Novel and simple test plating for screening relative transfructosylation activity of fungi

Fructooligosaccharides (FOS) have received particular attention recently because of their excellent biological and functional properties, namely, as prebiotic compounds that promote the growth of intestinal microflora. They are also low calorie non-carcinogenic sweeteners with numerous suggested health benefits. These include immune system activation, resistance to infections, synthesis of Bcomplex vitamins, calcium absorption. They can be used as a treatment for breast cancer, diarrhoea, and constipation. Although FOS are present in trace amounts in fruits, vegetables and honey as natural products, its mass production is limited by seasonal restrictions and the inherent inefficiencies of these systems. Hence, microbial FOS production by fungi in bioreactors is more realistic. Several microorganisms are reported to have transfructosylation activity due to fructosyltransferase (EC 2.4.1.9) and/or fructofuranosidase (EC 3.2.1.26) activities. However, the search for other fungi with higher transfructosylation activity is still a challenge. So, a presumptive and indirect colorimetric plate assay for the evaluation of transfructosylation activity in fungi was developed by the simultaneous determination in the same plate of glucose and fructose released from sucrose. The method entailed the coupling of two dye systems, namely the glucose oxidase-peroxidase coupled reaction using phenol and 4-aminoantipyrine for determination of glucose; and the fructose dehydrogenase oxidation in the presence of a tetrazolium salt for determination of fructose. In order to have a standard assay, the fungi were grown on Czapek Dox (CD) agar. 1 disc of mycelium (8 mm diameter) was cut from the edge of each colony and then put in contact with CD agar plates. After incubation at 25 ºC for 72 h each assay plate was overlaid with soft agar containing the reagents. The presence of enzymes with transfructosylation activity was identified by the formation of pink (presence of glucose) and blue (presence of fructose) halos around the discs. In conclusion, the results showed that the method is suitable for screening a large number of fungi due to its simplicity, reproducibility and rapidity.Agência de Inovação (AdI) - project BIOLIFE ref. PRIME 03/347

Gamma Irradiation of Magnetoresistive Sensors for Planetary Exploration

A limited number of Anisotropic Magnetoresistive (AMR) commercial-off-the-shelf (COTS) magnetic sensors of the HMC series by Honeywell, with and without integrated front-end electronics, were irradiated with gamma rays up to a total irradiation dose of 200 krad (Si), following the ESCC Basic Specification No. 22900. Due to the magnetic cleanliness required for these tests a special set-up was designed and successfully employed. Several parameters of the sensors were monitored during testing and the results are reported in this paper. The authors conclude that AMR sensors without front-end electronics seem to be robust against radiation doses of up to 200 krad (Si) with a dose rate of 5 krad (Si)/hour and up to a resolution of tens of nT, but sensors with an integrated front-end seem to be more vulnerable to radiation

Prebiotics : from concept to product : the BIOLIFE project

Nutrition has progressed from the discovery of essential nutrients and prevention of dietary deficiency to the promotion of a state of well-being and health and the reduction of the risk of disease. In this context the functional food concept emerges: a food or drink product that, when ingested, exerts particular benefits on some physiological functions. The gastrointestinal functions are associated with a balanced colonic microflora that plays a key role in individual health. This complex ecosystem can be modulated by the ingestion of dietary components that favour the growth and metabolism of beneficial indigenous species such as bifidobacteria and lactobacilli. These components of saccharidic nature are called prebiotics. The BIOLIFE project involves the development of novel biotechnological processes, enzymatic and fermentative, for the production of prebiotics, namely, galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS) from food industry by-products. Produced oligosaccharides will be incorporated in liquid and solid food formulations in order to obtain functional foods targeted towards gut function.Fundação para a Ciência e a Tecnologia (FCT)

Optimization of a fructooligosaccharides purification method using activated charcoal

Fructooligosaccharides (FOS) have gained large commercial interest due to its beneficial properties in the human health as prebiotics. FOS are produced industrially by fermentative processes. However, the result of such fermentations is a complex mixture containing salts and approximately 50% (w/w) of low molecular weight sugars that have to be eliminated. Among other techniques that have been studied, the adsorption onto activated carbon is still the most suitable one since activated carbon is cheap, has a large surface area and pore volume conducting to a good sorption capacity. Furthermore, this sorbent can be regenerated during desorption with ethanol. Based on the above discussion, in this work the adsorption and desorption characteristics of FOS on activated carbon, using a gradient of ethanol, were optimized. Initially, the activated carbon was loaded with fermentative broth. To remove the non adsorbed sugars, a washing step with pure water was included. Afterwards, the retained sugars were recovered by elution with a gradient of ethanol increased sequentially with specific volumes from 1 to 50% (v/v). Fractions collected at different time points were evaporated and subsequently freeze‐dried. This process was found to be very efficient in the demineralization of broth, and it was possible to recover 80% of the initial FOS loaded on the column with 89% of purity. Some of the fractions were found to contain 97% of pure FOS in total sugars. In summary, purification of FOS using an activated charcoal column is a very efficient process yielding high levels of purity from a fermentative broth

Modeling and simulation of fructo-oligosaccharides production

It is foreseen that Systems Biology will have a great impact not only in Metabolic Engineering and Drug Discovery efforts, but also in Bioprocess development and optimization. In fact, the computational tools developed in this area made possible to simulate a biochemical process with a mathematical model comprising dynamical equations based on first principles as well as empirical kinetic equations and parameters that can be estimated from experimental data. Fructo-oligosaccharides (FOS) have become important as healthy food ingredients because of their beneficial characteristics to the health of human (Lee, 1999). They can be obtained biotechnologically using the enzyme Betafructofuranosidase, produced by some fungi. The main purpose of this work was to develop a mathematical model able to simulate the formation of FOS in a bioreactor, in order to perform a faster optimization of the FOS production process, allowing to identify which parameters can influence the final amount of FOS. An empirical model presented by Kow Jen Duan et al (1994) was used to obtain the first set of reactions. Several hydrolysis equations were added in order to achieve a general mathematical model of the occurring enzymatic reactions. After the reformulation of the Kow Jen Duan model, the kinetic parameters were determined from experimental data using the System Biology toolbox [2] with the Simulated Annealing method for curve fitting. Several experiments in bioreactors were performed with two different FOS producing fungi for that purpose. The time evolution of 7 state variables (Sucrose, Glucose, Fructose, 1-Kestose, Nystose, 1-Fructosyl Nystose and Biomass) was considered. After parameter fitting, several simulations were performed in MatLab and the simulation results were compared with experimental data, exhibiting a very satisfactory correlation for both fungal cultures. Correlation coefficients of 0.9980 - 0.9549 between simulated and experimental data were obtained. This model will be used to optimize the production process, using an optimization tool that has been developed in our research group

Purification of fructo-oligosaccharides by adsorption onto charcoal filters

Production of fructo-oligosaccharides has received particular attention recently due to its beneficial effects as prebiotics. Fermentative processes appear to be a good technique for large scale production of FOS. However, the result of such fermentations is a complex mixture containing salts and low molecular weight sugars that do not contribute to the beneficial properties of the higher molecular weight oligosaccharides, and so have to be eliminated. Among the several strategies being studied to solve this problem, the adorption onto activated carbon is a promising technique that still requires further development. Thus, the aim of this work was the purification of FOS from a fermentation broth using charcoal filters. Microfiltration membranes (0,20 micrometers) were successfully used to separate the biomass; nevertheless salts and other small sugars had to be eliminated using a charcoal filter. Hence, 200 mL of fermentative broth (without cells) was passed through the filter. Subsequently, the filter was washed with water removing the salts and small sugars present in the mixture. The oligosaccharides adsorbed onto the charcoal filter were recovered using a 44% ethanol solution. An increased purity, from 30% to 97% (w/w) in total sugars, was achieved using this downstream process, as compared to the initial mixture. The results gathered in this work suggest that charcoal filters are a promising technology for the purification of FOS from fermentation mixtures at an industrial scale, making clear the need for further work in order to optimize this process