Optimizaciónde las variables tecnológicas en la producción de un probiótico para camarones

Aim: To optimize the technological variables engaged in the manufacture of a probiotic for shrimp. Methods: This research was based on experimental designs oriented to phenomenological mathematical modeling of the process to identify the main design variables. The product cost sheet was used to create the economic model that fits such variables. The objective-function was to minimize the unit cost of the product. Restrictions of design variables were established, and their optimal values were estimated through the use of multi-criteria computer optimizing tools. Results: The initial sucrose concentration produced in the culture medium was 132 g/L, at a shaking speed of 1.67 s-1, air flow equal to 0.025 L/L.h, and fermentation time 8.5 h, which minimized the unit cost of the product to 5.31 $/L of product. This value was quite below$ 31.50/L, which is the price of imported probiotic Epicin, used to breed shrimp larvae. Conclusions: The sale of this product produces a revenue of $478 483.20/year to the Center of Genetic Engineering and Biotechnology of Camagüey, leading to savings of approximately$ 40 000 US yearly in savings for Yaguacam Basic Production Company, through import substitution. Additionally, its use has led to a reduction in antibiotic use, increased quality and quantity of post larvae, and an overall positive impact for the company.Objetivo: Optimizar las variables tecnológicas que inciden en el proceso de producción de un probiótico para camarones. Métodos: Se emplearon diseños experimentales orientados a la modelación matemática fenomenológica del proceso para identificar las variables de diseño fundamentales. Se utilizó la ficha de costo del producto para confeccionar el modelo económico en función de estas variables. Se definió como función objetivo minimizar el costo unitario del producto. Se establecieron las restricciones de las variables de diseño y se determinaron sus valores óptimos mediante el empleo de herramientas computacionales de optimización multicriterio. Resultados:Se obtuvo que una concentración inicial de sacarosa en el medio de cultivo igual a 132 g/L, una velocidad de agitación de 1,67 s-1, un flujo de aire igual a 0,025 L/L.h y un tiempo de fermentación de 8,5 h minimizan el costo unitario del producto hasta 5,31 $/L de producto, valor muy inferior a los 31,50$/L que es el precio del probiótico de importación Epicin que se emplea en la cría de larvas de camarones. Conclusiones: La venta del producto genera una ganancia neta de 478 483,20 $/año para el Centro de Ingeniería Genética y Biotecnología de Camagüey, un ahorro de cerca de 40 000 USD anuales a la Unidad Empresarial de Base Yaguacam por concepto de sustitución de importaciones y su uso ha permitido la disminución del empleo de antibióticos, el incremento de la calidad y cantidad de las postlarvas y un impacto económico positivo en la entidad. &nbsp

Optimización de las variables tecnológicas en la producción de un probiótico para camarones

Objetivo: Optimizar las variables tecnológicas que inciden en el proceso de producción de un probiótico para camarones. Métodos: Se emplearon diseños experimentales orientados a la modelación matemática fenomenológica del proceso para identificar las variables de diseño fundamentales. Se utilizó la ficha de costo del producto para confeccionar el modelo económico en función de estas variables. Se definió como función objetivo minimizar el costo unitario del producto. Se establecieron las restricciones de las variables de diseño y se determinaron sus valores óptimos mediante el empleo de herramientas computacionales de optimización multicriterio. Resultados: Se obtuvo que una concentración inicial de sacarosa en el medio de cultivo igual a 132 g/L, una velocidad de agitación de 1,67 s-1, un flujo de aire igual a 0,025 L/L.h y un tiempo de fermentación de 8,5 h minimizan el costo unitario del producto hasta 5,31 $/L de producto, valor muy inferior a los 31,50$/L que es el precio del probiótico de importación Epicin que se emplea en la cría de larvas de camarones. Conclusiones: La venta del producto genera una ganancia neta de 478 483,20 $/año para el Centro de Ingeniería Genética y Biotecnología de Camagüey, un ahorro de cerca de 40 000 USD anuales a la Unidad Empresarial de Base Yaguacam por concepto de sustitución de importaciones y su uso ha permitido la disminución del empleo de antibióticos, el incremento de la calidad y cantidad de las postlarvas y un impacto económico positivo en la entidad

Influencia de aditivos en humectabilidad del bionematicida HeberNem-S obtenido mediante secado por atomización

Brevibacterium celere C-924 is a microorganism whose nematicidal activity has been demonstrated, through which powder formulations (HeberNem-S) have been obtained for application in protected cultivation houses. The objective of this work was to carry out sieving studies to determine the factors and additives that most influence the wettability of the HeberNem-S product during the spray-drying operation. A response surface experiment was elaborated where the following factors were evaluated: sucrose concentration, soluble solids of the exhausted fermentation medium, synchronous speed of the atomizing disc and the dry matter of the cream formulated on the wettability of the HeberNem-S product. The results of the experiment showed that the variable that most influences the wettability of the powder is the concentration of soluble solids from the spent fermentation medium that is added in the formulation. Based on these obtained results, the influence of 16 additives was evaluated, among which are sugars, gums, detergents and surfactants. It was determined that the additives that most influence wettability are soy lecithin and Glanapon, achieving a reduction in the wettability time of 19.18 times and 17.5 times, respectively. The result obtained is of vital importance for subsequent studies to optimize the composition of HeberNem-S, in order to guarantee continuous improvements in the production process and the physical properties of the powder. Keywords: Additives, Glanapon, Wettability, Soy Lecithin, Spray Drying, Brevibacterium celere C-924.Brevibacterium celere C-924 es un microorganismo cuya actividad nematicida ha sido demostrada, mediante el cual se ha obtenido formulaciones en polvo (HeberNem-S) para su aplicación en casas de cultivo protegidas. El objetivo de este trabajo consistió en realizar estudios de tamizado para determinar los factores y aditivos que más influencia tienen en la humectabilidad del producto HeberNem-S durante la operación de secado por atomización. Se realizó un experimento de superficie de respuesta dónde se evaluaron los siguientes factores: concentración de sacarosa, sólidos solubles del medio de fermentación agotado, velocidad sincrónica del disco atomizador y la materia seca de la crema formulada sobre la humectabilidad del producto HeberNem-S. Los resultados del experimento demostraron que la variable que más influye en la humectabilidad del polvo es la concentración de sólidos solubles del medio de fermentación agotado que se añade en la formulación. Con base en estos resultados obtenidos se evaluó la influencia de 16 aditivos, dentro de los cuales se encuentran azúcares, gomas, detergentes y agentes tensoactivos. Se determinó que los aditivos que más influyen en la humectabilidad son la lecitina de soya y el Glanapon, logrando una reducción del tiempo de humectabilidad de 19,18 veces y 17,5 veces, respectivamente. El resultado obtenido es de vital importancia para estudios posteriores de optimización de la composición del HeberNem-S, con el fin de garantizar las mejoras continuas del proceso de producción y las propiedades físicas del polvo. &nbsp

A Smartphone-Based System for Automated Bedside Detection of Crackle Sounds in Diffuse Interstitial Pneumonia Patients

In this work, we present a mobile health system for the automated detection of crackle sounds comprised by an acoustical sensor, a smartphone device, and a mobile application (app) implemented in Android. Although pulmonary auscultation with traditional stethoscopes had been used for decades, it has limitations for detecting discontinuous adventitious respiratory sounds (crackles) that commonly occur in respiratory diseases. The proposed app allows the physician to record, store, reproduce, and analyze respiratory sounds directly on the smartphone. Furthermore, the algorithm for crackle detection was based on a time-varying autoregressive modeling. The performance of the automated detector was analyzed using: (1) synthetic fine and coarse crackle sounds randomly inserted to the basal respiratory sounds acquired from healthy subjects with different signal to noise ratios, and (2) real bedside acquired respiratory sounds from patients with interstitial diffuse pneumonia. In simulated scenarios, for fine crackles, an accuracy ranging from 84.86% to 89.16%, a sensitivity ranging from 93.45% to 97.65%, and a specificity ranging from 99.82% to 99.84% were found. The detection of coarse crackles was found to be a more challenging task in the simulated scenarios. In the case of real data, the results show the feasibility of using the developed mobile health system in clinical no controlled environment to help the expert in evaluating the pulmonary state of a subject

Conformational and thermal stability improvements for the large-scale production of yeast-derived rabbit hemorrhagic disease virus-like particles as multipurpose vaccine.

Recombinant virus-like particles (VLP) antigenically similar to rabbit hemorrhagic disease virus (RHDV) were recently expressed at high levels inside Pichia pastoris cells. Based on the potential of RHDV VLP as platform for diverse vaccination purposes we undertook the design, development and scale-up of a production process. Conformational and stability issues were addressed to improve process control and optimization. Analyses on the structure, morphology and antigenicity of these multimers were carried out at different pH values during cell disruption and purification by size-exclusion chromatography. Process steps and environmental stresses in which aggregation or conformational instability can be detected were included. These analyses revealed higher stability and recoveries of properly assembled high-purity capsids at acidic and neutral pH in phosphate buffer. The use of stabilizers during long-term storage in solution showed that sucrose, sorbitol, trehalose and glycerol acted as useful aggregation-reducing agents. The VLP emulsified in an oil-based adjuvant were subjected to accelerated thermal stress treatments. None to slight variations were detected in the stability of formulations and in the structure of recovered capsids. A comprehensive analysis on scale-up strategies was accomplished and a nine steps large-scale production process was established. VLP produced after chromatographic separation protected rabbits against a lethal challenge. The minimum protective dose was identified. Stabilized particles were ultimately assayed as carriers of a foreign viral epitope from another pathogen affecting a larger animal species. For that purpose, a linear protective B-cell epitope from Classical Swine Fever Virus (CSFV) E2 envelope protein was chemically coupled to RHDV VLP. Conjugates were able to present the E2 peptide fragment for immune recognition and significantly enhanced the peptide-specific antibody response in vaccinated pigs. Overall these results allowed establishing improved conditions regarding conformational stability and recovery of these multimers for their production at large-scale and potential use on different animal species or humans

Analysis of the recombinant VLP extracted from vaccine formulations subjected to an accelerated stress treatment experiment.

<p>(<b>A</b>) Analysis in size-exclusion HPLC. The temperature at which the emulsion was kept for one week and the retention times corresponding to elution of multimeric VP1 are indicated in each chromatogram. The inner figures show Western blot analyses and detection of VP1 in the main peaks detected, which were designated as 1 and 2 according to their appearance in the chromatogram. An anti-RHDV hyperimmune serum was used. The 60 kDa band is indicated. (<b>B</b>) Electron micrographs of negatively stained samples containing the extracted VLP. The VLP structure was analyzed by TEM using 2% uranyl acetate. The bar indicates 200 nm. Magnification: x40 000. (<b>C</b>) Detection of RHDV VLP epitopes with the conformational-sensitive monoclonal antibodies 1H8, 6F9, 6H6, 5B2, and 6G2 after thermal stresses. Standard deviation bars are indicated.</p

Recovery of properly assembled RHDV VLP during the evaluation of different pH in the disruption and purification processes.

<p>Purification was performed by size exclusion chromatography using a preparative TSK G3000 column. Alternatively, an additional step known to induce instability and protein aggregation (rounds of freezing-thawing) was included. Quantification of recovered VLP was accomplished by sandwich ELISA with the use of mAbs.</p><p>VLP directly detected after cell disruption and clarification were expressed in the first raw as µg per mL of disruption supernatant. Starting from those values, percents of recovery are shown for the next two steps. All measurements were done in duplicate starting from two independent bioreactions. Protein amounts and percents of recovery refer only to properly assembled VLP.</p

Analysis of the RHDV VLP purified under different conditions.

<p>(<b>A</b>) Chromatographic profile of RHDV VLP after disruption and purification conducted at different pH values. Purification was performed by sec-HPLC using a TSK G-3000 preparative column. In all cases multimeric VP1 was detected in the first peak and quantified by sandwich ELISA using monoclonal antibodies. Retention times are indicated. (<b>B</b>) Electron micrographs of negatively stained samples containing VP1 multimers obtained after separation in sec-HPLC. According to measurements by ELISA, the highest recoveries were obtained at pH 4 and 7. The morphology and size of the VLP was analyzed by TEM using 2% uranyl acetate for negative stain. The bars indicate 200 nm. Magnifications: x30 000 and x40 000. (<b>C</b>) SDS-PAGE (lanes 1, 2) and Western blot analysis (lanes 3, 4) of purified VLP. Lane 1 corresponds to disruption supernatant from <i>P. pastoris</i> PVP12 strain. Lanes 2 and 3 were loaded with purified RHDV VLP. A minor band migrating close to 60 kDa was also detected in lane 3. For comparison, lane 4 shows glycosylated VP1 expressed associated to the yeast membranous system in PVP11 strain, in which various protein bands were recognized by the hyperimmune serum. The molecular weight of approximately 60 kDa is shown in the left and indicated by an arrow in the right. (<b>D</b>) Analysis with conformation dependent monoclonal antibodies of epitopes located on the VLP obtained after cell disruption, purification, and rounds of stressing steps at pH values of 4 and 7. RHDV VLP and native RHDV were detected using mAbs 1H8, 6F9, 5B2, and 6G2. Standard deviation bars of measurements performed are indicated.</p