14 research outputs found

    Specific Strains of Lactic Acid Bacteria Differentially Modulate the Profile of Adipokines In Vitro

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    Obesity induces local/systemic inflammation accompanied by increases in macrophage infiltration into adipose tissue and production of inflammatory cytokines, chemokines, and hormones. Previous studies have shown that probiotics could improve the intestinal dysbiosis induced by metabolic diseases such as obesity, diabetes, and metabolic syndrome. Microorganisms could (directly or indirectly) affect adipokine levels due to their capacity to induce translocation of several intestinal microbial antigens into systemic circulation, which could lead to metabolic endotoxemia or produce immunomodulation in different organs. The aim of the present study was to select non-inflammatory lactic acid bacteria (LAB) strains with the capacity to modulate adipokine secretion by the adipose tissue. We wish to elucidate the role of potential probiotic strains in the regulation of the cross talking between immune cells such as macrophages and adipose cells. Mouse macrophage cell line RAW 264.7 was used for evaluating the ability of 14 LAB strains to induce cytokine production. The LAB strains were chosen based on their previously studied beneficial properties in health. Then, in murine adipocyte culture and macrophage-adipocyte coculture, we determined the ability of these strains to induce cytokines and leptin secretion. Tumor necrosis factor alpha, interleukin 6 (IL-6), IL-10, monocyte chemoattractant protein-1, and leptin levels were measured in cell supernatants. We also performed the detection and quantification of leptin receptor (Ob-Rb) expression in macrophage cell lines stimulated by these LAB strains. Differential secretion profile of cytokines in macrophage cells induced by LAB strains was observed. Also, the levels of Ob-Rb expression diverged among different LAB strains. In LAB-stimulated coculture cells (adipocytes and macrophages), we observed differential production of leptin and cytokines. Furthermore, we detected lower production levels in single culture than cocultured cells. The principal component analysis showed an association between the four clusters of strains established according to their inflammatory profiles and leptin adipocyte production and leptin receptor expression in macrophages. We conclude that coculture is the most appropriate system for selecting strains with the ability to modulate adipokine secretion. The use of microorganisms with low and medium inflammatory properties and ability to modulate leptin levels could be a strategy for the treatment of some metabolic diseases associated with dysregulation of immune response.Fil: Fabersani Marrades, Mario Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Abeijon Mukdsi, Maria Claudia. Universidad del Norte Santo Tomás de Aquino; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Ross, Gloria Romina. Universidad del Norte Santo Tomás de Aquino; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: Medina, Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán; ArgentinaFil: González, Silvia. Universidad Nacional de Tucumán; ArgentinaFil: Gauffin-Cano, Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad del Norte Santo Tomás de Aquino; Argentin

    Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

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    IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

    Microencapsulation of probiotic strains for swine feeding

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    Probiotics are live microorganisms which have health-promoting attributes. These bacteria must overcome biological barriers, including acid in the stomach and bile in the intestine to exert beneficial effects. The encapsulation consists in a provision of an outer layer to protect the core material from damage. Microencapsulating in calcium alginate, nowadays, is being used to bacteria immobilization owing to its easy handling, nontoxic nature, and low cost. The aims of this study were to improve the microencapsulating method for probiotic bacteria and to investigate whether the material used as coating, afford an increase on strain survival under simulated gastrointestinal conditions. Lactic acid bacteria used in this work were isolated from feces of young and healthy pigs and they were selected because of their probiotic properties. Our results showed that the optimal encapsulation process was achieved using 1:1 (v/v) 20% non fat milk cell suspension mixed with 1.8% sodium alginate solution. Alginate capsules hardening was carried out using 0.1 M calcium chloride solution for 30 min. This microencapsulating technique could protect the probiotic bacteria against gastric environment, allowing viable cells get to the intestinal tract. So it could be a useful way to deliver these beneficial bacteria to host.Fil: Ross, Gloria Romina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Tucuman. Facultad de Bioquímica, Química y Farmacia. Instituto de Farmacia. Cátedra de Salud Publica; ArgentinaFil: Gusils Leon, Carlos Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gonzalez, Silvia Nelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentin

    Use of probiotics as dietary supplement in cattle goats and pigs

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    The application of potentially beneficial microorganisms to improve host defense is a new trend to increase health benefits. We developed the first specific host probiotics for goats and pigs from a mixture of lactic acid bacteria isolated from healthy animals. In both cases, the probiotic administration enhanced the body weight and was able to modify microflora balance by reducing Enterobacteria and increasing lactic acid bacteria population. The intestinal composition of fatty acids was modified, with a diminution of saturated fatty acid and an increase of the beneficial linoleic acid. On goats, the probiotics administration was correlated with a ten time diminution of fecal putrescine (cancer and bacterial disease marker) and a decrease of 60% mutagen fecal concentration, indicating the protective effect of this treatment. These results are encouraging towards the use of probiotic mixtures as functional food for goats and pigs.Fil: Ross, Gloria Romina. Universidad Nacional de Tucumán. Instituto de Biotecnología Farmacéutica y Alimentaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Biotecnología Farmacéutica y Alimentaria; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: Apas, Ana Lidia. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: Arena, Mario Eduardo. Universidad Nacional de Tucumán. Instituto de Biotecnología Farmacéutica y Alimentaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Biotecnología Farmacéutica y Alimentaria; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: Gonzalez, Silvia Nelina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina. Universidad Nacional de Tucumán. Instituto de Biotecnología Farmacéutica y Alimentaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Biotecnología Farmacéutica y Alimentaria; Argentin

    Inhibitory effects of Lactobacilli on enteropathogenic Salmonella

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    The major source of human salmonellosis are farm animals, which may frequently be intestinal carriers of the organism. Lactobacilli isolated from the intestinal tract with efficiency adhesion were selected and studied in assay competitions with different Salmonella serotypes. The growth and lectin production of lactobacilli remained unchanged in several mixed and single culture studies. However, in mixed cultures, the inhibition (bacteriostatic) of viable bacteria of salmonella strains was observed. The adhesion ratio showed significant values for L. animalis, L. fermentum, S. Gallinarum and S. Pullorum. These results indicate the remarkable importance of a specific host interaction in the colonization process by microorganisms. L. animalis was effective in reducing the attachment of S. Gallinarum, S. Pullorum and S. Enteritidis to host-specific epithelial cells, while L. fermentum was able to reduce the attachment of S. Pullorum and S. Gallinarum. Therefore, we suggest that chicken lactobacilli included in this work may be considered potential probiotic microorganisms, and used in the preparation of a probiotic food.Fil: Gusils Leon, Carlos Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ross, Gloria Romina. Universidad Nacional de Tucuman. Facultad de Bioquímica, Química y Farmacia. Instituto de Farmacia. Cátedra de Salud Publica; ArgentinaFil: Draksler, Diana. Universidad Nacional de Tucumán; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Pérez, C. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Tous, M. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentin

    Effect of Excess Iodide Intake on Salivary Glands in a Swiss Albino Mice Model

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    Iodine is an important micronutrient required for nutrition. Excess iodine has adverse effects on thyroid, but there is not enough information regarding its effect on salivary glands. In addition to food and iodized salt, skin disinfectants and maternal nutritional supplements contain iodide, so its intake could be excessive during pregnancy, lactation, and infancy. The aim of this work was to evaluate the effect of excess iodide ingestion on salivary glands during mating, gestation, lactation, and postweaning period in mouse. During assay, mice were allocated into groups: control and treatment groups (received distilled water with NaI 1 mg/mL). Water intake, glandular weight, and histology were analyzed. Treatment groups showed an increase in glandular weight and a significantly (p < 0.05) higher water intake than control groups. Lymphocyte infiltration was observed in animals of treatment groups, while there was no infiltration in glandular sections of control groups. Results demonstrated that a negative relationship could exist between iodide excess and salivary glands. This work is novel evidence that high levels of iodide intake could induce mononuclear infiltration in salivary glands. These results should be considered, especially in pregnant/lactating women, to whom a higher iodine intake is usually recommended

    Lactobacillus spp. impair the ability of listeria monocytogenes FBUNT to adhere to and invade Caco-2 cells

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    The objective of this study was to evaluate the ability of Lactobacillus curvatus CRL705, CRL1532, and CRL1533 and Lactobacillus sakei CRL1613 to survive under simulated gastrointestinal conditions. Moreover, a microencapsulation approach was proposed to improve gastrointestinal survival. Finally, experiments were performed to demonstrate that Lactobacillus spp. can modulate the ability of Listeria monocytogenes FBUNT to adhere to and invade Caco-2 cells. RESULTS:Lactobacillus strains were encapsulated in alginate beads to enhance the survival of bacteria under in vitro gastrointestinal conditions. All strains hydrolyzed bile salts using chenodeoxycholic acid as a substrate and adhered to Caco-2 cells. Cell-free supernatants (CFSs) showed antimicrobial activity against L. monocytogenes as demonstrated by agar diffusion assays. The average percentages of L. monocytogenes adhesion decreased from 67.74 to 41.75 and 38.7% in the presence of 50 and 90% (v/v), respectively, for all CFSs tested. The highest concentrations of CFSs completely inhibited the L. monocytogenes invasion of Caco-2 cells.CONCLUSIONS:The studied Lactobacillus strains have protective effects against the adhesion and invasion of L. monocytogenes FBUNT. Alginate encapsulation of these bacteria improved gastrointestinal tolerance such that they could be further studied as potential probiotics against intestinal pathogenic bacteria.Fil: Castellano, Patricia Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Pérez Ibarreche, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Longo Borges, Liliana. Universidade de Sao Paulo; BrasilFil: Niño Arias, Fabian. Universidade de Sao Paulo; BrasilFil: Ross, Gloria Romina. Universidad Nacional de Tucumán. Instituto de Biotecnología Farmacéutica y Alimentaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Biotecnología Farmacéutica y Alimentaria; ArgentinaFil: Pereira de Martinis, Elaine Cristina. Universidade de Sao Paulo; Brasi

    Lactic acid bacteria activities to promote health benefits

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    The activity of the probiotic lactic acid bacteria (LAB) has been widely documented and can be divided according to their nutritional, physiological and antimicrobial effects. These observations have led to the development of foods containing LAB to be used as human and animal probiotics. This chapter reviews the current results of the relevance of microencapsulation method of probiotic bacteria to resist the gastrointestinal conditions. Bacteria isolated from swine faeces have probiotic effects when they are administered in calcium alginate microcapsules. Other interesting property is the influence of these bacteria on leptin secretion. This hormone could be linked with the nutritional status and the immune system. In animal experimental models, we have demonstrated that probiotic microorganisms have different influences on leptin secretion of well-nourished and mild calorie restricted host. The presence of microbial enzymes like the cinnamoyl-esterase or the linoleic acid desaturase, were also studied. The cinnamoyl-esterase action could release, in intestinal tract, absorbable antioxidant compounds, such as caffeic or ferulic acid. Some foods contain these acids under ester forms. On the other hand, the linoleic acid desaturase allows the production of conjugated linoleic acid, a fatty acid with different biological functions. Strains from intestinal, ruminal and dairy origin that are able to produce CLA, may be included in foods or used as probiotic to offer health benefits to consumers.Fil: Ross, Gloria Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: Gauffin Cano, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Gusils Leon, Carlos Horacio. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Provincia de Tucumán. Ministerio de Desarrollo Productivo. Estación Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnología Agroindustrial del Noroeste Argentino; ArgentinaFil: Medina, Roxana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: Van Nieuwenhove, Carina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; ArgentinaFil: Gonzalez, Silvia Nelina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin

    Effect of Excess Iodide Intake on Salivary Glands in a Swiss Albino Mice Model

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    Iodine is an important micronutrient required for nutrition. Excess iodine has adverse effects on thyroid, but there is not enough information regarding its effect on salivary glands. In addition to food and iodized salt, skin disinfectants and maternal nutritional supplements contain iodide, so its intake could be excessive during pregnancy, lactation, and infancy. The aim of this work was to evaluate the effect of excess iodide ingestion on salivary glands during mating, gestation, lactation, and postweaning period in mouse. During assay, mice were allocated into groups: control and treatment groups (received distilled water with NaI 1 mg/mL). Water intake, glandular weight, and histology were analyzed. Treatment groups showed an increase in glandular weight and a significantly (p < 0.05) higher water intake than control groups. Lymphocyte infiltration was observed in animals of treatment groups, while there was no infiltration in glandular sections of control groups. Results demonstrated that a negative relationship could exist between iodide excess and salivary glands. This work is novel evidence that high levels of iodide intake could induce mononuclear infiltration in salivary glands. These results should be considered, especially in pregnant/lactating women, to whom a higher iodine intake is usually recommended.Fil: Ross, Gloria Romina. Universidad del Norte Santo Tomás de Aquino. Facultad de Ciencias de la Salud; Argentina. Universidad Nacional de Tucuman. Instituto de Biotecnología Farmaceutica y Alimentaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Instituto de Biotecnología Farmaceutica y Alimentaria.; ArgentinaFil: Fabersani Marrades, Mario Emanuel. Universidad Nacional de Tucumán. Facultad de Agronomía y Zootecnia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Russo, Matias Irineo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Gómez, Alba. Universidad Nacional de Tucumán. Facultad de Medicina; ArgentinaFil: Japaze, Hugo. Universidad Nacional de Tucumán. Facultad de Medicina; ArgentinaFil: Gonzalez, Silvia Nelina. Universidad Nacional de Tucuman. Instituto de Biotecnología Farmaceutica y Alimentaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Instituto de Biotecnología Farmaceutica y Alimentaria.; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: Gauffin Cano, María Paola. Universidad del Norte Santo Tomás de Aquino. Facultad de Ciencias de la Salud; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin

    Spray dried lactobacilli maintain viability and feruloyl esterase activity during prolonged storage and under gastrointestinal tract conditions

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    The use of lactobacilli with feruloyl esterase (FE) activity in the development of functional foods has gained considerable interest in recent years. Microencapsulation of FE-producing bacteria to facilitate their incorporation into food is a challenge. The aim of this study was to evaluate survival and maintenance of FE activity during storage at 4 °C and under simulated gastrointestinal tract (GIT) conditions of microcapsules of FE-producing Lactobacillus (Lb.) strains obtained by spray drying. Lb. fermentum CRL1446 and Lb. johnsonii CRL1231 powders maintained viability at concentrations ≥ 106 CFU/g (minimum probiotic dose) when stored at 4 °C for 12 months. Lb. acidophilus CRL1014 powders were only able to maintain ≥ 106 CFU/g during 4 months of storage. FE activity was conserved in three microencapsulated strains evaluated, an increase of specific activity being observed until month 12 of storage. Powders of the three strains incubated under GIT conditions maintained their viability (≥ 106 CFU/g), but specific FE activity was only detected in Lb. fermentum and Lb. johnsonii powders (0.80- 0.83 U/mg and 0.21- 0.56 U/mg, respectively). CRL1446 and CRL1231 microcapsules were able to resist prolonged storage and GIT conditions, retaining FE activity and preserving their probiotic potential and could be incorporated into functional foods.Fil: Russo, Matias Irineo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Abeijon Mukdsi, Maria Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Santacruz López, Yolanda Arlette. Instituto Tecnológico de Monterrey; MéxicoFil: Ross, Gloria Romina. Universidad Nacional de Tucumán. Instituto de Biotecnología Farmacéutica y Alimentaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto de Biotecnología Farmacéutica y Alimentaria; ArgentinaFil: López Malo, Aurelio. Universidad de las Américas. Departamento de Ingeniería Química, Alimentos y Ambiental; MéxicoFil: Gauffin Cano, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad del Norte Santo Tomás de Aquino; ArgentinaFil: Medina, Roxana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Agronomía y Zootecnia; Argentin
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