Waste soybean frying oil for the production, extraction, and characterization of cell-wall-associated lipases from Yarrowia lipolytica.

The lipolytic yeast Yarrowia lipolytica produces cell-wall-associated lipases, namely Lip7p and Lip8p, that could have interesting properties as catalyst either in free (released lipase fraction-RLF) or cell-associated (cell-bound lipase fraction-CBLF) forms. Herein, a mixture of waste soybean frying oil, yeast extract and bactopeptone was found to favor the enzyme production. Best parameters for lipase activation and release from the cell wall by means of acoustic wave treatment were defined as: 26 W/cm2 for 1 min for CBLF and 52 W/cm2 for 2 min for RLF. Optimal pH and temperature values for lipase activity together with storage conditions were similar for both the free enzyme and cell-associated one: pH 7.0; T = 37 °C; and > 70% residual activity for 60 days at 4, - 4 °C and for 15 days at 30 °C.

Production of bioactive films of carboxymethyl cellulose enriched with green coffee oil and its residues.

Carboxymethyl cellulose (CMC)-based films were developed by incorporating green coffee oil (GCO) obtained by cold pressing and hydroalcoholic extracts of its residues. The effect of cake (CE) and sediment extracts (SE) in different proportions (20-40%) and GCO on chemical, morphological, physical, mechanical, optical, and antioxidant properties of the films was investigated. Eight fatty acids and four major phenolic compounds were identified by High-Resolution Direct-Infusion Mass Spectrometry in GCO and residue extracts. FTIR indicated interactions among CMC, phenolic compounds, and fatty acids. Films enriched with residue extracts presented heterogeneous microstructure. The tensile strength of the films decreased from 58 to 3 MPa with the extracts concentration, while elongation increased from 28 to 156% (p < 0.05). The water vapor permeability (averaging 3.94 × 10-8 g mm/cm2 h Pa) was not significantly affected by the extracts and GCO. The surface color was influenced by the type and concentration of extracts (p < 0.05), the film with SE40% had remarkable UV-vis barrier properties. The incorporation of GCO residue extracts imparted high antioxidant capacity to the CMC-based films, especially with CE40% (643.8 µmol Trolox eq./g dried film; 51.3 mg GAE/g dried film). General observations indicated the potential of these films, mainly the ones containing CE, like active packaging material for food applications.

Microspheres of alginate encapsulated minocycline-loaded nanocrystalline carbonated hydroxyapatite: therapeutic potential and effects on bone regeneration.

Background and objective: Tetracycline and its derivatives, combined with calcium phosphates, have been proposed as a delivery system to control inflammatory processes and chronic infections. The objective of this study was to evaluate the microspheres of alginate encapsulated minocycline-loaded nanocrystalline carbonated hydroxyapatite (CHAMINO) as a biomimetic device to carry out target-controlled drug delivery for alveolar bone repair. Methods: CHAMINO microspheres were implanted in a rat central incisor socket after 7 and 42 days. New bone was formed in both groups between 7 and 42 days of implantation. However, the bone growth was significantly higher for the CHAMINO microspheres. Results: The minocycline (MINO) loading capacity of the nanocrystaline carbonated hydroxyapatite (CHA) nanoparticles was 25.1±2.2 µg MINO/mg CHA for adsorption over 24 hrs. The alginate microspheres containing minocycline-loaded CHA were biologically active and inhibited the Enterococcus faecalis culture growth for up to seven days of the MINO release. An osteoblastic cell viability assay based on the resazurin reduction was conducted after the cells were exposed to the CHAMINO powder and CHAMINO microspheres. Thus, it was found that the alginate extracts encapsulated the minocycline-loaded CHA microspheres and did not affect the osteoblastic cell viability, while the minocycline-doped CHA powder reduced the cell viability by 90%. Conclusion: This study concluded that the alginate microspheres encapsulating the minocycline-loaded nanocrystalline carbonated hydroxyapatite exhibited combined antibacterial activity against Enterococcus faecalis with cytocompatibility and osteoconduction properties. The significant improvement in the new bone formation after 42 days of implantation suggests that the CHAMINO microsphere has potential in clinical applications of bone regeneration.

Blueberry Residue Encapsulation by Ionotropic Gelation.

In the processing of fruits such as blueberry (Vaccinium sp), that has high levels of phenolic acid, the food industry produces tons of organic waste that causes harm to the environment. Encapsulation is a technique used to take advantage of these wastes. Several methods are used to encapsulate substances, among them ionotropic gelation proves to be a simple, precise, efficient and economical method for obtaining particles with encapsulated bioactives. In this manner, the aim of this study was to test sodium alginate as wall material to encapsulate blueberry residue by ionotropic gelation. The microbeads were characterized by scanning electron microscopy (SEM), x-ray diffraction (XRD), total phenolic compounds, antioxidant capacity and in vitro dissolution. The results showed that the microbeads had surface invagination; retention of 67.01% of the phenolic compounds after encapsulation and 68.2%, phenolic release 120 min after in vitro dissolution. The results suggest that the tested matrix was suitable for encapsulation. The produced microbeads are promising for applications in food products, once the phenolic compounds present in the blueberry residues were maintained after encapsulation.

Studies on the volatile fraction composition of three native Amazonian-Brazilian fruits: Murici (Byrsonima crassifolia L., Malpighiaceae), bacuri (Platonia insignis M., Clusiaceae), and sapodilla (Manilkara sapota L., Sapotaceae).

The volatile fraction of murici, bacuri and sapodilla are here studied because of their increasing interest for consumers, abundance of production in Brazil, and the general demand for new flavors and aromas. Their volatile profiles were studied by two High Concentration Capacity Headspace techniques (HCC-HS), Headspace Solid Phase Microextraction (HS-SPME) and Headspace Sorptive Extraction (HSSE), in combination with GC-MS. Murici volatile fraction mainly contains esters (38%), carboxylic acids (19%), aldehydes (11%), alcohols (14%), others (13%) and sulfur compounds; bacuri is characterized by terpenes (41%), non-terpenic alcohols (24%), esters (15%), aldehydes (6%), and others (12%); sapodilla consists of esters (33%), alcohols (27%), terpenes (18%) and others (21%). The GC-MS component co-elution was overcome by GC×GC-qMS. The adoption of modern analysis technologies afforded to achieve a better knowledge of the volatile fraction composition of these fruit pulps by increasing substantially the number of compounds identified.

Chlorhexidine-loaded hydroxyapatite microspheres as an antimicrobial delivery system and its effect on in vivo osteo-conductive properties.

Hydroxyapatite (HA) has been investigated as a delivery system for antimicrobial and antibacterial agents to simultaneously stimulate bone regeneration and prevent infection. Despite evidence supporting the bactericidal efficiency of these HA carriers, few studies have focused on the effect of this association on bone regeneration. In this work, we evaluated the physico-chemical properties of hydroxyapatite microspheres loaded with chlorhexidine (CHX) at two different concentrations, 0.9 and 9.1 µgCHX/cm2 HA, and characterized their effects on in vitro osteoblast viability and bone regeneration. Ultraviolet-visible spectroscopy, scanning and transmission electron microscopy associated with energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to characterize the association of CHX and HA nanoparticles. The high CHX loading dose induced formation of organic CHX plate-like aggregates on the HA surface, whereas a Langmuir film was formed at the low CHX surface concentration. Quantitative evaluation of murine osteoblast viability parameters, including adhesion, mitochondrial activity and membrane integrity of cells exposed to HA/CHX extracts, revealed a cytotoxic effect for both loading concentrations. Histomorphological analysis upon implantation into the dorsal connective tissues and calvaria of rats for 7 and 42 days showed that the high CHX concentration induced the infiltration of inflammatory cells, resulting in retarded bone growth. Despite a strong decrease in in vitro cell viability, the low CHX loading dose did not impair the biocompatibility and osteoconductivity of HA during bone repair. These results indicate that high antimicrobial doses may activate a strong local inflammatory response and disrupt the long-term osteoconductive properties of CHX-HA delivery systems.

Oxidative stability and sensory evaluation of microencapsulated flaxseed oil.

Cold pressed flaxseed oil was microencapsulated by spray drying using an emulsion containing modified starch. The fatty acid composition, moisture, water activity, wettability, water holding capacity, water solubility, crystallinity, and particle size distribution of the microcapsules were determined. The stability of the microcapsules and the crude oil were assessed. An acceptance test was used for the sensory evaluation of a powdered supplement containing the microcapsules. The fatty acid composition was not significantly affected by the microencapsulation. The moisture, water activity, wettability, water solubility and crystallinity were appropriate for dry powders. The microcapsules had no cracks and showed better oxidative stability compared with the crude oil. Storage under vacuum prevented oxidation of the microcapsules. In sensory evaluation, all quality scores of the supplement containing microcapsules were mid-range or higher. The microencapsulation improved the oxidative stability of the oil and this procedure was satisfactorily applied in powdered food.

Production of grape juice powder obtained by freeze-drying after concentration by reverse osmosis

This study aimed to evaluate the freeze-drying process for obtaining grape juice powder by reverse osmosis using 50% grape juice pre-concentrated (28.5 °Brix) and 50% hydrocolloids (37.5% maltodextrin and 12.5% arabic gum). The morphology of the glassy food showed the absence of crystalline structure, which was the amorphous wall that protected the contents of the powder. The samples were stored in clear and dark containers at room temperature, evaluated for their physical (X-ray diffraction) for 65 days and chemical (polyphenol content) stability for 120 days. During the storage time in plastic vessels, samples remained physically stable (amorphous) and the phenolic concentration was constant, indicating the potentiality of this technique to obtain a stable product with a high concentration of phenolic compounds. Therefore, the freeze-drying process promoted the encapsulation of concentrated grape juice increasing its stability and shelf life, as well as proving to be an applicable process to food industry.

Characterization of antibiotic-loaded alginate-OSA starch microbeads produced by ionotropic pregelation.

The aim of this study was to characterize the penicillin-loaded microbeads composed of alginate and octenyl succinic anhydride (OSA) starch prepared by ionotropic pregelation with calcium chloride and to evaluate their in vitro drug delivery profile. The beads were characterized by size, scanning electron microscopy (SEM), zeta potential, swelling behavior, and degree of erosion. Also, the possible interaction between penicillin and biopolymers was investigated by differential scanning calorimetry (DSC), powder X-ray diffraction (XRD), and Fourier transform infrared (FTIR) analysis. The SEM micrograph results indicated a homogeneous drug distribution in the matrix. Also, based on thermal analyses (TGA/DSC), interactions were detected between microbead components. Although FTIR spectra of penicillin-loaded microbeads did not reveal the formation of new chemical entities, they confirmed the chemical drug stability. XRD patterns showed that the incorporated crystalline structure of penicillin did not significantly alter the primarily amorphous polymeric network. In addition, the results confirmed a prolonged penicillin delivery system profile. These results imply that alginate and OSA starch beads can be used as a suitable controlled-release carrier for penicillin.

Study of trans-trans farnesol effect on hyphae formation by Yarrowia lipolytica.

Dimorphism is an ability of certain fungi related to its adaptation to the environment and provides a selective advantage under stress conditions and is associated to the development of human diseases. Hyphae inducing- and inhibitory-effect of farnesol on hyphae formation by the dimorphic yeast Yarrowia lipolytica was evaluated through digital image analysis. The agitation speed of the culture was the most effective hyphae inducer in comparison to bovine calf serum and N-acetylglucosamine. In low agitation system, bovine calf serum was more effective for hyphae formation inducing 57 % of hyphae transition. Farnesol inhibited hyphae formation even in low concentration (300 µM) and this effect increased with increasing concentrations. In the presence of N-acetylglucosamine, this effect was more evident in comparison to the presence of bovine calf serum, which might have protected the cells from farnesol. Digital image analysis was an important tool to evaluate this phenomenon.

Biocompatibility analysis of a novel reabsorbable alloplastic membrane composed of alginate-Capsul / Análise da biocompatibilidade de uma membrana aloplástica reabsorvível composta de alginato-capsul

Objective The aim of this study was to evaluate in vivo the biological response after implantation of a novel alginate-capsule membrane. Methods The material was implanted into subcutaneous tissue of mice (n=15) and after 1, 3 and 9 weeks, the animals were sacrificed and biopsies analyzed with light microscopy, using the stains hematoxylin-eosin, picrosirius and alcian blue pH 2.5. The parameters evaluated were: intensity and kind of inflammatory infiltrate, presence of connective tissue, foreign body reaction, vascularization and biodegradation. Results 1 week after implantation, the following was observed: mixed inflammatory infiltrate, absence of necrosis and beginnings of membrane fragmentation; after 3 weeks, discrete presence of multinuclear giant cells and beginnings of neovascularization; and after 9 weeks there was minor biodegradation associated with the presence of new connective tissue, and persistence of moderate inflammatory reaction observed from beginning to end of the experiment. Conclusion Considering the results obtained, it is possible to conclude that the novel alginate-capsule membrane is partially reabsorbable but with low biocompatibility, requiring more tests to validate its clinical use.

Objetivo Avaliar in vivo a resposta tecidual após a implantação de uma nova membrana de alginato-capsul. Métodos O material foi implantado no tecido subcutâneo de camundongos (n=15) e após 1, 3 e 9 semanas, os animais foram mortos e as biópsias analisadas à microscopia de luz, através de coloração com hematoxilina-eosina, picrosirius e azul de alcian pH 2,5. Os parâmetros avaliados foram: intensidade e tipo de infiltrado inflamatório, presença de tecido conjuntivo, reação de corpo estranho, vascularização e biodegradação. Resultados Após 1 semana da implantação, notou-se infiltrado inflamatório misto, ausência de necrose e início de fragmentação da membrana, em 3 semanas, observou-se presença discreta de células gigantes multinucleadas e início de neovascularização, e em 9 semanas houve pequena biodegradação associada com a presença de novo tecido conjuntivo e persistência de reação inflamatória moderada observada desde o início do experimento. Conclusão Considerando os resultados obtidos concluiu-se que a nova membrana de alginato-capsul é parcialmente reabsorvível, mas com baixa biocompatibilidade, necessitando de mais testes para validar seu uso clínico.

Factors influencing crude oil biodegradation by Yarrowia lipolytica

Yarrowia lipolytica is unique strictly aerobic yeast with the ability to efficiently degrade hydrophobic substrates such as n-alkenes, fatty acids, glycerol and oils. In the present work, a 2(4) full factorial design was used to investigate the influence of the independent variables of temperature, agitation, initial cell concentration and initial petroleum concentration on crude oil biodegradation. The results showed that all variables studied had significant effects on the biodegradation process. Temperature, agitation speed and initial cell concentration had positive effects, and initial petroleum concentration had a negative effect. Among the crude oil removal conditions studied, the best temperature and agitation conditions were 28ºC and 250 rpm, respectively.

Adsorption of chlorhexidine on synthetic hydroxyapatite and in vitro biological activity.

The kinetic of chlorhexidine digluconate (CHXDG) uptake from aqueous solution by hydroxyapatite (HA) was investigated by ultraviolet (UV) analysis performed in HA powder (UV-solid) after the CHX adsorption. Adsorption isotherm of chlorhexidine (CHX) uptake was modeled by a combination of Languimir and Langmuir-Freundlich mechanisms. Strong molecule-molecule interactions and positive cooperativity predominated in the surface when CHX concentration was above 8.6 µg(CHX)/mg(HA). UV-solid spectra (shape, intensity and band position) of CHX bound to HA revealed that long-range molecular structures, such as aggregates or micelles, started to be formed at low CHX concentrations (1.52 µg(CHX)/mg(HA)) and predominated at high concentrations. Grazing-incidence X-ray diffraction (GIXRD) analysis from synchrotron radiation discarded the formation of crystalline structures on HA surface or precipitation of CHX crystalline salts, as suggested in previous works. The effect of the HA/CHX association on HA in vitro bioactivity, cytotoxicity and CHX antimicrobial activity was evaluated. It was shown that CHX did not inhibit the precipitation of a poorly crystalline apatite at HA/CHX surface after soaking in simulating body fluid (SBF). Cell viability studies after exposure to extracts of HA and HA/CHX showed that both biomaterials did not present significant in vitro toxicity. Moreover, HA/CHX inhibited Enterococcus faecalis growth for up to 6 days, revealing that binding to HA did not affect antimicrobial activity of CHX and reduced bacterial adhesion. These results suggested that HA/CHX association could result in a potential adjuvant antimicrobial system for clinical use.

Microcapsules of alginate/chitosan containing magnetic nanoparticles for controlled release of insulin.

The challenge of this work was to investigate the potential of alginate/chitosan beads containing magnetite nanoparticles as a drug delivery system. The insulin beads were prepared by dripping a solution of sodium alginate containing insulin into a CaCl(2) solution. Magnetite nanoparticles of 5 nm mean size were synthesized inside the alginate egg-box structure by co-precipitation of Fe(III) and Fe(II) in the presence of NH(4)OH. Quantitative analysis revealed that insulin encapsulation depends on the initial protein content and 35% of insulin was entrapped by alginate beads for a protein concentration of 10 wt%. It was verified that approximately 50% of the insulin was released to Milli-Q water in 800 h release experiments. The application of oscillating magnetic field increased three fold the insulin release. The results suggest that the alginate/chitosan system containing magnetite nanoparticles is a promising system for clinical applications of controlled release of insulin in the presence of an oscillating magnetic field in a subcutaneous implant approach.

Optimization of oxygen mass transfer in a multiphase bioreactor with perfluorodecalin as a second liquid phase.

Oxygenation is an important parameter involved in the design and operation of mixing-sparging bioreactors and it can be analyzed by means of the oxygen mass transfer coefficient (k(L)a). The operational conditions of a stirred, submerged aerated 2-L bioreactor have been optimized by studying the influence of a second liquid phase with higher oxygen affinity (perfluorodecalin or olive oil) in the k(L)a. Using k(L)a measurements, the influence of the following parameters on the oxygen transfer rate was evaluated: the volume of working medium, the type of impellers and their position, the organic phase concentration, the aqueous phase composition, and the concentration of inactive biomass. This study shows that the best experimental conditions were achieved with a perfluorodecalin volume fraction of 0.20, mixing using two Rushton turbines with six vertical blades and in the presence of YPD medium as the aqueous phase, with a k(L)a value of 64.6 h(-1). The addition of 20% of perfluorodecalin in these conditions provided a k(L)a enhancement of 25% when pure water was the aqueous phase and a 230% enhancement when YPD medium was used in comparison to their respective controls (no perfluorodecalin). Furthermore it is shown that the presence of olive oil as a second liquid phase is not beneficial to the oxygen transfer rate enhancement, leading to a decrease in the k(L)a values for all the concentrations studied. It was also observed that the magnitude of the enhancement of the k(L)a values by perfluorodecalin depends on the biomass concentration present.

Comparison of alpha-tocopherol microparticles produced with different wall materials: pea protein a new interesting alternative.

Alpha-tocopherol is a radical chain breaking antioxidant that can protect the integrity of tissues and play an important role in life process. Microparticles containing alpha-tocopherol were produced by spray drying technique using pea protein (PP), carboxymethylcellulose(CMC) and mixtures of these materials with maltodextrin (PP-M and CMC-M) as wall materials. The microparticles produced were characterised as regards the core retention (high performance liquid chromatography), the morphology (scanning electron microscopy) and size distribution (laser diffraction). The retention of alpha-tocopherol within all microparticles was above 77%. They showed a spherical shape and roughness at varied degrees. Their mean particles size remained below 7 microm, and the smallest sizes were found in PP and CMC-M microparticles. The results obtained in this work show that the pea protein use for alpha-tocopherol microencapsulation is a promising system for further application in food.

New microencapsulation system for ascorbic acid using pea protein concentrate as coat protector.

Microencapsulation is essential to preserve biological activity of ascorbic acid (AA) and pea protein has not been used as a carrier in such processes. This work aimed to produce microparticles by a spray-drying process using pea protein (PPC) as wall material of AA and evaluate the retention of the core by HPLC, overall morphology SEM, size distribution by light scattering and release kinetics. Carboxymethylcellulose (CMC) and blends with maltodextrin (M) were produced for comparative analyses. The yields were compatible with the applied technology and the retention was above 84% for all materials. The PPC microparticles presented irregular and rough surfaces, CMC produced a regular and smooth surface and agglomeration was more intense in microparticles with M. Mean particle diameters were all below 8 microm. The microparticle release rates were lower than those with free AA, being best correlated to the Higuchi kinetic model. These results support the utilization of PPC for microencapsulation of AA.

Electrical stimulation of saccharomyces cerevisiae cultures

Modulação do potencial de membrana celular endógeno por um campo elétrico externo influencia a estrutura e função dos compartimentos da membrana, de suas proteínas e da bi-camada lipídica. Neste trabalho, os efeitos da aplicação de potencial no crescimento de Saccharomyces cerevisiae foram caracterizados por experimentos simples, mas conclusivos. O perfil temporal de crescimento celular e a divisão celular foram investigados como respostas macroscópicas ao estímulo elétrico. Experimentos controle foram conduzidos em condições idênticas, exceto pela ausência de potencial aplicado. Através de análise comparativa, verificou-se que o estímulo elétrico alterou o ciclo celular como foi possível observar através da medida da dispersão de tamanho celular de cada população, sugerindo um possível sincronismo na divisão celular. Análise do espectro de potência foi empregada para sustentar o aumento no sincronismo, e uma modelagem matemática foi conduzida para determinar mudanças na cinética de crescimento celular. Parâmetros cinéticos do modelo tipo Monod para crescimento foram determinados por regressão não-linear. A constante de afinidade (a saber, KS) apresentou uma dependência com o potencial aplicado, sugerindo mudanças no transporte através da membrana celular. Verificou-se, também, que o estresse promovido eletroquimicamente inibiu o crescimento e induziu mudanças na viabilidade celular.

A strategic study using mutant-strain entrapment in calcium alginate for the production of Saccharomyces cerevisiae cells with high invertase activity.

Entrapped cells and entrapped cells grown inside of a calcium alginate matrix as well as free cells have been investigated using Saccharomyces cerevisiae mutant strains with regard to their pattern of growth and invertase activity. The repression of invertase by glucose and glucose-consumption ability were considered in the selection process of the mutants. Efficient sucrose bioconversion due to high invertase activity was obtained when entrapped mutant strain Q6R2 cells were grown within calcium alginate gel beads using sucrose plus glucose as the carbon source. Under these conditions, 1 mg (dry weight) of entrapped cells is able to produce 20 micromol of inverted sugar in 3 min (the maximum activity obtained was 20 units x mg(-1)). The experiments were carried out for 6 months without appreciable loss of either bead integrity or invertase activity. The biocatalyst was also stored at 4 degrees C for 6 months without appreciable loss of the invertase activity. This work shows that entrapped yeast cells with a weak ability to consume sugar may be used to produce inverted sugar.