Valorización de residuos agroindustriales para la obtención de liquenisina por Bacillus licheniformis AL 1.1

[spa] La cepa bacteriana AL 1.1 se aisló de una muestra de suelo de la isla Decepción del continente Antártico. Inicialmente se observó que dicha cepa tenía la capacidad de reducir la tensión superficial del medio, por lo que fue seleccionada para realizar la presente tesis doctoral. Después de confirmar la identificación del aislado como un Bacillus licheniformis se caracterizó el biotensioactivo (BT) producido como liquenisina (LchAL1.1). El estudio de las propiedades físico-químicas de la LchAL1.1 mostró que es capaz de reducir la tensión superficial del agua de 72 a 30 mN/m y posee una concentración micelar crítica de 15 mg/L, indicando una elevada eficacia y eficiencia. La LchAL1.1 tiene la capacidad de formar cristales líquidos del tipo de cruces de malta, lo cual sugiere una importante capacidad de agregación y abre un campo importante en la encapsulación de compuestos de interés. La primera parte del trabajo se centró en el estudio de la producción del BT. Al analizarse diversas fuentes de carbono se observó que la cepa AL 1.1 solo producía liquenisina con hidratos de carbono. Dicha información fue de utilidad para la selección de las melazas como fuente de carbono ya que es un sustrato rico en dichos componentes y además es una fuente residual de bajo coste. Utilizando dicho sustrato se diseñó el medio de producción óptimo mediante la aplicación de la metodología de superficie de respuesta (107,82 g/L de melazas, 6,47 g/L de NaNO3 y 9,7 g/L de K2HPO4/KH2PO4), obteniendo rendimientos cercanos a los 3,2 g/L de liquenisina, lo que representa un incremento de 15 veces con respecto al medio inicial. Se han estudiado las propiedades biológicas de la LchAL1.1 para conocer su posible aplicación en diferentes ámbitos. A pesar de su débil acción antimicrobiana, la LchAL1.1 se presenta como una alternativa interesante para evitar la adherencia y eliminación de biofilms microbianos, encontrando una mayor eficacia para evitar la adherencia microbiana y una alta eficiencia para la eliminación del biofilm pre-formado. Adicionalmente se estudió la interacción de la LchAL1.1 con membranas biológicas y vesículas fosfolipídicas con el objetivo de conocer el mecanismo molecular de su acción. La LchAL1.1 produce hemólisis a concentraciones por debajo de su concentración micelar crítica. Las medidas cinéticas muestran que con la adición de la LchAL1.1, la salida de K+ precede a la de la hemoglobina, y que la adición de protectores osmóticos de tamaño apropiado al medio externo hace posible disminuir y hasta evitar la hemólisis. Estos resultados sugieren que la hemólisis de los eritrocitos humanos ocurre por un proceso coloide-osmótico. Por otro lado, la adición de la LchAL1.1 a vesículas unilamelares (LUV) de POPC produce la salida selectiva de solutos atrapados en su interior al medio externo, encontrando que la composición de la membrana modula el proceso de salida en gran medida. Esto nos indica que la incorporación de la LchAL1.1 en membranas tanto experimentales como modelos, provoca la formación de dominios laterales, los cuales forman defectos o “poros” a través de los cuales tiene lugar la salida de solutos con un tamaño determinado. Con respecto a su acción sobre células eucariotas, la liquenisina induce apoptosis en un 96,5% de las células epiteliales de cáncer de colón (Caco-2). Dicho efecto puede estar relacionado con la interacción de las micelas con la bicapa lipídica, ya que el efecto ocurre a concentraciones de la LchAL1.1 que están por encima de su concentración micelar crítica (50 – 100 µg/mL). Un nuevo enfoque para potenciar las propiedades biológicas de los compuestos bioactivos, es la posibilidad de utilizarlos en combinación. En este sentido, se evaluó la interacción de la LchAL1.1 con tensioactivos sintéticos geminales derivados de aminoácidos como el C3(LA)2 y C6(LL)2, encontrando que con el C3(LA)2 se produce una respuesta sinérgica en sus propiedades antimicrobianas, mejorando la acción de ambos compuestos especialmente contra bacterias Gram negativas.[eng] Biosurfactants are of great interest due to the demand for natural products with low toxicity. Strain AL 1.1, isolated on Deception Island (Antarctica) and identified as Bacillus licheniformis, produce lipopeptide when grow under a variety of carbohydrates. The lipopeptide was characterised as lichenysin (LchAL1.1). The LchAL1.1 reduced surface tension to 30 mN/m and had a critical micelle concentration of 15 mg/L. This highly effective and efficient characterized the product as a powerful surfactant. Besides, the formation of liquid crystalline by LchAL1.1 in form of Maltese crosses was revealed. This suggested important properties to self-aggregate and opens an important field in the compounds encapsulation. Nevertheless, their production is not competitive when cost is a limiting factor. For this reason, an economical medium, containing molasses, was optimized to enhance lichenysin production by response surface methodology (RSM). A production of 3.2 g l-1 of lichenysin was achieved with an optimum medium containing 107.82 g l-1 of molasses, 6.47 g l-1 of NaNO3 and 9.7 g l-1 of K2HPO4/KH2PO4, in which, molasses and phosphate salts had a significant effect on biosurfactant production. This medium resulted in a fifteen times increase in production compared with the non-optimized medium. The LchAL1.1 showed a weak antimicrobial activity, however, it had notable anti-adhesion activity, and was able to prevent and eliminate the biofilm formation by pathogenic strains associated with foodborne illness. Lychenysin was effectively applied in a surface pre-treatment to avoid microbial biofilm. It was also very efficient in removing biofilms in surface post-treatment. In addition, the molecular mechanism underlying permeabilization of model and biological membranes by the lipopeptide lichenysin was evaluated. The LchAL1.1 caused hemolysis of human erythrocytes, which varied with LchAL1.1 concentration in a sigmoidal manner. The LchAL1.1-induced K+ release from red blood cells preceded the leakage of hemoglobin, and in addition, hemolysis could be impeded by the presence of compounds in the external medium having a size larger than PEG 4000, indicating a colloid-osmotic mechanism for hemolysis. Lichenysin also caused permeabilization of model phospholipid membranes, as monitored by the release of carboxyfluorescein, from large unilamellar vesicles (LUV). Lipid membrane composition plays a role in the target membrane selectivity of lichenysin. The experimental results support a pore-type behaviour that can be explained by the formation of enhanced permeability domains in the erythrocyte membrane, as observed in model membranes. Furthermore, the LchAL1.1 induced apoptosis in non-differentiated intestinal Caco-2 cells (96.5%). The action can be due to the micelles interaction with lipid bilayer, because the effect occurred to LchAL1.1 concentration above critical micelle concentration (50-100 µg ml-1). Finally, the interaction between lichenysin, arginine (C3(LA)2) and lysine (C6(LL)2) surfactants was evaluated. Thus, when the LchAL1.1 act as co-surfactant improved the antimicrobial activity of arginine (C3(LA)2) surfactant, showing a synergistic effect, mainly in front to Gram negatives bacteria

Optimizing the production of the biosurfactant lichenysin and its application in biofilm control

Aims: Apply response surface methodology (RSM) to develop and optimize an economical medium for lichenysin production, which is a surfactant produced by Bacillus licheniformis and evaluate the application of lichenysin in the prevention and disruption of pathogenic micro-organism biofilm that creates health problems in the food industry and hospitals. Results: An economical medium containing molasses was optimized to enhance lichenysin production by RSM. A production of 3.2 g l 1 of lichenysin was achieved with an optimum medium containing 107.82 g l 1 of molasses, 6.47 g l 1 of NaNO3 and 9.7 g l 1 of K2HPO4/KH2PO4, in which molasses and phosphate salts had a significant effect on biosurfactant production. Lichenysin was effectively applied in a surface pre-treatment to avoid microbial biofilm development of methicillin-resistant Staphylococcus aureus (MRSA) (68.73%) and Candida albicans (74.35%), with ED50 values of 8.3 and 17.2 lg ml 1 respectively. It was also very efficient in a surface posttreatment to remove biofilms of MRSA (55.74%) and Yersinia enterocolitica (51.51%), with an ED50 of 2.79 and 4.09 lg ml 1 respectively. Conclusions: Lichenysin was found to have notable anti-adhesion activity, being able to prevent and eliminate the biofilm formation by pathogenic strains associated with foodborne illness. This new medium resulted in a fourfold increase in production compared with the nonoptimized medium. Significance and Impact of the Study: Molasses can be regarded as a useful resource for biotechnological applications, such as the production of lichenysin. The use of agro-industrial substrates has an important role in the sustainable and competitive development of several industrial sectors, as well as in industrial residues management. Additionally, lichenysin is particularly effective in preventing biofilm formation by strains problematic for the food industry and in the hospital environment. Lichenysin also efficiently disrupts biofilm

Lichenysin production and application in the pharmaceutical field

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/103042Lipopeptides such as lichenysin are biosurfactants of great interest, due to the demand for natural surface-active agents with low toxicity. Bacillus licheniformis AL 1.1 produces a lipopeptide characterized as lichenysin (LchAL1.1), which acts as a powerful surfactant, able to reduce surface tension to 28.5 mN m-1 and with a critical micelle concentration of 15 mg L-1. LchAL1.1 is particularly effective in preventing biofilm formation by pathogenic strains, has an emulsifying capacity and permeabilizes membranes by a colloid-osmotic process. The production of lipopeptides from agro-industrial residues, particularly molasses, is a sustainable process of great potential for the development of economic bioprocesses

Biosurfactant from Bacillus subtilis DS03: Properties and Application in Cleaning Out Place System in a Pilot Sausages Processing

Microbial surfactants (MS) or biosurfactants (BS) are amphiphilic molecules composed of a hydrophilic and a hydrophobic segment, which align at the interface between polar and non-polar compounds, reducing the surface tension. BS production is developed as an alternative to synthetic surfactants because they are biodegradable, with low toxicity and high specificity. Several BS applications are published in this research proposing using a biosurfactant crude extract (BCE) as part of cleaning products. This paper reported the BCE production from Bacillus subtilis DS03 using a medium with molasses. The BCE reduced surface tension from 72 mN/m (water) to 34 mN/m and achieved a critical micelle concentration of 24.66 ppm. The highly effective and efficient behavior characterized the product as a powerful surfactant with stability under a wide pH range, high temperatures, and emulsifying properties, suggesting potential applications in food industry cleaning operations. BCE was also applied in a surface pre-treatment to avoid microbial biofilm development, showing inhibition in more than 90% of Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes above 2000 ppm of BCE. It was also tested on a surface post-treatment to remove biofilms reporting a significant reduction of Escherichia coli (50.10%), Staphylococcus aureus (55.77%), and Listeria monocytogenes (59.44%) in a concentration higher than 250 ppm of BCE. Finally, we compared the functionality between sodium lauryl ether sulfate (SLES) and BCE. The results suggested that BCE is a promising ingredient for cleaning formulations for industrial food applications

Lichenysin-geminal amino acid-based surfactants: Synergistic action of an unconventional antimicrobial mixture

Recently it has been demonstrated that catanionic mixtures of oppositely charged surfactants have improved physicochemical-biological properties compared to the individual components. Isotherms of mixtures of an anionic biosurfactant (lichenysin) and a cationic aminoacid surfactant (C-3(LA)(2)) indicate a strong interaction suggesting the formation of a new 'pseudo-surfactant'. The antimicrobial properties of the mixture lichenysin and C-3(LA)(2) M80:20, indicate a synergistic effect of the components. The mechanism of action on the bacterial envelope was assessed by flow cytometry and Transmission Electron Microscopy. (C) 2016 Elsevier B.V. All rights reserved. Keywords: Antimicrobial properties; Arginine; Escherichia coli; Flow cytometry; Gemini surfactants; Lichenysin; Listeria nonocytogenes; Potassium leakage; Transmission electron microscopy

Alternativas de mejora en el manejo postcosecha de tomate de riñón cultivados en la provincia de Santa Elena

Investigación desarrollada en los laboratorios del centro de investigación biotecnológica del Ecuador conjuntamente con el laboratorio de bromatología de la carrera de ingeniería en alimentos de la espol, para estudiar aquellos factores que directa o indirectamente influyen sobre la disminución de la calidad del tomate riñon y su vida útil; actividad realizada post-cosecha a nivel de campo en la provincia de Santa Elena. Así mismo, se complementa con la caracterización de las propiedades físico químicas bajo ciertas condiciones experimentales, para lo cual se evalua las zonas tomateras de Atahualpa y Chanduy parroquias rurales del cantón Santa Elena.GuayaquilIngeniero de Alimento

Alternativas de mejora en el manejo postcosecha de tomate riñón cultivados en la provincia de Santa Elena

La presente investigación se enmarcó en el estudio de las diferentes actividades post-cosechas del tomate riñón realizadas a nivel de campo, en la provincia de Santa Elena. Así mismo, se complemento con la caracterización de las propiedades físico químicas bajo ciertas condiciones experimentales, para lo cual se evaluó las zonas tomateras de Atahualpa y Chanduy parroquias rurales del cantón Santa Elena. La primera parte de la investigación se basó en la evaluación de aspectos básicos relacionados con el cultivo del tomate en la zona de Santa Elena, además se logró conocer y cuantificar las causas por las que se producían perdidas de tomate. A nivel de laboratorio se trabajó con un diseño experimental bifactorial, donde los factores de estudio fueron la temperatura y el material de embalaje. Para la temperatura se trabajó con dos sub-niveles: temperatura ambiente (25ºC), temperatura de refrigeración (15ºC), en cambio los materiales de plástico, cartón y madera fueron los sub-niveles pertenecientes a material de embalaje. Una vez planteados los diferentes ensayos se estudió el comportamiento de las características del tomate como pH, acidez titulable, grados Brix y pérdida de peso, posteriormente se evaluó los daños producidos en el tomate mediante la escala de daño propuesta por Marcos D. Ferreira, 2005. Por otra parte mediante el Textura Analyzer C3, se simuló las fuerzas de Impacto, Compresión y Penetración para establecer la relación entre estas y el daño mecánico que se presenta, y se determinó como esto influyó en la vida útil del tomate riñón. Esta investigación se desarrolló en los laboratorios del Centro de Investigación Biotecnológica del Ecuador conjuntamente con el laboratorio de Bromatología de la carrera de Ingeniería en Alimentos de la Escuela Superior Politécnica del Litoral, Campus “Gustavo Galindo” ubicado en el Km 30,5 de la vía Perimetral de la ciudad de Guayaquil. Los resultados obtenidos muestran que los parámetros de pH, acidez titulable, grados brix, pérdida de peso retardan su desarrollo cuando están bajo la acción de temperaturas de refrigeración. De igual manera se pudo determinar que la caja de madera provoca la mayor cantidad de perdidas de tomate, en comparación con los materiales propuestos como alternativas, al finalizar el trabajo se proporcionan algunas opciones en cuanto a las operaciones realizadas en campo con el fin de aumentar la producción de tomate riñón

Alternativas de mejora en el manejo postcosecha de tomate riñón cultivados en la provincia de Santa Elena

This research was part of the study of different post field level and its influence on the life and functional quality of it. The purpose is to recommend management operations tomato post currently affecting the life loss and tomato crops from the Santa Elena Peninsula. For the development of this is implemented the use of alternative materials in the process of packing the same way we stu influence of temperature on tomato quality and better packaging was determined as the temperature where the smallest losses were quantified

9. Lichenysin production and application in the pharmaceutical field

Abstract. Lipopeptides such as lichenysin are biosurfactants of great interest, due to the demand for natural surface-active agents with low toxicity. Bacillus licheniformis AL 1.1 produces a lipopeptide characterized as lichenysin (Lch AL1.1 ), which acts as a powerful surfactant, able to reduce surface tension to 28.5 mN m -1 and with a critical micelle concentration of 15 mg L -1 . Lch AL1.1 is particularly effective in preventing biofilm formation by pathogenic strains, has an emulsifying capacity and permeabilizes membranes by a colloid-osmotic process. The production of lipopeptides from agro-industrial residues, particularly molasses, is a sustainable process of great potential for the development of economic bioprocesses

Lichenysin production and application in the pharmaceutical field

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/103042Lipopeptides such as lichenysin are biosurfactants of great interest, due to the demand for natural surface-active agents with low toxicity. Bacillus licheniformis AL 1.1 produces a lipopeptide characterized as lichenysin (LchAL1.1), which acts as a powerful surfactant, able to reduce surface tension to 28.5 mN m-1 and with a critical micelle concentration of 15 mg L-1. LchAL1.1 is particularly effective in preventing biofilm formation by pathogenic strains, has an emulsifying capacity and permeabilizes membranes by a colloid-osmotic process. The production of lipopeptides from agro-industrial residues, particularly molasses, is a sustainable process of great potential for the development of economic bioprocesses