Ruptura celular, extração e encapsulamento de Astaxantina de Haematococcus pluvialis (Volvocales, Chlorophyta)

O interesse na produção de astaxantina de fontes naturais vem aumentando significativamente, devido principalmente à sua capacidade como potente agente antioxidante. Na obtenção da astaxantina por via biotecnológica, a microalga Haematococcus pluvialis é um dos micro-organismos industrialmente mais interessantes. Entretanto, como a maioria dos carotenoides, a astaxantina é uma molécula altamente insaturada que pode ser facilmente degradada por processos térmicos. Em função desta instabilidade, uma possibilidade que se abre, a fim de proteger sua atividade biológica de fatores ambientais e reforçar a sua estabilidade física, é o encapsulamento. Neste sentido, este trabalho vem contribuir em inovações relacionadas ao desenvolvimento de tecnologia para ruptura celular, extração e nanoencapsulamento de astaxantina produzida por via biotecnológica, mais especificamente de astaxantina obtida através do cultivo de H. pluvialis. Neste estudo, os cultivos foram realizados em meio BBM e acetato de sódio e conduzidos a temperatura constante de 25±1 ºC em fotobiorreatores de 1 L com aeração por borbulhamento de ar de 300 mL.min-1 , agitação manual diária e sob iluminância constante de 444 µmol fótons.m-2 s -1 durante 15 dias, sendo inoculados com suspensão de microalgas previamente preparada, na proporção de 10%, e pH ajustado em 7,0. A biomassa foi recuperada dos cultivos por centrifugação e seca a 35 °C por 48 h. Em seguida, foram empregadas diferentes técnicas de ruptura celular (química, mecânica e enzimática). Após a ruptura, foi realizada a extração dos carotenoides e a quantificação dos carotenoides totais (µg.g-1 ) e da extratibilidade (%). Entre os solventes testados no método de ruptura química, o diclorometano foi o selecionado para a extração dos pigmentos carotenoides. Dentre as técnicas mecânicas de ruptura celular, a maceração da biomassa congelada com terra diatomácea resultou na maior extratibilidade e carotenoides totais (66,01% e 972,35 μg.g-1 ). A melhor condição de lise da parede celular de H. pluvialis, utilizando o preparado enzimático Glucanex® , ocorreu em pH do meio reacional de 4,5 a 55 ºC, com atividade inicial de β-1,3-glucanase de 0,6 U.mL-1 e um tempo de reação de 30 min, alcançando-se 17,73% de atividade lítica relativa. Nestas condições, com a reação enzimática assistida por ultrassom sem congelamento prévio da biomassa, atingiu-se 83,90% e 1235,89 µg.g -1 , respectivamente, para extratibilidade e carotenoides totais. Dentre as técnicas combinadas testadas, a maceração com terra diatomácea associada à lise enzimática apresentou valores de extratibilidade e carotenoides totais de, respectivamente, 93,83% e 1382,12 µg.g-1 . No encapsulamento do extrato contendo astaxantina obtido por lise enzimática associada por ultrassom, envolvendo a coprecipitação com PHBV (poli(3-hidroxibutirato-cohidroxivalerato)) em fluidos supercríticos, o aumento da pressão tendeu a reduzir o diâmetro da partícula formada, enquanto que o aumento da relação biomassa contendo astaxantina:diclorometano usada na etapa de extração incrementou o percentual de encapsulamento e a eficiência de encapsulamento para ambas pressões testadas (80 e 100 bar). Os maiores valores de percentual de encapsulamento (17,06%) e eficiência de encapsulamento (51,21%) foram obtidos nas condições de 80 bar e relação biomassa:diclorometano de 10 mg.mL -1 . Nestas condições, o diâmetro médio de partícula foi de 0,228 µm. Com base nos resultados obtidos, técnicas para a obtenção de astaxantina de H. pluvialis e seu encapsulamento foram desenvolvidas com sucesso, podendo ser extendidas a outros produtos intracelulares de microalgas.

Encapsulation of Local Anesthetic Bupivacaine in Biodegradable Poly(DL-lactide-co-glycolide) Nanospheres: Factorial Design, Characterization and Cytotoxicity Studies

Local anesthetic agents cause temporary blockade of nerve impulses productiong insensitivity to painful stimuli in the area supplied by that nerve. Bupivacaine (BVC) is an amide-type local anesthetic widely used in surgery and obstetrics for sustained peripheral and central nerve blockade. in this study, we prepared and characterized nanosphere formulations containing BVC. To achieve these goals, BVC loaded poly(DL-lactide-co-glycolide) (PLGA) nanospheres (NS) were prepared by nanopreciptation and characterized with regard to size distribution, drug loading and cytotoxicity assays. The 2(3-1) factorial experimental design was used to study the influence of three different independent variables on nanoparticle drug loading. BVC was assayed by HPLC, the particle size and zeta potential were determined by dynamic light scattering. BVC was determined using a combined ultrafiltration-centrifugation technique. The results of optimized formulations showed a narrow size distribution with a polydispersivity of 0.05%, an average diameter of 236.7 +/- 2.6 nm and the zeta potential -2.93 +/- 1,10 mV. In toxicity studies with fibroblast 3T3 cells, BVC loaded-PLGA-NS increased cell viability, in comparison with the effect produced by free BVC. In this way, BVC-loaded PLGA-NS decreased BVC toxicity. The development of BVC formulations in carriers such as nanospheres could offer the possibility of controlling drug delivery in biological systems, prolonging the anesthetic effect and reducing toxicity.

Conjugation of bioactive groups to poly(lactic acid) and poly[(lactic acid)-co-(glycolic acid)] films

A novel PLA-based polymer containing reactive pendent ketone or hydroxyl groups was synthesized by the copolymerization of L-lactide with epsilon-caprolactone-based monomers. The polymer was activated with NPC, resulting in an amine-reactive polymer which was then cast into thin polymeric films, either alone or as part of a blend with PLGA, before immersion into a solution of the cell adhesion peptide GRGDS in PBS buffer allowed for conjugation of GRGDS to the film surfaces. Subsequent 3T3 fibroblast cell adhesion studies demonstrated an increase in cellular adhesion and spreading over films cast from unmodified PLGA. Hence the new polymer can be used to obtain covalent linkage of amine-containing molecules to polymer surfaces.

Thermal and mechanical properties of poly(butylene terephthalate)/epoxy blends

In this study, melt blends of poly(butylene terephthalate) (PBT) with epoxy resin were characterized by dynamic mechanical analysis, differential scanning calorimetry, tensile testing, Fourier transform infrared spectroscopy, and wide-angle X-ray diffraction. The results indicate that the presence of epoxy resin influenced either the mechanical properties of the PBT/epoxy blends or the crystallization of PBT. The epoxy resin was completely miscible with the PBT matrix. This was beneficial to the improvement of the impact performance of the PBT/epoxy blends.

Role of Nonplanar Peptide Unit in Regular Polypeptide Helices - New Model for Pply-Beta-Benzyl-L-Aspartate

The effect of non-planarity of the peptide unit on helical structures stabilized by intrachain hydrogen bonds is discussed. While the present calculations generally agree with those already reported in the literature for right-handed helical structures, it is found that the most stable left-handed structure is a novel helix, called the delta-helix. Its helical parameters are close to these reported for poly-beta-benzyl-L -aspartate. Conformational energy calculations show that poly-beta-benzyl-L -aspartate with the delta-helical structure is considerably more stable than the structure it is generally believed to take up (the omega-helix) by about 15 kcal/mol-residue.

Effect of nucleating agent on the isothermal crystallization kinetics of PHBV

The crystallization behavior of PHBV, poly(beta -hydroxybutyrate-co-beta -hydrxyvalerate), with nucleating agents under isothermal conditions was investigated. A differential scanning calorimeter was used to monitor the crystallization process from the melt. During isothermal crystallization, the dependence of relative degree of crystallinity on time was described by the Avrami equation. It has been shown that the addition of BN and Tale causes a considerable increase in the overall crystallization rate of PHBV but does not influence the Avrami exponent n, mechanism of nucleation and spherulite growth mode of PHBV. A little of nucleating agent will increase the crystallization rate and decrease the fold surface free energy sigma (e), remarkably. The effect of BN is more significant than that of Talc.

Polyhydroxyalkanoates production by actinobacteria isolated from soil

Polyhydroxyalkanoates (PHAs) are biodegradable and renewable polymers produced by a wide range of bacterial groups. New microbial bioprospection approaches have become an important way to find new PHA producers and new synthesized polymers. Over the past years, bacteria belonging to actinomycetes group have become known as PHA producers, such as Nocardia and Rhodococcus species, Kineosphaera limosa Liu et a]. 2002, and, more recently, Streptomyces species. In this paper, we disclose that there are more actinobacteria PHA producers in addition to the genera cited. Some unusual genera, such as Streptoalloteichus, and some genera frequently present in soil, such as Streptacidiphilus, have been found. Thirty-four isolates were able to accumulate poly(3-hydroxybutyrate) and a number of these have traces of poly(3-hydroxyvalerate) when cultivated on glucose or glucose and casein as carbon source. Furthermore, some strains showed traces of medium chain length PHA. Transmission electron microscopy demonstrated that the PHA accumulation occurs in hyphae and spores.

Block Copolymers Containing (R)-3-Hydroxybutyrate and Isosorbide Succinate or (S)-Lactic Acid Mers

A new aliphatic block copolyester was synthesized in bulk from transesterification techniques between poly((R)-3-hydroxybutyrate) (PHB) and poly(isosorbide succinate) (PIS). Additionally, other two block copolyesters were synthesized in bulk either from transesterification reactions involving PHB and poly(l-lactide) (PLLA) or from ring-opening copolymerization of l-lactide and hydroxyl-terminated PHB, as result of a previous transesterification reactions with isosorbide. Two-component blends of PHB and PIS or PLLA were also prepared as comparative systems. SEC, MALDI-TOF mass spectrometry (MALDI-TOFMS), (1)H and (13)C NMR spectroscopy, WAXD, solubility tests, and TG thermal analysis were used for characterization. The block copolymer structures of the products were evidenced by MALDI-TOFMS, (13)C NMR, and WAXD data. The block copolymers and the corresponding binary blends presented different solubility properties, as revealed by solubility tests. Although the incorporation of PIS sequences into PHB main backbone did not enhance the thermal stability of the product, it reduced its crystallinity, which could be advantageous for faster biodegradation rate. These products, composed of PHB and PIS or PLLA sequences, are an interesting alternative in biomedical applications.

Efeitos do calor e da ação microbiana em filmes de PHBV / PP-co-PE aditivados em solo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Biodegradação de filmes de polietileno de baixa densidade (PEBD), polihidroxibutirato-co-hidroxivalerato (PHBV) e blenda de PEBD/PHBV (70/30), em coluna de solo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Understanding the mechanical and biodegradation behaviour of poly(hydroxybutyrate)/rubber blends in relation to their morphology

In this work poly(hydroxybutyrate/poly(vinyl butyral)- co-(vinyl alcohol)-co(vinyl acetate) (or ethylene propylene diene monomer rubber) blends were prepared by conventional processing techniques (extrusion and injection moulding). A droplet type morphology was obtained for P(3HB)/PVB blends whereas P(3HB)/EPDM blends presented some extent of co-continuous morphology. In addition, rubbery domains were much smaller in the case of PVB. These differences in morphology are discussed taking into account solubility parameters and rheological behaviours of each component. For both blends, the increase of elastomer ratio led to a decrease of Young's modulus but an increase in elongation at break and impact strength. The latter increased more in the case of P(3HB)/EPDM blends although the rubbery domains were larger. These results are explained in the light of the glass transition of the rubber and the presence of plasticizer in the case of PVB. The addition of elastomer also resulted in an increase of P(3HB) biodegradation rate, especially in the case of EPDM. It is assumed that, in this case, the size and morphology of the rubbery domains induce a geometrical modification of the erosion front which leads to an increase of the interface between P(3HB) phase and the degradation medium and consequently to an apparently faster biodegradation kinetics of PHB/rubber blends. Copyright (C) 2011 Society of Chemical Industry

Novel three-component blends based on poly(L-lactide)

A significant number of poly a-ester homologues of poly(L-lactide) (PLLA) have been synthesized and used in miscibility studies together with conventional isomeric diacid-diol polyester variants, poly ß-esters (based on ß-hydroxybutyrate (HB) and ß-hydroxyvalerate (HV)), poly e-caprolactone (PCL), poly e-caprolactone copolymers (e.g. poly(L-lactide-co-caprolactone), and a series of cellulose-based polymers (e.g. cellulose acetate butyrate (CAB), cellulose acetate propionate (CAP)). A combinatorial approach to rapid miscibility screening using 96-well plates and a uv-visible multi-wavelength plate reader has been developed enabling the clarity of PLLA-based multi-component blend films to be observed. Using these techniques and materials, the ternary phase compatibility diagrams of a range of three-component blend films was prepared, illustrating ranges of behavior varying from miscible blends giving rise to clear films to immiscible blends which are opaque. In this way, novel three-component blends of PLLA/CAB/PCL were developed which are miscible when the CAB content is more than 30%, PLLA less than 80% and PCL less than 60%.

Properties of lignin and poly(hydroxybutyrate) blends

The Queensland University of Technology (QUT) allows the presentation of a thesis for the Degree of Doctor of Philosophy in the format of published or submitted papers, where such papers have been published, accepted or submitted during the period of candidature. This thesis is composed of Seven published/submitted papers and one poster presentation, of which five have been published and the other two are under review. This project is financially supported by the QUTPRA Grant. The twenty-first century started with the resurrection of lignocellulosic biomass as a potential substitute for petrochemicals. Petrochemicals, which enjoyed the sustainable economic growth during the past century, have begun to reach or have reached their peak. The world energy situation is complicated by political uncertainty and by the environmental impact associated with petrochemical import and usage. In particular, greenhouse gasses and toxic emissions produced by petrochemicals have been implicated as a significant cause of climate changes. Lignocellulosic biomass (e.g. sugarcane biomass and bagasse), which potentially enjoys a more abundant, widely distributed, and cost-effective resource base, can play an indispensible role in the paradigm transition from fossil-based to carbohydrate-based economy. Poly(3-hydroxybutyrate), PHB has attracted much commercial interest as a plastic and biodegradable material because some its physical properties are similar to those of polypropylene (PP), even though the two polymers have quite different chemical structures. PHB exhibits a high degree of crystallinity, has a high melting point of approximately 180°C, and most importantly, unlike PP, PHB is rapidly biodegradable. Two major factors which currently inhibit the widespread use of PHB are its high cost and poor mechanical properties. The production costs of PHB are significantly higher than for plastics produced from petrochemical resources (e.g. PP costs $US1 kg-1, whereas PHB costs $US8 kg-1), and its stiff and brittle nature makes processing difficult and impedes its ability to handle high impact. Lignin, together with cellulose and hemicellulose, are the three main components of every lignocellulosic biomass. It is a natural polymer occurring in the plant cell wall. Lignin, after cellulose, is the most abundant polymer in nature. It is extracted mainly as a by-product in the pulp and paper industry. Although, traditionally lignin is burnt in industry for energy, it has a lot of value-add properties. Lignin, which to date has not been exploited, is an amorphous polymer with hydrophobic behaviour. These make it a good candidate for blending with PHB and technically, blending can be a viable solution for price and reduction and enhance production properties. Theoretically, lignin and PHB affect the physiochemical properties of each other when they become miscible in a composite. A comprehensive study on structural, thermal, rheological and environmental properties of lignin/PHB blends together with neat lignin and PHB is the targeted scope of this thesis. An introduction to this research, including a description of the research problem, a literature review and an account of the research progress linking the research papers is presented in Chapter 1. In this research, lignin was obtained from bagasse through extraction with sodium hydroxide. A novel two-step pH precipitation procedure was used to recover soda lignin with the purity of 96.3 wt% from the black liquor (i.e. the spent sodium hydroxide solution). The precipitation process is presented in Chapter 2. A sequential solvent extraction process was used to fractionate the soda lignin into three fractions. These fractions, together with the soda lignin, were characterised to determine elemental composition, purity, carbohydrate content, molecular weight, and functional group content. The thermal properties of the lignins were also determined. The results are presented and discussed in Chapter 2. On the basis of the type and quantity of functional groups, attempts were made to identify potential applications for each of the individual lignins. As an addendum to the general section on the development of composite materials of lignin, which includes Chapters 1 and 2, studies on the kinetics of bagasse thermal degradation are presented in Appendix 1. The work showed that distinct stages of mass losses depend on residual sucrose. As the development of value-added products from lignin will improve the economics of cellulosic ethanol, a review on lignin applications, which included lignin/PHB composites, is presented in Appendix 2. Chapters 3, 4 and 5 are dedicated to investigations of the properties of soda lignin/PHB composites. Chapter 3 reports on the thermal stability and miscibility of the blends. Although the addition of soda lignin shifts the onset of PHB decomposition to lower temperatures, the lignin/PHB blends are thermally more stable over a wider temperature range. The results from the thermal study also indicated that blends containing up to 40 wt% soda lignin were miscible. The Tg data for these blends fitted nicely to the Gordon-Taylor and Kwei models. Fourier transform infrared spectroscopy (FT-IR) evaluation showed that the miscibility of the blends was because of specific hydrogen bonding (and similar interactions) between reactive phenolic hydroxyl groups of lignin and the carbonyl group of PHB. The thermophysical and rheological properties of soda lignin/PHB blends are presented in Chapter 4. In this chapter, the kinetics of thermal degradation of the blends is studied using thermogravimetric analysis (TGA). This preliminary investigation is limited to the processing temperature of blend manufacturing. Of significance in the study, is the drop in the apparent energy of activation, Ea from 112 kJmol-1 for pure PHB to half that value for blends. This means that the addition of lignin to PHB reduces the thermal stability of PHB, and that the comparative reduced weight loss observed in the TGA data is associated with the slower rate of lignin degradation in the composite. The Tg of PHB, as well as its melting temperature, melting enthalpy, crystallinity and melting point decrease with increase in lignin content. Results from the rheological investigation showed that at low lignin content (.30 wt%), lignin acts as a plasticiser for PHB, while at high lignin content it acts as a filler. Chapter 5 is dedicated to the environmental study of soda lignin/PHB blends. The biodegradability of lignin/PHB blends is compared to that of PHB using the standard soil burial test. To obtain acceptable biodegradation data, samples were buried for 12 months under controlled conditions. Gravimetric analysis, TGA, optical microscopy, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), FT-IR, and X-ray photoelectron spectroscopy (XPS) were used in the study. The results clearly demonstrated that lignin retards the biodegradation of PHB, and that the miscible blends were more resistant to degradation compared to the immiscible blends. To obtain an understanding between the structure of lignin and the properties of the blends, a methanol-soluble lignin, which contains 3× less phenolic hydroxyl group that its parent soda lignin used in preparing blends for the work reported in Chapters 3 and 4, was blended with PHB and the properties of the blends investigated. The results are reported in Chapter 6. At up to 40 wt% methanolsoluble lignin, the experimental data fitted the Gordon-Taylor and Kwei models, similar to the results obtained soda lignin-based blends. However, the values obtained for the interactive parameters for the methanol-soluble lignin blends were slightly lower than the blends obtained with soda lignin indicating weaker association between methanol-soluble lignin and PHB. FT-IR data confirmed that hydrogen bonding is the main interactive force between the reactive functional groups of lignin and the carbonyl group of PHB. In summary, the structural differences existing between the two lignins did not manifest itself in the properties of their blends.

Mechanical and biological characterization of a porous poly-L-lactic acid-co-ε-caprolactone scaffold for tissue engineering

This article presents a method for making highly porous biodegradable scaffold that may ultimately be used for tissue engineering. Poly(L-lactic-co-1-caprolactone) acid (70:30) (PLCL) scaffold was produced using the solvent casting/leaching out method, which entails dissolving the polymer and adding a porogen that is then leached out by immersing the scaffold in distillated water. Tensile tests were performed for three types of scaffolds, namely pre-wetted, dried, and UV-irradiated scaffolds and their mechanical properties were measured. The prewetted PLCL scaffold possessed a modulus of elasticity 0.92+0.09 MPa, a tensile strength of 0.12+0.03 MPa and an ultimate strain of 23+5.3%. No significant differences in the modulus elasticity, tensile strength, nor ultimate strain were found between the pre-wetted, dried, and UV irradiated scaffolds. The PLCL scaffold was seeded by human fibroblasts in order to evaluate its biocompatibility by Alamar bluew assays. After 10 days of culture, the scaffolds showed good biocompatibility and allowed cell proliferation. However, the fibroblasts stayed essentially at the surface. This study shows the possibility to use the PLCL scaffold in dynamic mechanical conditions for tissue engineering