Oxygen Transfer Effect on the Growth of Limosilactobacillus reuteri ATCC 53608 and on Its Metabolic Capacity.

Limosilactobacillus reuteri is a probiotic microorganism used in the treatment of gastrointestinal disorders. The effect of oxygen transfer on cultures of L. reuteri ATCC 53608 at shake flask and stirred tank bioreactor scales was studied, using MRS and molasses-based media. At shake flask scale, in MRS medium, a maximum bacterial concentration of 2.01 ± 0.02 g L-1 was obtained; the oxygen transfer coefficient was 2.01 ± 0.04 h-1. Similarly, in a 7.5 L bioreactor, in MRS, a maximum bacterial concentration of 2.46 ± 0.16 g L-1 was achieved (kLa = 2.64 ± 0.06 h-1). In contrast, using a molasses-based medium, bacterial concentration reached 3.13 ± 0.17 g L-1 in the 7.5 L bioreactor. A progressive reduction in lactic acid concentration and yield was observed as the oxygen transfer coefficient increased, at shake flask scale. Also, the oxygen transfer coefficient strongly affected the growth of L. reuteri in shake flask and bioreactor and allowed us to successfully scale up L. reuteri culture, producing similar maximum bacterial concentrations in both scales (2.01 g L-1 and 2.46 g L-1 in MRS). This is the first study on oxygen transfer coefficients in L. reuteri, and it is a valuable contribution to the field as it provides important insights about how this organism tolerates oxygen and adapts its metabolism for larger biomass production.

Production of Limonoids with Insect Antifeedant Activity in a Two-Stage Bioreactor Process with Cell Suspension Culture of Azadirachta indica.

Neem tree (Azadirachta indica) cell suspension culture is an alternative for the production of limonoids for insect control that overcomes limitations related to the supply of neem seeds. To establish conditions for cell growth and azadiracthin-related limonoid production, the effect of different sucrose concentrations, nitrate and phosphate in Murashige and Skoog (MS) medium, and the addition of one precursor and three elicitors was evaluated in shake flasks. The process was scaled up to a 3-l stirred tank bioreactor in one- and two-stage batch cultivation. In shake flasks, more than fivefold increase in the production of limonoids with the modified MS medium was observed (increase from 0.77 to 4.52 mg limonoids/g dry cell weight, DCW), while an increase of more than fourfold was achieved by adding the elicitors chitosan, salicylic acid, and jasmonic acid together (increase from 1.03 to 4.32 mg limonoids/g DCW). In the bioreactor, the volumetric production of limonoids was increased more than threefold with a two-stage culture in day 18 (13.82 mg limonoids/l in control single-stage process and 41.44 mg/l in two-stage process). The cultivation and operating mode of the bioreactor reported in this study may be adapted and used in optimization and process plant development for production of insect antifeedant limonoids with A. indica cell suspension cultures.

Biotechnological valorization of agro industrial and household wastes for lactic acid production / Valorización biotecnológica de residuos agroindustriales y domésticos para la producción de ácido láctico

ABSTRACT Lactic acid (LA) is an organic compound used in several industries, such as food, textile, chemical, and pharmaceutical. The global interest in this product is due to its use for the synthesis of numerous chemical compounds, including polylactic acid, a biodegradable thermoplastic and substitute for petroleum-derived plastics. An in-depth overview of the use of industrial and household wastes as inexpensive substrates in order to reduce the cost of LA production is presented. A review is carried out of the biotechnological aspects that must be taken into account when using some wastes with high transformation potential to produce LA in a submerged culture, as well recommendations for their use. The advantages and disadvantages of different types of treatments used for the transformation of waste into suitable substrates are considered. Several methods of fermentation, as well as genetic strategies for increasing the production, are summarized and compared. It is expected that in a few years there will be many advances in these areas that will allow greater large-scale production of LA using agroindustrial or household wastes, with potential positive economic and environmental impact in some regions of the planet.

RESUMEN El ácido láctico (AL) es un compuesto orgánico utilizado en diferentes industrias como la alimentaria, textil, química y farmacéutica. El interés mundial en este producto se debe a su uso para la síntesis de numerosos compuestos químicos, entre los que se incluye el ácido poliláctico, un termoplástico biodegradable y substituto del plástico derivado del petróleo. En este artículo se presenta una descripción general y en profundidad, del uso de residuos agroindustriales y domésticos como sustratos económicos para reducir los costos de producción del AL. La revisión aborda los aspectos biotecnológicos que deben ser considerados al utilizar algunos residuos con alto potencial de transformación para producir AL en un cultivo sumergido, así como algunas recomendaciones para su uso. Además, se consideran las ventajas y desventajas de diferentes tipos de tratamientos empleados para la transformación de residuos en sustratos adecuados. Finalmente, se resumen y comparan varios métodos de fermentación, así como estrategias genéticas para incrementar la producción de ácido láctico. Se espera que en pocos años existan más avances en esta área, que permitan una mayor producción de AL a gran escala usando residuos agroindustriales y domésticos, con un impacto económico y ambiental positivo, en algunas regiones del planeta.

ANTIFUNGAL ACTIVITY OF NEEM (Azadirachta indica: MELIACEAE) EXTRACTS AGAINST DERMATOPHYTES / Actividad antifúngica de extractos de neem (Azadirachta indica: Meliaceae) sobre hongos dermatofitos

In order to assess the antifungal activity of methanolic extracts from neem tree (Azadirachta indica A. Juss.), several bioassays were conducted following M38-A2 broth microdilution method on 14 isolates of the dermatophytes Trichophytonmentagrophytes, Trichophyton rubrum, Microsporum canis and Epidermophyton floccosum. Neem extracts were obtained through methanol-hexane partitioning of mature green leaves and seed oil. Furthermore, high performance liquid chromatography (HPLC) analyses were carried out to relate the chemical profile with their content of terpenoids, ofwidely known antifungal activity. The antimycotic Terbinafine served as a positive control. Results showed that there was total growth inhibition of the dermatophytes isolates at minimal inhibitory concentrations (MIC) between 50 μg/mL and 200 μg/mL for leaves extract, and between 625 μg/mL and 2500 μg/mL for seed oil extract. The MIC of positive control (Terbinafine) ranged between 0.0019 μg/mL and 0.0313 μg/mL. Both neem leaves and seed oil methanol extracts exhibited different chromatographic profiles by HPLC, which could explain the differences observed in their antifungal activity. This analysis revealed the possible presence of terpenoids in both extracts, which are known to have biological activity. The results of this research are a new report on the therapeutic potential of neem to the control of dermatophytosis.

Se determinó la actividad antifúngica de extractos metanólicos de la especie Azadirachta indica A. Juss. (Meliaceae), conocida comúnmente como neem, empleando el método de microdilución en caldo M38-A2 de referencia para hongos filamentosos y dermatofitos. Se evaluaron 14 aislamientos de los dermatofitos Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis y Epidermophyton floccosum. Los extractos de neem fueron obtenidos mediante partición metanol-hexano a partir de aceite de semillas y hojas verdes maduras. Adicionalmente, se analizaron por cromatografía líquida de alta resolución (CLAR) con el fin de relacionar su perfil químico con el contenido de terpenoides, de conocida actividad antifúngica. Se empleó como control positivo el antimicótico Terbinafina. Los resultados mostraron inhibición total del crecimiento de los aislamientos de dermatofitos a concentraciones mínimas inhibitorias (CMI) entre 50 μg/mL y 200 μg/mL para el extracto de hojas y entre 625 μg/mL y 2500 μg/mL para el extracto de aceite de semillas. La CMI encontrada para el control positivo (Terbinafina) fluctuó entre 0,0078 μg/mL y 0,0313 μg/mL. Los extractos metanólicos de hojas y aceite de semillas de neem exhibieron diferentes perfiles cromatográficos en CLAR, lo cual podría explicar las diferencias observadas en su actividad antifúngica. Éste análisis químico reveló la posible presencia de compuestos terpenoides en ambos extractos, los cuales se conocen por su actividad biológica. Los resultados de esta investigación son un nuevo aporte sobre el potencial terapéutico del neem para el control de dermatofitosis.

Actividad antifúngica de extractos de biomasa celular de neem sobre aislamientos de dermatofitos / Antifungal activity of neem cellular biomass extracts on dermatophytes isolates

Las líneas celulares de neem (Azadirachta indica A. Juss.) cultivadas en suspensión líquida han demostrado producir metabolitos secundarios bioactivos, particularmente triterpenoides. En consecuencia, se han realizado estudios para el control de microorganismos de importancia médica, como los hongos dermatofitos. El objetivo principal de este trabajo fue evaluar a través de un método de referencia in vitro la actividad antifúngica de diferentes extractos de cultivos celulares de neem sobre varios aislamientos de Trichophyton mentagrophytes, Trichophyton rubrum y Epidermophyton floccosum. Se realizó un escalado de cultivos de suspensiones celulares de neem, a partir de los cuales se obtuvo un extracto crudo metanólico. Éste extracto fue fraccionado posteriormente por cromatografía en columna de silica gel. Con los extractos obtenidos se determinó la Concentración Mínima Inhibitoria (CMI), siguiendo el método de microdilución en caldo M38-2, con cinco aislamientos de T. mentagrophytes, cinco de T. rubrum y tres de E. floccosum. Se usó como control positivo el antimicótico Terbinafina. Los resultados mostraron que el extracto crudo de biomasa celular de neem inhibe el crecimiento hasta en 100 % de T. mentagrophytes, T. rubrum y E. floccosum. Al evaluar las fracciones por separado, se observó que las de menor polaridad exhibieron en general mayor actividad antifúngica (CMI=109 μg/mL) que el extracto crudo per se (CMI=2500 μg/ mL) y las fracciones más polares (CMI=7000 μg/mL). Lo anterior indica que las células de neem cultivadas en suspensión producen compuestos con actividad antifúngica, siendo más bioactivos los presentes en las fracciones de menor polaridad.

Cell lines of neem (Azadirachta indica A. Juss.) grown in liquid suspension have shown to produce bioactive secondary metabolites, particularly triterpenoids. In consequence, its use as a control of medical microorganisms (like dermatophytes) is proposed. The main goal of this study was to assess the antifungal activity of methanolic extracts from neem cultured cell suspensions on several isolates of Trichophyton mentagrophytes (five isolates), Trichophyton rubrum (five isolates) and Epidermophyton floccosum (three isolates). Neem cell suspension cultures were scaled up, from which a raw methanolic extract was obtained. This extract was fractionated by silica gel column chromatography. The raw methanolic extract and its fractions were used in order to determine the Minimal Inhibitory Concentration (MIC) on the dermatophytes isolates by following M38-A2 broth microdilution method. Antimycotic Terbinafine was used as positive control. The results shown that neem raw cellular biomass extract inhibits the growth of T. mentagrophytes, T. rubrum and E. floccosum in at least 100%. In the evaluations of the separated fractions, it was observed that the low polarity fractions had higher antifungal activity (MIC=109 μg/mL) than the raw extract per se (MIC=2500 μg/mL) and the most polar ones (MIC=7000 μg/mL). The latter suggest that neem cells cultured in liquid suspension produces compounds with antifungal activity, being more active those present in the low polarity fractions.