Evaluating glucose and xylose as cosubstrates for lipid accumulation and c-linolenic acid biosynthesis of Thamnidium elegans

Aims: To study the biotechnological production of lipids containing rich amounts of the medically and nutritionally important c-linolenic acid (GLA), during cultivation of the Zygomycetes Thamnidium elegans, on mixtures of glucose and xylose, abundant sugars of lignocellulosic biomass. Methods and Results: Glucose and xylose were utilized as carbon sources, solely or in mixtures, under nitrogen-limited conditions, in batch-flask or bioreactor cultures. On glucose, T. elegans produced 31.9 g/L of biomass containing 15.0 g/L lipid with significantly high GLA content (1014 mg/L). Xylose was proved to be an adequate substrate for growth and lipid production. Additionally, xylitol secretion occurred when xylose was utilized as carbon source, solely or in mixtures with glucose. Batch-bioreactor trials on glucose yielded satisfactory lipid production, with rapid substrate consumption rates. Analysis of intracellular lipids showed that the highest GLA content was observed in early stationary growth phase, while the phospholipid fraction was the most unsaturated fraction of T. elegans. Conclusions: Thamnidium elegans represents a promising fungus for the successful valorization of sugar-based lignocellulosic residues into microbial lipids of high nutritional and pharmaceutical interest.

Production of acid whey hydrolysates applying an integrative process: effect of calcium on process performance

High ionic calcium concentration and the absence of caseinmacropeptides (CMP) in acid whey could influence the production of angiotensin-I-converting enzyme (ACE)-inhibitory hydrolysate and its bioactivity through the application of the integrative process. Therefore, the aim of the present study was to produce a hydrolysate from acid whey applying the integrative process. Process performance was evaluated based on protein adsorption capacity and conversion in relation to ACE-inhibitory activity (ACEi%) and ionic calcium concentration. Hydrolysates with high potency of their biological activity were produced (IC50 = 206-353 μg mL-1). High ionic calcium concentration in acid whey contributed to ACE-inhibitory activity. However, low β-lactoglobulin adsorption and conversion was observed. Optimisation of the resin volume increased the adsorption of β-lactoglobulin significantly but with lower selectivity. The changes in conversion value were not significant even at higher concentration of enzyme. Several ACE inhibitors derived from β-lactoglobulin that were identified before in sweet whey hydrolysates such as, IIAEKT, IIAE, IVTQ, LIVTQ, LIVTQT, LDAQ and LIVT were found. New peptides such as, SNICNI and ECCHGD derived from α-lactalbumin and BSA respectively were identified.

Rhodosporidium toruloides cultivated in NaCl-enriched glucose-based media: adaptation dynamics and lipid production

In the present report and for the first time in the international literature, the impact of the addition of NaCl upon growth and lipid production on the oleaginous yeast Rhodosporidium toruloides was studied. Moreover, equally for first time, lipid production by R. toruloides was performed under non-aseptic conditions. Therefore, the potentiality of R. toruloides DSM 4444 to produce lipid in media containing several initial concentrations of NaCl with glucose employed as carbon source was studied. Preliminary batch-flask trials with increasing amounts of NaCl revealed the tolerance of the strain against NaCl content up to 6.0% (w/v). However, 4.0% (w/v) of NaCl stimulated lipid accumulation for this strain, by enhancing lipid production up to 71.3% (w/w) per dry cell weight. The same amount of NaCl was employed in pasteurized batch-flask cultures in order to investigate the role of the salt as bacterial inhibiting agent. The combination of NaCl and high glucose concentrations was found to satisfactorily suppress bacterial contamination of R. toruloides cultures under these conditions. Batch-bioreactor trials of the yeast in the same media with high glucose content (up to 150 g/L) resulted in satisfactory substrate assimilation, with almost linear kinetic profile for lipid production, regardless of the initial glucose concentration imposed. Finally, fed-batch bioreactor cultures led to the production of 37.2 g/L of biomass, accompanied by 64.5% (w/w) of lipid yield. Lipid yield per unit of glucose consumed received the very satisfactory value of 0.21 g/g, a value amongst the highest ones in the literature. The yeast lipid produced contained mainly oleic acid and to lesser extent palmitic and stearic acids, thus constituting a perfect starting material for “second generation” biodiesel

In situ gelling systems based on Pluronic F127/Pluronic F68 formulations for ocular drug delivery

This study evaluated the use of Pluronic F127 and Pluronic F68 as excipients for formulating in situ gelling systems for ocular drug delivery. Thermal transitions have been studied in aqueous solutions of Pluronic F127, Pluronic F68 as well as their binary mixtures using differential scanning calorimetry, rheological measurements, and dynamic light scattering. It was established that the formation of transparent gels at physiologically relevant temperatures is observed only in the case of 20 wt % of Pluronic F127. The addition of Pluronic F68 to Pluronic F127 solutions increases the gelation temperature of binary formulation to above physiological range of temperatures. The biocompatibility evaluation of these formulations using slug mucosa irritation assay and bovine corneal erosion studies revealed that these polymers and their combinations do not cause significant irritation. In vitro drug retention study on glass surfaces and freshly excised bovine cornea showed superior performance of 20 wt % Pluronic F127 compared to other formulations. In addition, in vivo studies in rabbits demonstrated better retention performance of 20 wt % Pluronic F127 compared to Pluronic F68. These results confirmed that 20 wt % Pluronic F127 offers an attractive ocular formulation that can form a transparent gel in situ under physiological conditions with minimal irritation.

Using NMR-based metabolomics to evaluate postprandial urinary responses following consumption of minimally processed wheat bran or wheat aleurone by men and women

Wheat bran, and especially wheat aleurone fraction, are concentrated sources of a wide range of components which may contribute to the health benefits associated with higher consumption of whole-grain foods. This study used NMR metabolomics to evaluate urine samples from baseline at one and two hours postprandially, following the consumption of minimally processed bran, aleurone or control by 14 participants (7 Females; 7 Males) in a randomized crossover trial. The methodology discriminated between the urinary responses of control, and bran and aleurone, but not between the two fractions. Compared to control, consumption of aleurone or bran led to significantly and substantially higher urinary concentrations of lactate, alanine, N-acetylaspartate acid and N-acetylaspartylglutamate and significantly and substantially lower urinary betaine concentrations at one and two hours postprandially. There were sex related differences in urinary metabolite profiles with generally higher hippurate and citrate and lower betaine in females compared to males. Overall, this postprandial study suggests that acute consumption of bran or aleurone is associated with a number of physiological effects that may impact on energy metabolism and which are consistent with longer term human and animal metabolomic studies that used whole-grain wheat diets or wheat fractions.

Gelatin-based films containing hydrophobic plasticizers and saponin from Yucca schidigera as the surfactant

The aim of this study was to evaluate the incorporation of hydrophobic plasticizers (acetyltributyl citrate - ATB, tributyl citrate - TB and acetyltriethyl citrate - ATC) in a matrix of gelatin, using the saponin extracted from Yucca schidigera (yucca) as emulsifier, in the production of biodegradable emulsified films using the casting technique. High levels of hydrophobic plasticizers were incorporated, reaching up to 75% of plasticizer in relation to the protein (w/w) for ATB and TB, and up to 60% for ATC. The minimum values of water vapor permeability were 0.08, 0.07 and 0.06 g mm m(-2) h(-1) kPa(-1) for ATB, TB and ATC respectively, with no significant differences (p > 0.05). The water solubility of the films ranged from 21% to 59.5%. Although the WVP decreased, both scanning electron microscopy and laser scanning confocal microscopy indicated that the incorporation of the hydrophobic plasticizers did not occur homogeneously in the film matrix. (C) 2010 Elsevier Ltd. All rights reserved.

Effect of hydrophobic plasticizers on functional properties of gelatin-based films

The aim of this study was the production and characterization of gelatin-based films using hydrophobic plasticizers derived from citric acid and soy lecithin as emulsifier. The films were characterized as to their mechanic properties, permeability to water vapor, opacity, morphology and possible interactions using Fourier transform infrared spectroscopy. Tensile strength values (TS) varied from 36 to 103 MPa, how-ever, the increase in the concentration of plasticizers (acetyltributyl citrate and tributyl citrate) reduced TS by 57% and no relation was observed between plasticizer quantities and the elongation in the quantities tested. Permeability to water vapor varied between 0.17 and 0.34 (g mm/m(2) h kPa), slightly increasing with the increase in concentration of plasticizers. The effectiveness in the use of soy lecithin emulsifier in the homogenization between the compounds could be proven by microscopic observation using confocal laser microscopy. (C) 2009 Elsevier Ltd. All rights reserved.

Biodegradable Films Based on Blends of Gelatin and Poly (Vinyl Alcohol): Effect of PVA Type or Concentration on Some Physical Properties of Films

The aim of this work was to develop biodegradable films based on blends of gelatin and poly (vinyl alcohol) (PVA), without a plasticizer. Firstly, the effect of five types of PVA with different degree of hydrolysis (DH) on the physical properties of films elaborated with blends containing 23.1% PVA was studied. One PVA type was then chosen for the study of the effect of the PVA concentration on the mechanical properties, color, opacity, gloss, and water solubility of the films. The five types of PVA studied allowed for films with different characteristics, but with no direct relationship with the DH of the PVA. Therefore, the PVA Celvol (R) 418 with a DH = 91.8% was chosen for the second part, because they produced films with greater tensile strength. The PVA concentration affected all studied properties of films. These results could be explained by the results of the DSC and FTIR analyses, which showed that some interactions between the gelatin and the PVA occurred depending on the PVA concentration, affecting the crystallinity of the films.

Development of edible films based on differently processed Atlantic halibut (Hippoglossus hippoglossus) skin gelatin

The gelatin prepared from the skins of the Atlantic halibut (Hippoglossus hippoglossus) was investigated for the development of edible films plasticized with 30g sorbitol/100g gelatin. Two types of dry gelatin preparations were obtained depending on whether an intermediate evaporation step at 60 degrees C in the drying procedure is included or not. The amino acid composition, molecular weight distribution (determined by SDS-polyacrylamide gel electrophoresis) and glass transition temperature (determined by differential scanning calorimetry) of the gelatins were determined and related to some physical properties of the resulting films. The gelatin extracted from the halibut skins showed a suitable filmogenic capacity, leading to transparent, weakly colored, water-soluble and highly extensible films. The intermediate evaporation step at 60 degrees C induced thermal protein degradation, causing the resulting films to be significantly less resistant and more extensible. No differences in water vapor permeability, viscoelasticity, glass transition or color properties were evidenced between the two gelatins tested. (C) 2007 Elsevier Ltd. All rights reserved.

Polymorphism, crystallinity and hydrophilic-lipophilic balance of stearic acid and stearic acid-capric/caprylic triglyceride matrices for production of stable nanoparticles

There is an increasing interest in lipid nanoparticles because of their suitability for several administration routes. Thus, it becomes even more relevant the physicochemical characterization of lipid materials with respect to their polymorphism, lipid miscibility and stability, as well as the assessment of the effect of surfactant on the type and structure of these nanoparticles. This work focuses on the physicochemical characterization of lipid matrices composed of pure stearic acid or of mixtures of stearic acid-capric/caprylic triglycerides, for drug delivery. The lipids were analyzed by Differential Scanning Calorimetry (DSC), Wide Angle X-ray Diffraction (WAXD), Polarized Light Microscopy (PLM) and hydrophilic-lipophilic balance (HLB) in combination with selected surfactants to determine the best solid-to-liquid ratio. Based on the results obtained by DSC and WAXD, the selected qualitative and quantitative composition contributed for the production of stable nanoparticles, since the melting and the tempering processes provided important information on the thermodynamic stability of solid lipid matrices. The best HLB value obtained for stearic acid-capric/caprylic triglycerides was 13.8, achieved after combining these lipids with accepted surfactants (trioleate sorbitan and polysorbate 80 in the ratio of 10:90). The proposed combinations were shown useful to obtain a stable emulsion to be used as intermediate form for the production of lipid nanoparticles. (C) 2011 Elsevier B.V. All rights reserved.

A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 mu M benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 mu M 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog`s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.

Role of glutaredoxin 2 and cytosolic thioredoxins in cysteinyl-based redox modification of the 20S proteasome

The yeast 20S proteasome is subject to sulfhydryl redox alterations, such as the oxidation of cysteine residues (Cys-SH) into cysteine sulfenic acid (Cys-SOH), followed by S-glutathionylation (Cys-S-SG). Proteasome S-glutathionylation promotes partial loss of chymotrypsin-like activity and post-acidic cleavage without alteration of the trypsin-like proteasomal activity. Here we show that the 20S proteasome purified from stationary-phase cells was natively S-glutathionylated. Moreover, recombinant glutaredoxin 2 removes glutathione from natively or in vitro S-glutathionylated 20S proteasome, allowing the recovery of chymotrypsin-like activity and post-acidic cleavage. Glutaredoxin 2 deglutathionylase activity was dependent on its entry into the core particle, as demonstrated by stimulating S-glutathionylated proteasome opening. Under these conditions, deglutathionylation of the 20S proteasome and glutaredoxin 2 degradation were increased when compared to non-stimulated samples. Glutaredoxin 2 fragmentation by the 20S proteasome was evaluated by SDS-PAGE and mass spectrometry, and S-glutathionylation was evaluated by either western blot analyses with anti-glutathione IgG or by spectrophotometry with the thiol reactant 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. It was also observed in vivo that glutaredoxin 2 was ubiquitinated in cellular extracts of yeast cells grown in glucose-containing medium. Other cytoplasmic oxido-reductases, namely thioredoxins 1 and 2, were also active in 20S proteasome deglutathionylation by a similar mechanism. These results indicate for the first time that 20S proteasome cysteinyl redox modification is a regulated mechanism coupled to enzymatic deglutathionylase activity.

Effect of olive oil-based emulsion on human lymphocyte and neutrophil death

Background The incorporation of lipid emulsions in parenteral diets is a requirement for energy and essential fatty acid supply to critically ill patients. The most frequently used IV lipid emulsions (LE) are composed with long-chain triacylglycerols rich in omega-6 polyunsaturated fatty acids (PUFA) from soybean oil, but these LE promote lymphocyte and neutrophil death. A new emulsion containing 20% soybean oil and 80% olive oil rich in (omega-9 monounsaturated fatty acids (MUFA) has been hypothesized not to cause impairment of immune function. In this study, the toxicity of an olive oil-based emulsion (OOE) on lymphocytes and neutrophils from healthy volunteers was investigated. Methods: Twenty volunteers were recruited and blood was. collected before a 6-hour infusion of an OOE, immediately after infusion, and again 18 hours postinfusion. Lymphocytes and neutrophils were isolated by gradient density. The cells were studied immediately after isolation and after 24 hours or 48 hours in culture. The following determinations were carried out: triacylglycerol levels and fatty acid composition and levels in plasma, lymphocyte proliferation, production of reactive oxygen species, and parameters of lymphocyte and neutrophil death (viability, DNA fragmentation, phosphatidylserine externalization, mitochondrial depolarization, and neutral lipid accumulation). Results: OOE decreased lymphocyte proliferation, provoked lymphocyte necrosis, and had no effect on the proportion of viable neutrophils. The mechanism of cell death induced by OOE involved neutral lipid accumulation but had no effect on mitochondrial membrane depolarization. Conclusions: The OOE given as a single dose of 500 mL induced low toxicity to lymphocytes from healthy volunteers, probably by necrosis.

Dietary Free Oleic and Linoleic Acid Enhances Neutrophil Function and Modulates the Inflammatory Response in Rats

The high ingestion of oleic (OLA) and linoleic (LNA) acids by Western populations, the presence of inflammatory diseases in these populations, and the importance of neutrophils in the inflammatory process led us to investigate the effects of oral ingestion of unesterified OLA and LNA on rat neutrophil function. Pure OLA and LNA were administered by gavage over 10 days. The doses used (0.11, 0.22 and 0.44 g/kg of body weight) were based on the Western consumption of OLA and LNA. Neither fatty acid affected food, calorie or water intake. The fatty acids were not toxic to neutrophils as evaluated by cytometry using propidium iodide (membrane integrity and DNA fragmentation). Neutrophil migration in response to intraperitoneal injection of glycogen and in the air pouch assay, was elevated after administration of either OLA or LNA. This effect was associated with enhancement of rolling and increased release of the chemokine CINC-2 alpha beta. Both fatty acids elevated l-selectin expression, whereas no effect on beta(2)-integrin expression was observed, as evaluated by flow cytometry. LNA increased the production of proinflammatory cytokines (IL-1 beta and CINC-2 alpha beta) by neutrophils after 4 h in culture and both fatty acids decreased the release of the same cytokines after 18 h. In conclusion, OLA and LNA modulate several functions of neutrophils and can influence the inflammatory process.

Effect of medium/omega-6 long chain triglyceride-based emulsion on leucocyte death and inflammatory gene expression

Lipid emulsion (LE) containing medium/omega-6 long chain triglyceride-based emulsion (MCT/omega-6 LCT LE) has been recommended in the place of omega-6 LCT-based emulsion to prevent impairment of immune function. The impact of MCT/omega-6 LCT LE on lymphocyte and neutrophil death and expression of genes related to inflammation was investigated. Seven volunteers were recruited and infusion of MCT/omega-6 LCT LE was performed for 6 h. Four volunteers received saline and no change was found. Blood samples were collected before, immediately afterwards and 18 h after LE infusion. Lymphocytes and neutrophils were studied immediately after isolation and after 24 and 48 h in culture. The following determinations were carried out: plasma-free fatty acids, triacylglycerol and cholesterol concentrations, plasma fatty acid composition, neutral lipid accumulation in lymphocytes and neutrophils, signs of lymphocyte and neutrophil death and lymphocyte expression of genes related to inflammation. MCT/omega-6 LCT LE induced lymphocyte and neutrophil death. The mechanism for MCT/omega-6 LCT LE-dependent induction of leucocyte death may involve changes in neutral lipid content and modulation of expression of genes related to cell death, proteolysis, cell signalling, inflammatory response, oxidative stress and transcription.