Physical–chemical parameters and validation of a colorimetric method for deoxycholic and ursodeoxycholic acids: kit reagent and optical sensor

AbstractThe simple and low cost β-cyclodextrin (β-CD)–phenolphthalein (PHP) inclusion complex was used for both the study of physical–chemical parameters and validation of analytical procedures for deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) determinations in different formulations. The usefulness of this inclusion complex is proposed either in the form of kit reagent and as an original optical sensor for DCA and UDCA. The results showed that temperature had a negative effect on the equilibrium constant resulting in high negative values of enthalpy and positive values of entropy. The half-life values for DCA and UDCA measurements were 68.71 and 294.71 days, respectively. The method was validated showing limits of detection and quantification of 4.92×10−5molL−1 and 1.64×10−4molL−1 for DCA, 1.14×10−5molL−1 and 3.79×10−5molL−1 for UDCA, respectively. The developed optical sensor also showed response linearity, ease of implementation and potential application in fast screening tasks even out of the laboratory

The microbial culture collections of the Federal University of Pernambuco (UFPE) and the new consortium towards the establishment of BRC-UFPE

The UFPE from Recife in Brazil hosts a bacterial (UFPEDA) and a fungal (URM) collections since 1951 and 1954, respectively. The UFPEDA was established by Prof. Oswaldo Gonçalves de Lima and is register in WDCM as 114. It is hosted at Antibiotic Department (DA) of UFPE and started out with 200 species mainly of the genus Streptomyces. Nowadays this collection holds 4000 strains of actinomycetes isolated from all the Brazilian places and from the International Streptomyces Project (ISP). The URM – University of Recife Mycology was established by Prof. Augusto Chaves Batista and is register in WDCM as 604. Actual it holds 9000 identified species including 1400 yeasts and 7600 filamentous fungi. All major fungal taxonomic groups are cover by this collection. The collections preserve each strain at least by two different techniques. Water and mineral oil storage were used for long operation time while freeze-drying and freezing at -80 ºC become the main techniques used at this stage. Special care is taken to test whether cultures recovered from preserved material conform to the original deposit. These collections have a range of services which are acceptance of free and confidential deposits, supply strains for academia, industry and services, support research and education (graduate and post-graduate students, as well as advanced training courses), identification services and confidential contracts (e.g. fungal medical diagnosis, starters for agro-industry companies, etc.). The OECD initiative related to guidance for the operation of Biological Resource Centres (BRC) is now a key reference for these collections. The right management of biological resources and their associate information including quality control are perused by these collections. The recent national projects, with reasonable budgets to support their activities, either on networking activities or requalification and management create a new breath and responsibilities to these collections. Taking advantage of good and well equipped premises of LIKA these collections are now open new avenues working in consortium to improve the quality control of their holdings using new tools from molecular biology and spectral analysis (MALDI-TOF) to achieve in the future a certified BRC for the UFPE microbial culture collections

Production and characterization of protease from Penicillium aurantiogriseum URM 4622

Proteases with new properties are required due to their increasing industrial importance. In this work, the optimal fermentation conditions for the production of a protease from Penicillium aurantiogriseum dierchx (URM-4622) are presented together with partial characterization of the protease catalytic properties. The batch fermentation conditions that allow for the highest specific proteolytic activity are 26 ºC, pH 7.0, and 25 % saturation dissolved O2 concentration. The obtained protease is stable over a wide range of pH (5.8 to 9.5) and temperature (25 to 40 ºC) values. In the presence of Zn2+ a 26 % reduction in the enzyme proteolytic activity occurs and, in contrast, Mn2+ enhances its activity by 28.9 %. 96.2 % and 70.8 % of the protease activity are maintained after 90 min incubation in 5 and 10 % (v/v) H2O2 aqueous solutions, respectively. PMSF inhibition reveals that this enzyme is a serine protease. Protease is able to hydrolyze different proteins

Optimization of clavulanic acid production by Streptomyces daufpe 3060 by response surface methodology

Clavulanic acid is a β-lactam antibiotic which has a potent β-lactamase inhibiting activity. In order to optimize its production by the new isolate Streptomyces DAUFPE 3060, the influence of two independent variables, temperature and soybean flour concentration, on clavulanic acid and biomass concentrations was investigated in 250 mL-Erlenmeyers according to a 2² central composite design. To this purpose, temperature and soybean flour (SF) concentration were varied in the ranges 26-34°C and 10-50 g/L, respectively, and the results evaluated utilizing the Response Surface Methodology. The experimental maximum production of clavulanic acid (629 mg/L) was obtained at 32°C and 40 g/L SF after 48 h, while the maximum biomass concentration (3.9 g/L) at 30°C and 50 g/L soybean flour, respectively. These values are satisfactorily close to those (640 mg/L and 3.75 g/L, respectively) predicted by the model, thereby demonstrating the validity of the mathematical approach adopted in this study.Brazilian Research Funding InstitutionsCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

Influence of pH on cellular growth of Pichia pastoris KM71H by fed-batch process

Pichia pastoris is a methylotrophic yeast that can be genetically engineered to express proteins for industrial use. One of the most important advantages of protein expression in P. pastoris is its capability of growing on minimal medium and efficiently secreting heterologous proteins with low secretion levels of endogenous proteins. Operational variables such as pH, temperature, stirring rate, among others, usually affect the microorganism’s growth during the fermentation processes. Therefore, the present work aimed to evaluate the influence of pH on cellular growth of P. pastoris KM71H by fed‐batch process. The fermentation run was carried out in a 1.6 L (total volume) bioreactor, being performed in two phases: In the first stage (24 h), the yeast was batchcultured in BMGH medium; while in the second stage (72 h), it was cultivated by feed‐batch operation with a feeding medium containing 50% glycerol and 12ml/l of trace metal solution. During the overall process, which lasted after 96 h, the aeration and temperature conditions were fixed at 10 ml\L.h, 1.5 vvm and 30°C, respectively. Different pH values were evaluated: 5.0, 5.5 and 6.0. Cellular growth was determined by measuring the fermentation broth UVspectrophotometric absorbance at 600 nm, which was correlated to a calibration curve (dry weight ´ optical density). Glycerol consumption was detected by HPLC analysis. P. pastoris KM71H successfully grew in all the evaluated pH values; but the highest biomass production was observed at pH 5.0 (98.79 g/L). Although P. pastoris is reported as being a microorganism able to grow over a wide pH range (from 3 to 7); it was not observed high cell density of P. pastoris KM71H strain when cultivated at pHs 5.5 and 6.0. High cellular growth is especially important for proteins secretion, as the concentration of product in the medium is roughly proportional to the concentration of cells in culture. Finally, these results reveal the possibility of obtaining high cell density of P. pastoris KM71H by fed‐bach cultivation at pH 5.0, which can be a suitable condition for the yeast application in heterologous proteins production.Conselho Nacional de Desenvolvimento Científico e Tecnológico Brazil (CNPq)Improving Skills Across Continents (ISAC ) - Erasmus Mundus External Cooperation Window (ERASMUS

Regenerative collagen biomembrane: Interim results of a Phase I veterinary clinical trial for skin repair [version 1; referees: 2 approved, 1 approved with reservations]

Background: The availability of commercial tissue engineering skin repair products for veterinary use is scarce or non-existent. To assess features of novel veterinary tissue engineered medical devices, it is therefore reasonable to compare with currently available human devices. During the development and regulatory approval phases, human medical devices that may have been identified as comparable to a novel veterinary device, may serve as predicate devices and accelerate approval in the veterinary domain. The purpose of the study was to evaluate safety and efficacy of the biomembrane for use in skin repair indications. Methods: In the study as a whole (3 year total length), 15 patients (animals), dogs and cats (male/female, 2 cm), with a wound depth equivalent to 2nd/3rd degree burns are to be studied from Day 0 to Day 120-240, post-application of the biomembrane. This interim report covers the 5 patients assessed to date and deemed eligible, of which 3 enrolled, and 2 have completed the treatment. Wound beds were prepared and acellular collagen biomembranes (Eva Scientific Ltd, São Paulo, Brazil) applied directly onto the wounds, and sutured at the margins to the patient's adjacent tissue. Wound size over time, healing rate, general skin quality and suppleness were assessed as outcomes. Qualitative (appearance and palpation) and quantitative (based on Image Analysis of photographs) wound assessment techniques were used. Results: Both patients’ wounds healed fully, with no adverse effects, and the healing rate was comparable in both, maxing out at approximately 1 cm2/day. Conclusions: Early results on the biomembrane's safety and efficacy indicate suitability for skin repair usage in veterinary patients

Production and characterization of protease from Penicillium aurantiogriseum URM 4622

Abstract Proteases with new properties are required due to their increasing industrial importance. In this work, the optimal fermentation conditions for the production of a protease from Penicillium aurantiogriseum dierchx (URM-4622) are presented together with partial characterization of the protease catalytic properties. The batch fermentation conditions that allow for the highest specific proteolytic activity are 26 ºC, pH 7.0, and 25 % saturation dissolved O 2 concentration. The obtained protease is stable over a wide range of pH (5.8 to 9.5) and temperature (25 to 40 ºC) values. In the presence of Zn 2+ a 26 % reduction in the enzyme proteolytic activity occurs and, in contrast, Mn 2+ enhances its activity by 28.9 %. 96.2 % and 70.8 % of the protease activity are maintained after 90 min incubation in 5 and 10 % (v/v) H 2 O 2 aqueous solutions, respectively. PMSF inhibition reveals that this enzyme is a serine protease. Protease is able to hydrolyze different proteins