ABSTRACT
Objetivo: se diseñó un medio de cultivo a base de peptonas vegetales para la producción de biomasa de Clostridium chauvoei. Métodos: se seleccionaron nueve peptonas vegetales y dos caldos con peptona vegetal para sustituir la peptona de origen animal en el medio Clostridium modificado. Se desarrolló una fermentación relacionada con la producción de masa celular, verificando la absorbancia y el porcentaje de transmitancia (%T), la segunda etapa involucró los ensayos de patogenicidad, determinando el titulo de DL50. Resultados: las peptonas vegetales 1-B, 8-M, 10-M, 11-M fueron seleccionadas por presentar un % T cercano al del medio Clostridium modificado. Luego se realizó una prueba de letalidad (DL50) en cobayos. Los cultivos con estas peptonas vegetales presentaron DL50 entre diluciones 10-8 y 10-9, en contraste con los cultivos realizados con el medio control que fue de 106.5 Conclusiones: las peptonas vegetales 1-B, 8-M, 10-M y 11-M favorecieron la producción de biomasa de Clostridium chauvoei a pH 8,2. Las peptonas vegetales probadas mostraron una letalidad mayor comparada con la del medio base.
Objective: a culture medium based on vegetable peptones was designed for the production of Clostridium chauvoei biomass.Methods: nine vegetable peptones and two vegetable peptone broths were selected to substitute animal peptone in the modified Clostridium medium. A fermentation process was developed which was related to the production of cell mass, and absorbance and transmittance percentage (%T) were verified. The second stage included pathogenicity assays to determine LD50 titers. Results: vegetable peptones 1-B, 8-M, 10-M, 11-M were selected, for their % T was close to that of the modified Clostridium medium. A lethality test (LD50) was then performed on guinea pigs. Cultures with these vegetable peptones presented LD50 between 10-8 and 10-9 dilutions, in contrast to the 106.5 obtained in cultures with the control medium. Conclusions: vegetable peptones 1-B, 8-M, 10-M and 11-M stimulated the production of Clostridium chauvoei biomass at pH 8.2. The vegetable peptones tested showed greater lethality than the base medium.
ABSTRACT
S. pneumoniae é um importante patógeno humano que afeta sobretudo crianças. Vacinas são a principal estratégia de combate e o polissacarídeo (PS) da cápsula é o antígeno. Dentre os sorotipos do patógeno, o tipo 1 é prevalente em crianças e o PS1 componente obrigatório das vacinas. Neste trabalho objetivou-se: estabelecer um método de dosagem do PS1, selecionar a melhor cepa produtora de PS1, avaliar 3 peptonas, investigar 4 componentes do meio de cultura em planejamento experimental (PE) em reator e propor um novo meio e estratégia de cultivo. A cepa ST595/01 foi selecionada e o Phytone (Phy) foi a peptona com maior produção de PS1, 298mg/L, medido por m-hidroxidifenil com sulfamato. O PE 24-1 com extrato de levedura (EL), Phy, Asn e Gln mostrou os maiores efeitos positivos do Phy para produção de biomassa (Cx) e PS1. EL foi positivo para Cx, Asn não apresentou efeitos e Gln menor efeito positivo para PS1. Assim, testou-se o novo meio com Phy 15g/L e EL 2g/L sob duas estratégias: a batelada alimentada superou a batelada simples em 2x para Cx e 2,5x para PS1.
S. pneumoniae is an important human pathogen that affects mainly children. Vaccines are the main strategy to fight the disease and capsular polysaccharide (PS) is the antigen. Among pneumococcal serotypes, type 1 is prevalent in children. The aims of this work were: establish a method to measure PS1, screen strains for the best PS1 producer, evaluate 3 peptones, investigate the effects of 4 medium components by a design of experiment (DoE) and propose a new medium/strategy for cultivation. The strain ST595/01 was selected. Phytone (Phy) was the peptone with the highest PS1 production (298mg/L, measured by m-hydroxydiphenyl + sulfamate method). DoE 24-1 was carried out to assess the effects of yeast extract (YE), Phy, Asn and Gln. Phy showed the major positive effects for biomass (Cx) and PS1. YE demonstrated effects for Cx. Gln showed minor effect on PS1 production and Asn did not present effects. A new medium based on 15g/L Phy and 2g/L YE was evaluated by 2 strategies: fed-batch showed Cx production 2-fold higher and PS1 2.5-fold higher than simple batch.
Subject(s)
Humans , Child , Culture Media , Streptococcus pneumoniae , Pneumococcal Vaccines/immunology , BrazilABSTRACT
Las bases nutritivas empleadas para el cultivo microbiano deben reunir una serie de características esenciales como aportantes de nutrientes, en especial, de componentes nitrogenados de origen proteico, tales como aminoácidos, péptidos y polipéptidos; macroelementos y microelementos, así como vitaminas y carbohidratos. Otro grupo de indicadores comprende características fisicoquímicas como pH, densidad aparente, propiedades organolépticas (color) y tecnológicas (rendimiento). Los índices biológicos y sanitarios son fundamentales para su funcionalidad en los medios, entre ellos, la reactividad biológica, que incluye la producción de indol, acetilmetilcarbinol y gas sulfhídrico y presencia de carbohidratos fermentables; el límite microbiano y la promoción del crecimiento de microorganismos específicos.
Nutritive bases for microbial growth, as nutrient sources, should comply with a group of essential characteristics, especially as sources of nitrogenous compounds derivedfrom proteins, such as aminoacids, peptides andpolypeptides, as well as sources of vitamins andcarbohydrates. Other group of characteristics includesphysico-chemical parameters such as pH, apparentdensity, organoleptic (color) and technological (yield)properties. Biological and sanitary characteristics are fundamentalfor their funcionality in culture media, amongthem, the biological reactivity that includes indole, acethylmethylcarbinol and hydrogen sulphide productionand the presence of fermentable carbohydrates, microbiallimit and the growth promotion of specific microorganisms.
Subject(s)
Culture Media , Peptones , Amino Acids , Nutrients , Micronutrients , Nitrogen , Sodium ChlorideABSTRACT
Los medios de cultivo pueden ser definidos como conjuntos de elementos o sustancias que garantizan a los microorganismos u otras células los nutrientes necesarios para su conservación o desarrollo. Las bases nutritivas son ingredientes fundamentales de muchos de estos medios y aportan, para el crecimiento de los microorganismos, los compuestos nitrogenados de naturaleza proteica, tales como péptidos, polipéptidos, aminoácidos, vitaminas, carbohidratos y otros elementos nutritivos. Se obtienen mediante extracción o hidrólisis del material biológico, por diferentes vías, seguido de clarificación mediante la filtración, concentración, secado y tamizado. Especial interés ocupa la búsqueda de diferentes sustratos proteicos alternativos, debido al incremento de la frecuencia de los brotes de encefalopatía espongiforme bovina y fiebre aftosa en diferentes países, el peligro de transmisión de estas enfermedades al hombre a través de las proteínas derivadas de los sub productos de los animales y las regulaciones para el comercio, importación y uso de estos productos.
Culture media could be defined as a group of elements or substances, which provide, to microorganisms or toother cells, the necessary nutrients for their preservation or growth. Nutritive bases are main ingredients for muchof these media and they provide for the growth of microorganisms, protein derived nitrogen compoundssuch as peptides, polypeptides, aminoacids; vitamins, carbohydrates and other nutritive elements. They areobtained by extraction or hydrolysis of biological rawmaterial by different ways, and then undergo processesof clarification, filtration, concentration, drying andsieving. It is of great interest to identify different alternative protein substrates, due to the increase of Bovine Spongiform Encephalopathy and Aftose Feveroutbreaks in different countries, the possible spread ofthese diseases to humans by proteins derived from animalby-products and due to trading, importation and consumption regulations for these products.