por citometría de flujo), que constituye una estrategia ?natural? y, como en el caso anterior, permite obtener cepas ?food grade?. Son métodos simples y rápidos, salvo el de citometría, que requiere equipamiento específico y costoso.Fil: Reinheimer, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; Argentin

Phage adsorption and lytic propagation in Lactobacillus plantarum: Could host cell starvation affect them?

Background: Bacteriophages constitute a great threat to the activity of lactic acid bacteria used in industrial processes. Several factors can influence the infection cycle of bacteriophages. That is the case of the physiological state of host cells, which could produce inhibition or delay of the phage infection process. In the present work, the influence of Lactobacillus plantarum host cell starvation on phage B1 adsorption and propagation was investigated. Result: First, cell growth kinetics of L. plantarum ATCC 8014 were determined in MRS, limiting carbon (S-N), limiting nitrogen (S-C) and limiting carbon/nitrogen (S) broth. L. plantarum ATCC 8014 strain showed reduced growth rate under starvation conditions in comparison to the one obtained in MRS broth. Adsorption efficiencies of > 99 % were observed on the starved L. plantarum ATCC 8014 cells. Finally, the influence of cell starvation conditions in phage propagation was investigated through one-step growth curves. In this regard, production of phage progeny was studied when phage infection began before or after cell starvation. When bacterial cells were starved after phage infection, phage B1 was able to propagate in L. plantarum ATCC 8014 strain in a medium devoid of carbon source (S-N) but not when nitrogen (S-C broth) or nitrogen/carbon (S broth) sources were removed. However, addition of nitrogen and carbon/nitrogen compounds to starved infected cells caused the restoration of phage production. When bacterial cells were starved before phage infection, phage B1 propagated in either nitrogen or nitrogen/carbon starved cells only when the favorable conditions of culture (MRS) were used as a propagation medium. Regarding carbon starved cells, phage propagation in either MRS or S-N broth was evidenced. Conclusions: These results demonstrated that phage B1 could propagate in host cells even in unfavorable culture conditions, becoming a hazardous source of phages that could disseminate to industrial environments.Fil: Briggiler Marcó, Mariángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Reinheimer, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Quiberoni, Andrea del Lujan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; Argentin

PCR method for detection and identification of Lactobacillus casei/paracasei bacteriophages in dairy products

Bacteriophage infections of starter lactic acid bacteria (LAB) pose a serious risk to the dairy industry. Nowadays, the expanding use of valuable Lactobacillus strains as probiotic starters determines an increase in the frequency of specific bacteriophage infections in dairy plants. This work describes a simple and rapid Polymerase Chain Reaction (PCR) method that detects and identifies bacteriophages infecting Lactobacillus casei/paracasei, the main bacterial species used as probiotic. Based on a highly conserved region of the NTP-binding genes belonging to the replication module of L. casei phages φA2 and φAT3 (the only two whose genomes are completely sequenced), a pair of primers was designed to generate a specific fragment. Furthermore, this PCR detection method proved to be a useful tool for monitoring and identifying L. casei/paracasei phages in industrial samples since specific PCR signals were obtained from phage contaminated milk (detection limit: 104 PFU/mL milk) and other commercial samples (fermented milks and cheese whey) that include L. casei/paracasei as probiotic starter (detection limit: 106 PFU/mL fermented milk). Since this method can detect the above phages in industrial samples and can be easily incorporated into dairy industry routines, it might be readily used to earmark contaminated milk for use in processes that do not involve susceptible starter organisms, or processes which involve phage-deactivating conditions.Fil: Binetti, Ana Griselda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Capra, María Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Alvarez, Miguel. Instituto de Productos Lácteos de Asturias; EspañaFil: Reinheimer, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; Argentin

Lactobacillus casei/paracasei: aislamiento de mutantes espontáneos fagorresistentes para uso industrial

ResumenLos fagos de bacterias probióticas son causa de enormes pérdidas económicas y severas limitaciones a las estrategias de control. En este trabajo se aislaron y caracterizaron mutantes de L. casei/paracasei empleando el primer fago L. paracasei aislado en Sudamérica en una planta industrial argentina. Se caracterizaron fenotipo (eficiencias de recuperación de resistentes verdaderos, estabilidad, resistencia frente a otros fagos específicos) y mecanismos de fagorresistencia (nivel de fagorresistencia, liberación espontánea de fagos virulentos, adsorción con y sin calcio). Se demostró que el aislamiento de mutantes espontáneos resistentes desde bacterias probióticas sensibles, resulta una alternativa válida para obtener cepas para rotar en procesos industriales. Summary Phage attacks to probiotic strains cause enormous economic losses and limit common control strategies. In this work, spontaneous bacteriophage insensitive mutants were isolated from sensitive L. casei/paracasei strains against the first Sudamerican L. paracasei phage isolated in an Argentinean dairy plant. Phage resistance phenotype (efficiency of phage-resistant recovery, stability, sensitivity against other specific phages) and phage resistance mechanisms (phage resistance level, spontaneous release of virulent phages, phage adsorption with or without cacium ions) were characterized. The isolation of spontaneous phage-resistant mutants, from sensitive probiotic bacteria, was demonstrated to be a useful tool to obtain strains to rotate in industrial processes.Fil: Capra, María Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Mercanti, Diego Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Reinheimer, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Quiberoni, Andrea del Lujan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; Argentin

Study of the effects of spray-drying on the functionality of probiotic lactobacilli

Three probiotic lactobacilli strains were spray-dried in 20% (w/v) skim milk and submitted to a simulated gastrointestinal digestion. Fresh or spray-dried cultures were administered to mice for 5 and 10 days, and Immunoglobulin A (IgA)-producing cells were enumerated in the small intestine by immunohistochemistry. Spray-drying significantly enhanced the resistance of Lactobacillus paracasei A13 and Lactobacillus casei Nad to a simulated gastrointestinal digestion (0.96 and 1.95 log orders, respectively), compared with fresh cultures. Also, a significant higher number of IgA-producing cells were induced by spray-dried cultures compared with fresh cultures. Spray-drying is a suitable, but strain-dependent, technological process for the development of probiotic cultures in skim milk with increased functionality.Fil: Paez, Roxana. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria, Rafaela; ArgentinaFil: Lavari, Luisina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria, Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Audero, Gabriela. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria, Rafaela; ArgentinaFil: Cuatrin, Alejandra. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria, Rafaela; ArgentinaFil: Zaritzky, Noemi Elisabet. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Reinheimer, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Vinderola, Celso Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; Argentin

High pressure homogenization versus heat treatment: effect on survival, growth, and metabolism of dairy Leuconostoc strains.

The effect of high pressure homogenization (HPH) with respect to a traditional heat treatment on the inactivation, growth at 8°C after treatments, and volatile profile of adventitious Leuconostoc strains isolated from Cremoso Argentino spoiled cheeses and ingredients used for their manufacture was evaluated. Most Leuconostoc strains revealed elevated resistance to HPH (eight passes, 100 MPa), especially when resuspended in skim milk. Heat treatment was more efficient than HPH in inactivating Leuconostoc cells at the three initial levels tested. The levels of alcohols and sulfur compounds increased during incubation at 8°C in HPH-treated samples, while the highest amounts of aldehydes and ketones characterized were in heated samples. Leuconostoc cells resuspended in skim milk and subjected to one single-pass HPH treatment using an industrial-scale machine showed remarkable reductions in viable cell counts only when 300 and 400 MPa were applied. However, the cell counts of treated samples rose rapidly after only 5 days of storage at 8°C. The Leuconostoc strains tested in this work were highly resistant to the inactivation treatments applied. Neither HPH nor heat treatment assured their total destruction, even though they were more sensitive to the thermal treatment. To enhance the inhibitory effect on Leuconostoc cells, HPH should be combined with a mild heat treatment, which in addition to efficient microbial inactivation, could allow maximal retention of the physicochemical properties of the product

Inhibitory activity of phosphates on moulds isolated from foods and food processing plants

Inhibition by six commercial phosphates on the growth of 17 molds isolated from food sources was investigated. The assays were performed at neutral and natural (without pH adjustment) pH values, streaking the molds on PCA-phosphate (Plate Count Agar with added phosphates). Concentrations of 0.1%, 0.3%, 0.5%, 1.0% and 1.5% (w/v) of phosphates were used and the Minimal Inhibitory Concentration (MIC) values were determined. The resistance of molds to phosphates depended on the species. At a neutral pH, Aspergillus ochraceus and Fusarium proliferatum were resistant to all phosphates and concentrations assayed, while Byssochlamys nivea, Aureobasidium pullulans and Penicillium glabrum were most sensitive. The most inhibitory phosphates were those with chain lengths greater than 15 phosphate units and the highest sequestering power. At natural pH values (resulting from dissolving the phosphate in the medium), inhibitory activity changed dramatically for phosphates that produced alkaline or acidic pH values in the medium. Phosphates with alkaline pH values (sodium tripolyphosphate–high solubility, sodium tripolyphosphate and sodium neutral pyrophosphate) showed greatly increased inhibition compared with that observed at a neutral pH, while sodium acid pyrophosphate (acidic pH) showed decreased activity. Our results demonstrated that some phosphates could be used in the food industry to inhibit molds linked to food spoilage.Fil: Suárez, Viviana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Frisón, Laura Noemí. Universidad Nacional del Litoral. Facultad de Ingeniería Química; ArgentinaFil: Zapata de Basilico, Maria de la Luz. Universidad Nacional del Litoral. Facultad de Ingeniería Química; ArgentinaFil: Riveras, Mauricio. Universidad Nacional del Litoral. Facultad de Ingeniería Química; ArgentinaFil: Reinheimer, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; Argentin

Evaluation of lactobacillus paracasei LP11 and lactobacillus rhamnosus 64 potential as candidates for use as probiotics in functional foods

Desirable characteristics for probiotic microorganisms were evaluated in Lactobacillus rhamnosus 64 and Lactobacillus paracasei LP11. Both strains were tested for resistance to lysozyme and simulated gastrointestinal fluids, determination of cell wall hydrophobicity, evaluation of antibiotic susceptibility, antagonist activity against pathogens and the ability to use prebiotics. The safety determination was performed by an in vivo test, where the occurrence of bacterial translocation to liver was evaluated after daily oral administration of the strains for 10 days to mice. All results were promising, with the exception of resistance to simulated gastrointestinal fluids, that was unsatisfactory, with a decrease in viable cell concentrations of up to 5.08 log CFU/mL. The hydrophobicity profile of the strains ranged between 8.47 and 19.19%. Both showed satisfactiry resistance to lysozyme, with survival rates above 80%, strains were able to antagonize Escherichia coli V517, Salmonella enteritidis OMS-Ca, Staphylococcus aureus 76 and Listeria monocytogenes ATCC 15313, and displayed satisfactory use of lactulose, inulin and P95 raftilose as prebiotics. No bacterial translocation was observed after the administration of the strains to mice. The results obtained herein justify the use of L. rhamnosus 64 and L. paracasei LP11 in new investigations for the future application of these microorganisms as probiotics.Fil: de Matos, Fernando Eustáquio. Universidade de Sao Paulo; Brasil. Universidade Federal de Juiz de Fora; BrasilFil: Santos, Tizá Teles. Universidade Estadual de Santa Cruz; BrasilFil: Burns, Patricia Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Reinheimer, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Vinderola, Celso Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Trindade, Carmen Sílvia Fávaro. Universidade de Sao Paulo; Brasi

Spray-drying process preserves the protective capacity of a breast milk-derived Bifidobacterium lactis strain on acute and chronic colitis in mice

Gut microbiota dysbiosis plays a central role in the development and perpetuation of chronic inflammation in inflammatory bowel disease (IBD) and therefore is key target for interventions with high quality and functional probiotics. The local production of stable probiotic formulations at limited cost is considered an advantage as it reduces transportation cost and time, thereby increasing the effective period at the consumer side. In the present study, we compared the anti-inflammatory capacities of the Bifidobacterium animalis subsp. lactis (B. lactis) INL1, a probiotic strain isolated in Argentina from human breast milk, with the commercial strain B. animalis subsp. lactis BB12. The impact of spray-drying, a low-cost alternative of bacterial dehydration, on the functionality of both bifidobacteria was also investigated. We showed for both bacteria that the spray-drying process did not impact on bacterial survival nor on their protective capacities against acute and chronic colitis in mice, opening future perspectives for the use of strain INL1 in populations with IBD.Fil: Burns, Patricia Graciela. Université de Lille. Centre D'infection Et D'immunite de Lille; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Alard, Jeanne. Universite Lille; FranciaFil: Hrdy, Jiri. Universite Lille; FranciaFil: Boutillier, Denise. Universite Lille; FranciaFil: Paéz, Roxana. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Reinheimer, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Pot, Bruno. Universite Lille; FranciaFil: Vinderola, Celso Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Lactología Industrial. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Grangette, Corinne. Universite Lille; Franci

Thermal, Chemical, and Photocatalytic Inactivation of <i>Lactobacillus plantarum</i> Bacteriophages

The effect of several biocides, thermal treatments, and photocatalysis on the viability of four Lactobacillus plantarum phages was investigated. Times to achieve 99% inactivation (T₉₉) of phages at 63, 72, and 90 degrees C were evaluated in four suspension media: deMan Rogosa Sharpe broth, reconstituted skim milk, a commercial EM-glucose medium, and Tris magnesium gelatin buffer. The four phages studied were highly resistant to 63 degrees C (T₉₉ > 45 min); however, counts < 10 PFU/ml were achieved by heating at 90 degrees C for 5 min. Higher thermal resistance at 72 degrees C was observed when reconstituted skim milk and EM-glucose medium were assayed. Peracetic acid (0.15%, vol/vol) was an effective biocide for the complete inactivation of all phages studied within 5 min of exposure. Sodium hypochlorite (800 ppm) inactivated the phages completely within 30 min. Ethanol (100%) did not destroy phage particles even after 45 min. Isopropanol did not have any effect on phage viability. Phage counts < 50 PFU/ml were obtained within 180 min of photocatalytic treatment. The results obtained in this work are important for establishing adequate methods for inactivating phages in industrial plants and laboratory environments.Centro de Investigación y Desarrollo en Criotecnología de Alimento