Miguel Anxo Murado

Hyaluronic acid production by Streptococcus zooepidemicus in marine by-products media from mussel processing wastewaters and tuna peptone viscera

BackgroundHyaluronic acid is one of the biopolymers most commonly used by the pharmaceutical industry. Thus, there is an increasing number of recent works that deal with the production of microbial hyaluronic acid. Different properties and characteristics of the fermentation process have been extensively optimised; however, new carbon and protein sources obtained from by-products or cheap substrates have not yet been studied.ResultsMussel processing wastewater (MPW) was used as a sugar source and tuna peptone (TP) from viscera residue as a protein substrate for the production of hyaluronic acid (HA), biomass and lactic acid (LA) by Streptococcus zooepidemicus in batch fermentation. Commercial medium formulated with glucose and tryptone was used as the control. The parametric estimations obtained from logistic equations and maintenance energy model utilized for modelling experimental data were compared in commercial and low-cost media. Complete residual media achieved high production (3.67, 2.46 and 30.83 g l-1 of biomass, HA and LA respectively) and a high molecular weight of HA (approximately 2500 kDa). A simple economic analysis highlighted the potential viability of this marine media for reducing the production costs by more than 50%.ConclusionsThe experimental data and mathematical descriptions reported in this article demonstrate the potential of media formulated with MPW and TP to be used as substrates for HA production by S. zooepidemicus. Furthermore, the proposed equations accurately simulated the experimental profiles and generated a set of interesting parameters that can be used to compare the different bacterial cultures. To the best of our knowledge, this is the first work in which a culture media formed by marine by-products has been successfully used for microbial HA production.

Growth and metabolic features of lactic acid bacteria in media with hydrolysed fish viscera. An approach to bio-silage of fishing by-products

The reparameterization of two classic growth models (the logistic and Gompertz equations), and the dynamic modification of the integrated form of the first of these, was applied to the description of the kinetics and metabolic behaviour of six strains of lactic acid bacteria in four media: three of these from visceral waste from fishing products, and one commercial medium. The descriptions obtained -always consistent models and statistically significant parameters- provided a wide range of reliable numerical values on notable characteristics of microbial growth and bioproductions, which allowed the assessment of the individual systems by direct comparison, and also the suggesting of the potentially most suitable groups (of species of fish and of bacteria) for bio-silage processes.

Dose–response modelling with two agents: Application to the bioassay of oil and shoreline cleaning agents

Single and joint effects of hydrocarbons and a shoreline cleaning agent (SCA) were studied by measuring the inhibition of the larval growth of sea urchin. Different dosage methods of hydrophobic compounds were compared. The results obtained in the evaluation of CytoSol toxicity revealed that the method of variable dilution of water accommodated fraction (WAF) led to the more conservative toxicological approach. Regarding to Libyan oil, the use of DMSO as carrier allowed us the evaluation of its potential toxicity in comparison with the limitations imposed to the use of WAF method. A reparametrised form of the Weibull equation was slightly modified to be useful for dose-response analysis. This was the basis for modelling single sigmoid responses, which were used to simulate biphasic profiles with addition of effects and to describe both the concentration addition (CA) and independent action (IA) hypotheses. In all cases, its descriptive ability was graphically and statistically satisfactory. The IA model was the best option to explain the combined experimental responses obtained.

Mathematical Model for the Characterization and Objective Comparison of Antioxidant Activities

The available data about the interference of antioxidants in the kinetics of lipid oxidation are abundant, but often they allow only semiquantitative conclusions, not always with sufficient basis. One of the causes of this problem is the absence of formal models able to guide the experimental design and to calculate characterizing parameters. In this regard, the model which we propose allows us to obtain the simultaneous solution of a series of oxidation kinetics in the presence of any number of antioxidant concentrations. It describes satisfactorily simulations in which substrate and antioxidant compete for oxygen in a second order kinetic scheme, as well as experimental results from other authors, in different systems and under different conditions. Its application is simple, it provides parametric estimates which characterize both the oxidative process and the antioxidant activity, and it facilitates rigorous comparisons among the effects of different compounds and experimental approaches. In all experimental data tested, the calculated parameters were always statistically significant (Students t test, alpha = 0.05), the equations were consistent (Fishers F-test), and the goodness of fit parameters (adj r(2), adjusted coefficients of multiple determination) were up to 0.97.

Comparison of several mathematical models for describing the joint effect of temperature and ph on glucanex activity

The aim of the present work was to evaluate with different statistical criteria the suitability of nine equations for describing and optimizing the simultaneous effect of temperature and pH on glucanex activity using two characteristic polysaccharides (curdlan and laminarin) as substrates. The most satisfactory solutions were found with an empirical equation constituted with parameters of practical interest (Rosso model), and a hybrid model between the Arrhenius equation and the mathematical expression generated by the protonation‐hydroxylation mechanism (Tijskens model). The joint optimal values of pH and temperature calculated with the Rosso model were obtained at 4.64 and 50°C with curdlan and 4.64 and 48°C using laminarin as substrate.

Evaluation of toxic effects of several carboxylic acids on bacterial growth by toxicodynamic modelling

BackgroundEffects of organic acids on microbial fermentation are commonly tested in investigations about metabolic behaviour of bacteria. However, they typically provide only descriptive information without modelling the influence of acid concentrations on bacterial kinetics.ResultsWe developed and applied a mathematical model (secondary model) to capture the toxicological effects of those chemicals on kinetic parameters that define the growth of bacteria in batch cultures. Thus, dose-response kinetics were performed with different bacteria (Leuconostoc mesenteroides, Carnobacterium pisicola, Escherichia coli, Bacillus subtilis and Listonella anguillarum) exposed at increasing concentrations of individual carboxylic acids (formic, acetic, propionic, butyric and lactic). In all bioassays the acids affected the maximum bacterial load (Xm) and the maximum growth rate (vm) but only in specific cases the lag phase (λ) was modified. Significance of the parameters was always high and in all fermentations the toxicodynamic equation was statistically consistent and had good predictability. The differences between D and L-lactic acid effects were significant for the growth of E. coli, L. mesenteroides and C. piscicola. In addition, a global parameter (EC50,τ) was used to compare toxic effects and provided a realistic characterization of antimicrobial agents using a single value.ConclusionsThe effect of several organic acids on the growth of different bacteria was accurately studied and perfectly characterized by a bivariate equation which combines the basis of dose-response theory with microbial growth kinetics (secondary model). The toxicity of carboxylic acids was lower with the increase of the molecular weight of these chemicals.

Crocin bleaching antioxidant assay revisited: application to microplate to analyse antioxidant and pro-oxidant activities.

The crocin bleaching assay (CBA) is a common method for evaluating the antioxidant activity of hydrosoluble samples. It is criticised due to its low reproducibility, problematic quantification of results, differences in reagent preparation, doubtful need for a preheating phase and sensitivity to factors such as temperature, pH, solvents and metals. Here, the critical points of the method were extensively revised, and a highly reproducible procedure for microplate readers redeveloped. The problems of using quantification procedures, disregarding kinetic considerations, are discussed in detail and a model is proposed for quantifying simultaneously anti- and pro-oxidant activities as function of concentration and time. Thus, the combined use of a reproducible procedure and robust mathematical modeling produced consistent and meaningful criteria for comparative characterization of any oxidation modifier, taking into account the dose-time-dependent behaviour. The method was verified by characterising several commercial antioxidants and some metal compounds using the parametric values of the proposed models. The activity of the tested antioxidants decreased in the order ETX>TR>PG>AA>TBHQ>BHA. Others, such as the lipophilic antioxidants of BHT and α-Tocopherol did not show any activity. Interference from metals were for Fe(2+), Fe(3+), Cd(2+), Ni(2+), Mg(2+), Zn(2+) and Sr(2+), slightly antioxidant for Cu(1+) and Cu(2+), and strongly antioxidant for Mn(2+). None of the tested metals showed a pro-oxidant activity.

Biphasic toxicodynamic features of some antimicrobial agents on microbial growth: a dynamic mathematical model and its implications on hormesis.

BackgroundIn the present work, we describe a group of anomalous dose-response (DR) profiles and develop a dynamic model that is able to explain them. Responses were obtained from conventional assays of three antimicrobial agents (nisin, pediocin and phenol) against two microorganisms (Carnobacterium piscicola and Leuconostoc mesenteroides).ResultsSome of these anomalous profiles show biphasic trends which are usually attributed to hormetic responses. But they can also be explained as the result of the time-course of the response from a microbial population with a bimodal distribution of sensitivity to an effector, and there is evidence suggesting this last origin. In light of interest in the hormetic phenomenology and the possibility of confusing it with other phenomena, especially in the bioassay of complex materials we try to define some criteria which allow us to distinguish between sensu stricto hormesis and biphasic responses due to other causes. Finally, we discuss some problems concerning the metric of the dose in connection with the exposure time, and we make a cautionary suggestion about the use of bacteriocins as antimicrobial agents.ConclusionsThe mathematical model proposed, which combines the basis of DR theory with microbial growth kinetics, can generate and explain all types of anomalous experimental profiles. These profiles could also be described in a simpler way by means of bisigmoidal equations. Such equations could be successfully used in a microbiology and toxicology context to discriminate between hormesis and other biphasic phenomena.

Alcoholic chestnut fermentation in mixed culture. Compatibility criteria between Aspergillus oryzae and Saccharomyces cerevisiae strains

The main objective of the present work consisted in the transfer to the case of the chestnut of a rice fermentative process that carried out to the Japanese traditional way to lead to an alcoholic bagasse, the moromi, capable of obtaining distilled. This way, selection assays of amylolitic Aspergillus oryzae strains and studies of compatibility between microfungi and yeast were carried out. These mixed cultivations were performed operating in batch submerged culture. Later on, using solid state system (chestnut, microfungi, yeast), a fermentative fed-batch process (koji, moto, moromi) was defined. By means of this approach a yield of 70% was reached in the conversion of total carbohydrates in ethanol. Also, the time required by the traditional operation was reduced in half.

Effects of spill-treating agents on growth kinetics of marine microalgae.

The effects of four spill-treating agents (STAs) (CytoSol, Finasol(®) OSR 51, Agma OSD 569 and OD4000) on the growth kinetics of three marine microalgae (Isochrysis galbana, Chaetoceros gracilis, Phaeodactylum tricornutum) were studied. Chlorophyll a concentration and optical density at 700 nm were assessed to describe the logistic growth of algae in batch cultures. The optical density data were initially analyzed as described for standard algal growth inhibition tests and subsequently modelled by a bivariate model, as a function of time and dose, to assess the toxic effects on growth parameters. Increasing trends in EC50 and EC10 values with time were found with the standard approach. In 8 of the 11 tests, the lag phase (λ) or the time required to achieve half the maximum biomass (τ) was significantly dependent on the STA concentration. A global parameter (EC50,τ) was calculated to summarize the effects of STAs on growth parameters in the bivariate model. The ranking of sensitivity as EC50,τ values was I. galbana>C. gracilis>P. tricornutum. For all species tested, the least toxic agent was Agma OSD 569, followed by CytoSol. The mathematical model allowed successful ecotoxicological evaluation of chemicals on microalgal growth.