Importance of the agar-media in the evaluation of bacteriocin activity against the same test-microorganisms

Abstract Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria, which exhibit a large spectrum of action against spoilage bacteria and foodborne pathogens. Successful application of techniques for quantitative or qualitative bacteriocin determination relies not only on the sensitivity of the test-microorganisms, but also on the agar-medium employed. Cell free supernatants are routinely used to preliminary screen for antimicrobial activity of bacteria by means of the agar well diffusion method, but the supernatant may also include other molecules (such as medium components and/or intracellular compounds) accidentally released during cell free supernatant preparation, which may interfere with the assay. Reproducibility of bacteriocin activity against the same test-microorganisms is an important factor to be considered. Unfortunately, no specific information about bioassays standardization to determine bacteriocin activity is available in the literature. In this work, growth inhibition by means of the agar well diffusion assays were carried out on different agar-media showing a strong dependence on the agar-medium used, indicating that the inhibitory effects could also depend on the diffusion of exudates that are included in the cell-free supernatant. The results presented in this communication show that selection of the agar-medium is crucial for the bioassay response.

Kinetic analysis of batch ethanol acetylation in isothermal non-stationary multiphase systems by lyophilized mycelium of Aspergillus oryzae

A relatively complex network of reactions has been investigated, using as a network model the isothermal batch esterification of acetic acid with ethanol in n-heptane catalyzed by lyophilized mycelium of Aspergillus oryzae. The kinetic analysis was firstly carried out on the whole system, without any simplification, by means of the well-known integral method. Owing to the poor results obtained by this way, we developed an alternative approach, combining initial rates and integral analysis and reducing the number of empirical parameters to be determined by the use of equilibrium data. All the values of the parameters calculated according to this "composite" approach to kinetic analysis well correlate with experimental data.

Bio-synthesis and hydrolysis of ethyl phenylacetate and ethyl 2-phenylpropionate in organic solvent by lyophilized mycelia

To select the best biocatalysts for ethanol acylations with phenylacetic and 2-phenylpropionic acids, lyophilized mycelia of Aspergillus oryzae CBS 10207, A. oryzae MIM, Rhizopus oryzae CBS 11207, R. oryzae CBS 39134, R. oryzae CBS 26028 and R. oryzae CBS 32847 were tested in this study. The carboxylesterase activities of A. oryzae MIM and R. oryzae 11207, which revealed to be the best biocatalysts, were investigated either in 0.1 M phosphate buffer or in n-heptane to catalyze the hydrolysis or the synthesis of ethyl esters of these acids, respectively. A. oryzae proved more effective than R. oryzae, probably due to more favorable microenvironment conditions and thermodynamic scenario. The results in terms of product formation and substrate consumption versus time were used to estimate the maximum conversion yields, the equilibrium constants and the times needed to reach half maximum conversion, thus providing sufficient information about these equilibria.

Micélios liofilizados de Aspergillus oryzae CBS 10207, A. oryzae MIM, Rhizopus oryzae CBS 11207, R. oryzae CBS 39134, R. oryzae CBS 26028 e R. oryzae CBS 32847 foram testados neste estudo com vista à seleção do melhor biocatalisador para efetuar a acilação de etanol com ácidos fenilacético e 2-fenilpropiônico. As atividades carboxilesterásicas de A. oryzae MIM e R. oryzae 11207, que resultaram ser os melhores biocatalisadores, foram investigadas tanto em tampão fosfato 0,1 M como em n-heptano para catalisar a hidrólise ou a síntese dos ésteres etílicos destes ácidos. A. oryzae pareceu ser mais eficaz que R. oryzae, provavelmente devido a condições micro-ambientais e a um cenário termodinâmico mais favoráveis. Os resultados obtidos em termos de formação do produto e consumo dos substratos em função do tempo foram usados para a estimativa dos rendimentos de conversão máximos, as constantes de equilíbrio e os tempos necessários para alcançar metade da conversão máxima, fornecendo desta forma suficientes informações sobre esses equilíbrios.