José Carlos Andrade

Regulation of Carbon and Electron Flow in Clostridium butyricum VPI 3266 Grown on Glucose-Glycerol Mixtures

The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146-6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H(+) ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.

Effect of cryoprotectants on the porosity and stability of insulin-loaded PLGA nanoparticles after freeze-drying

PLGA nanoparticles are useful to protect and deliver proteins in a localized or targeted manner, with a long-term systemic delivery pattern intended to last for a period of time, depending on polymer bioerosion and biodegradability. However, the principal concern regarding these carriers is the hydrolytic instability of polymer in aqueous suspension. Freeze-drying is a commonly used method to stabilize nanoparticles, and cryoprotectants may be also used, to even increase its physical stability. The aim of the present work was to analyze the influence of cryoprotectants on nanoparticle stability and porosity after freeze-drying, which may influence protein release and stability. It was verified that freeze-drying significantly increased the number of pores on PLGA-NP surface, being more evident when cryoprotectants are added. The presence of pores is important in a lyophilizate to facilitate its reconstitution in water, although this may have consequences to protein release and stability. The release profile of insulin encapsulated into PLGA-NP showed an initial burst in the first 2 h and a sustained release up to 48 h. After nanoparticles freeze-drying the insulin release increased about 18% in the first 2 h due to the formation of pores, maintaining a sustained release during time. After freeze-drying with cryoprotectants, the amount of insulin released was higher for trehalose and lower for sucrose, glucose, fructose and sorbitol comparatively to freeze-dried PLGA-NP with no cryoprotectant added. Besides the porosity, the ability of cryoprotectants to be adsorbed on the nanoparticles surface may also play an important role on insulin release and stability.

Effect of freeze-drying, cryoprotectants and storage conditions on the stability of secondary structure of insulin-loaded solid lipid nanoparticles.

This study aims to monitor the secondary structure behaviour of insulin when it is encapsulated into solid lipid nanoparticles (SLN), under the influence of several critical processing parameters. Insulin was used as a therapeutic protein model. Physicochemical properties of insulin-loaded SLN (Ins-SLN) were assessed, with special focus on the insulin secondary structure after its encapsulation into SLN and after freeze-drying using different cryoprotectants (glucose, fructose and sorbitol). Additionally, a 6-month stability study was performed to evaluate the maintenance of insulin secondary structure over time at different storage conditions (4 °C/60% RH, 25 °C/60% RH, 40 °C/75% RH). Ins-SLN were successfully produced with a mean and narrow particle size around 400 nm, zeta potential around -13 mV, an insulin association efficiency of 84%. Physical-chemical properties of SLN were maintained after freeze-drying. FTIR results showed that encapsulated insulin maintained a native-like structure in a degree of similarity around 92% after production, and 84% after freeze-drying. After 6 months, freeze-dried Ins-SLN without cryoprotectant stored at 40 °C/75% RH presented the same degree of structure preservation and morphology. Results revealed that insulin structure can be significantly protected by SLN matrix itself, without a cryoprotectant agent, even using a non-optimized freeze-drying process, and under the harsher storage conditions. Multivariable experimental settled the process parameters to fit with the desired product quality attributes regarding protein and nanoparticle stability.

Antioxidative Peptides: Trends and Perspectives for Future Research

In recent years, much attention has been given to dietary antioxidants, especially polyphenols. Several peptides derived from protein molecules have also been found to show antioxidant capacity along with other biological properties and thus there is an increasing interest in these compounds as health promoters. This review summarizes and discusses the main sources of antioxidative peptides with focus on food-derived peptides (animal, plant and marine sources), methods of preparation, antioxidant capacity evaluation as well as their proposed mechanisms of action. A discussion of the potential health effects and comments on the different applications for these antioxidants and their potential research interest are also subject of this review.

Drug-delivery systems of green tea catechins for improved stability and bioavailability.

Numerous studies in humans, animal models and cell lines have suggested the potential benefits from the consumption of green tea polyphenols, including prevention of cancer and heart diseases. However these potential effects have been strongly limited by green tea catechins low bioavailability, which hinders the development of therapeutic applications. In this review formulations that are being proposed for delivery of green tea catechins are discussed. New delivery systems are presented as valid alternatives to overcome the limitations such as green tea catechins poor stability or intestinal absorption.

Preparation methods and applications behind alginate-based particles

ABSTRACT Introduction: Alginate-based particles have emerged as one of the most extensively searched drug delivery platforms due to their inherent properties, including good biocompatibility and biodegradability. Moreover, the low price, easy availability, natural origin, versatility and sol-gel transition properties, make alginate an ideal candidate to produce particles with different applications. Several techniques have been developed and optimized to prepare microparticles and nanoparticles in order to achieve more rational, coherent, efficient and cost-effective procedures. Alginate represents a suitable choice concerning delivery systems’ safety, and therefore alginate-based particles have shown to be useful in the field of drug delivery with a special focus on biological encapsulants. Area covered: This review will provide an overview of alginate-based delivery systems, covering the innovative preparation methods of the last decade, the advantages and disadvantages of the most used methods, their wide diversity of applications and safety concerns. Expert opinion: The progression of nanotechnology over the last decades has stimulated the refinement of former microencapsulation methods and the exploration of new approaches towards the submicron scale with increased attention being focused on the safety of nanoparticles and product performance. Therefore, the design and optimization of the preparation methods of alginate-based microparticles and nanoparticles as well as their nontoxicity, biocompatibility and biodegradability to reach the desired application have been widely explored.

Chitosan-Based Nanoparticles as Delivery Systems of Therapeutic Proteins

Therapeutic proteins represent a significant part of the new pharmaceuticals coming on the market every year and are now widely spread in therapy to treat or relief symptoms related to many metabolic and oncologic diseases. The parenteral route remains as a primary strategy for protein administration essentially due to its specific physicochemical properties. However, the research on alternative nonparenteral delivery routes continues. The high molecular weight (MW), hydrophilicity, and charged nature of therapeutically valued proteins render transport through membranes very difficult. In this regard, chitosan arises as a promising candidate for the development of protein-containing drug formulations, due to its exceptional biological properties. Chitosan-based delivery systems have been proposed as valid approaches to provide protective conditions to proteins from denaturation and loss of activity, during preparation and delivery, as well as during long-term storage of the prepared formulation. In this chapter, one production method of a chitosan-based nanoparticle formulation is presented, as well as several characterization techniques to assess both nanoparticles and proteins characteristics and stability.

Biotechnological Production of Conjugated Fatty Acids With Biological Properties

Abstract Conjugated derivatives of fatty acids, namely conjugated linoleic acid (CLA) and conjugated α-linolenic acid (CLNA), have attracted much attention of the scientific community over the last two decades due to their biological properties. In fact, several studies realized in animal models and/or cell cultures have shown anticarcinogenic, antiobesity, antiatherogenic, antidiabetic, and immunomodulatory activities. CLA and CLNA isomers are commonly present in ruminant’s derived foods mainly because of the action of microorganisms on linoleic acid (LA) and α-linolenic acid (LNA), respectively. However, the natural concentrations of CLA and CLNA found in these food products do not seem to be sufficient to have any significant therapeutic effect and thus there are efforts to obtain CLA- and/or CLNA-enriched foods. Several food-grade microorganisms, such as bifidobacteria, lactic acid bacteria (LAB), and propionibacteria, are capable of producing CLA and CLNA from LA and LNA, respectively, given their linoleate isomerase activity. These microorganisms could thus be used to produce CLA-enriched foods either as starter or adjunct cultures or as biocatalysts producing CLA and/or CLNA that can be used as natural food additives. This chapter presents a comprehensive outlook of the biotechnological production of CLA and CLNA and a discussion of its technical issues, limitations, challenges, and potential food and nutraceutical applications based on nutritional value and biological properties.