Chemical Composition and In Vitro Bioactivities of Extracts from Cones of P. halepensis, P. brutia, and P. pinea: Insights into Pharmaceutical and Cosmetic Potential.

Various parts of the Pinaceae species, a traditional plant, have potential health benefits and exhibit antibacterial, anti-cancer, and antioxidant activities. This study aims to investigate the biochemical properties of both petal (P) and core (C) fractions from pinecones of P. halepensis (PA), P. brutia (PB), and P. pinea (PP). Pinecones were manually separated into P and C, which were then milled to investigate maceration with solvents of increasing polarity: cyclohexane (1SV), ethyl acetate (2SV), and methanol (3SV) at 20 °C. Spectrophotometry was utilized to quantify the total phenolic content (TPC) and to assess bioactivities. Gas chromatography with mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) were employed to identify the chemical composition. 3SV extracts demonstrated the highest TPC and a significant anti-oxidant potential. PA-P-3SV exhibited the highest TPC (460.66 mg GAE/g DW) and PP-P-3SV displayed the best IC50 (10.54 µg/mL) against DPPH. 1SV and 2SV extracts showed interesting anticancer activity against Hela and HepG2 cells. No significant toxic effect of P and C extracts from pinecones was observed on HEK-293 cells. GC-MS analysis unveiled 46 volatile compounds, of which 32 were detected for the first time in these species. HPLC analysis identified 38 compounds, of which 27 were not previously detected in these species. This study highlights the significant potential of pinecones as a rich source of bioactive compounds.

Complete genome sequences of two Bacillus thuringiensis serovar kurstaki strains isolated from Lebanon and Tunisia, highly toxic against lepidopteran larvae.

Bacillus thuringiensis-based products are key in the biopesticides market. Bacillus thuringiensis kurstaki strains Lip and BLB1 were isolated from Lebanese and Tunisian soils, respectively. These strains are highly toxic against lepidopteran larvae, Ephestia kuehniella. Here, we report Lip and BLB1 complete genomes, including their plasmid and toxin contents.

Production of recombinant scorpion antivenoms in E. coli: current state and perspectives.

Scorpion envenomation is a serious health problem in tropical and subtropical zones. The access to scorpion antivenom is sometimes limited in availability and specificity. The classical production process is cumbersome, from the hyper-immunization of the horses to the IgG digestion and purification of the F(ab)'2 antibody fragments. The production of recombinant antibody fragments in Escherichia coli is a popular trend due to the ability of this microbial host to produce correctly folded proteins. Small recombinant antibody fragments, such as single-chain variable fragments (scFv) and nanobodies (VHH), have been constructed to recognize and neutralize the neurotoxins responsible for the envenomation symptoms in humans. They are the focus of interest of the most recent studies and are proposed as potentially new generation of pharmaceuticals for their use in immunotherapy against scorpion stings of the Buthidae family. This literature review comprises the current status on the scorpion antivenom market and the analyses of cross-reactivity of commercial scorpion anti-serum against non-specific scorpion venoms. Recent studies on the production of new recombinant scFv and nanobodies will be presented, with a focus on the Androctonus and Centruroides scorpion species. Protein engineering-based technology could be the key to obtaining the next generation of therapeutics capable of neutralizing and cross-reacting against several types of scorpion venoms. KEY POINTS: • Commercial antivenoms consist of predominantly purified equine F(ab)'2fragments. • Nanobody-based antivenom can neutralize Androctonus venoms and have a low immunogenicity. • Affinity maturation and directed evolution are used to obtain potent scFv families against Centruroides scorpions.

Biochemical limitations of Bacillus thuringiensis based biopesticides production in a wheat bran culture medium.

Bacillus thuringiensis, a gram-positive sporulating bacteria found in the environment, produces, during its sporulation phase, crystals responsible for its insecticidal activity, constituted of an assembly of pore-forming δ-endotoxins. This has led to its use as a biopesticide, an eco-friendly alternative to harmful chemical pesticides. To minimize production cost, one endemic Bacillus thuringiensis sv. kurstaki (Btk) strain Lip, isolated from Lebanese soil, was cultivated in a wheat bran (WB) based medium (IPM-4-Citrus project EC n° 734921). With the aim of studying the biochemical limitations of Btk biopesticide production in a wheat bran based medium, the WB was sieved into different granulometries, heat treated, inoculated with Btk Lip at flask scale, then filtered and separated into an insoluble and a permeate fractions. Several biochemical analyses, ie. bio performances, starch, elemental composition, total nitrogen and ashes, were then conducted on both fractions before and after culture. On a morphological level, two populations were distinguished, the fine starch granules and the coarse lignocellulosic particles. The biochemical analyses showed that both the raw and sieved WB have a similar proteins content (0.115 g/gdm WB), water content (0.116 g/gdm WB) and elemental composition (carbon: 45%, oxygen: 37%, nitrogen: 3%, hydrogen: 6%, ashes: 5%). The starch content was 17%, 14% and 34% and the fermentable fraction was estimated to 32.1%, 36.1% and 51.1% respectively for classes 2, 3 and 4. Both the elemental composition and Kjeldahl analyses showed that the nitrogen is the limiting nutrient of the culture.

Structure of heteroxylans from vitreous and floury endosperms of maize grain and impact on the enzymatic degradation.

Heteroxylans (HX) from vitreous and floury parts of maize endosperm were isolated. Structural analysis showed a xylan backbone with few unsubstituted xylose residues (<9%) demonstrating the high content in side chains in both fractions. HX from floury endosperm contained more arabinose and galactose than vitreous HX. The mono-substitution rate was 15% higher in the vitreous endosperm HX. Similar amounts of uronic acids were present in both fractions (~7% DM). Galactose in the floury endosperm HX was present exclusively in terminal position. A xylanase preparation solubilized more material from floury (40.5%) than from vitreous endosperm cell walls (15%). This could be a consequence of the structural differences between the two fractions and/or of the impact of structure on the interaction abilities of these fractions with other cell wall polysaccharides. Our study advances the understanding of cell wall polysaccharides in maize endosperm and their role in enzymatic susceptibility of maize grain.

Effect of temperature on the production of a recombinant antivenom in fed-batch mode.

In the pharmaceutical industry, nanobodies show promising properties for its application in serotherapy targeting the highly diffusible scorpion toxins. The production of recombinant nanobodies in Escherichia coli has been widely studied in shake flask cultures in rich medium. However, there are no upstream bioprocess studies of nanobody production in defined minimal medium and the effect of the induction temperature on the production kinetics. In this work, the effect of the temperature during the expression of the chimeric bispecific nanobody CH10-12 form, showing high scorpion antivenom potential, was studied in bioreactor cultures of E. coli. High biomass concentrations (25 g cdw/L) were achieved in fed-batch mode, and the expression of the CH10-12 nanobody was induced at temperatures 28, 29, 30, 33, and 37°C with a constant glucose feed. For the bispecific form NbF12-10, the induction was performed at 29°C. Biomass and carbon dioxide yields were reported for each culture phase, and the maintenance coefficient was obtained for each strain. Nanobody production in the CH10-12 strain was higher at low temperatures (lower than 30°C) and declined with the increase of the temperature. At 29°C, the CH10-12, NbF12-10, and WK6 strains were compared. Strains CH10-12 and NbF12-10 had a productivity of 0.052 and 0.021 mg/L/h of nanobody, respectively, after 13 h of induction. The specific productivity of the nanobodies was modeled as a function of the induction temperature and the specific growth rates. Experimental results confirm that low temperatures increase the productivity of the nanobody.Key points• Nanobodies with scorpion antivenom activity produced using two recombinant strains.• Nanobodies production was achieved in fed-batch cultures at different induction temperatures.• Low induction temperatures result in high volumetric productivities of the nanobody CH10-12.

A protocol for recombinant protein quantification by densitometry.

The protein purity is generally checked using SDS-PAGE, where densitometry could be used to quantify the protein bands. In literature, few studies have been reported using image analysis for the quantification of protein in SDS-PAGE: that is, imaged with Stain-Free™ technology. This study presents a protocol of image analysis for electrophoresis gels that allows the quantification of unknown proteins using the molecular weight markers as protein standards. Escherichia coli WK6/pHEN6 encoding the bispecific nanobody CH10-12 engineered by the Pasteur Institute of Tunisia was cultured in a bioreactor and induced with isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 28°C for 12 hr. Periplasmic proteins extracted by osmotic shock were purified by immobilized metal affinity chromatography (IMAC). Images of the SDS-PAGE gels were analyzed using ImageJ, and the lane profiles were obtained in grayscale and uncalibrated optical density. Protein load and peak area were linearly correlated, and optimal image processing was then performed by background subtraction using the rolling ball algorithm with radius size 250 pixels. No brightness and contrast adjustment was applied. The production of the nanobody CH10-12 was obtained through a fed-batch strategy and quantified using the band of 50 kDa in the marker as reference for 750 ng of recombinant protein. The molecular weight marker was used as a sole protein standard for protein quantification in SDS-PAGE gel images.

Combined in situ Physical and ex-situ Biochemical Approaches to Investigate in vitro Deconstruction of Destarched Wheat Bran by Enzymes Cocktail Used in Animal Nutrition.

Wheat bran is a foodstuff containing more than 40% of non-starch polysaccharides (NSPs) that are hardly digestible by monogastric animals. Therefore, cocktails enriched of hydrolytic enzymes (termed NSPases) are commonly provided as feed additives in animal nutrition. However, how these enzymes cocktails contribute to NSPs deconstruction remains largely unknown. This question was addressed by employing an original methodology that makes use of a multi-instrumented bioreactor that allows to dynamically monitor enzymes in action and to extract in-situ physical and ex-situ biochemical data from this monitoring. We report here that the deconstruction of destarched wheat bran by an industrial enzymes cocktail termed Rovabio® was entailed by two concurrent events: a particles fragmentation that caused in <2 h a 70% drop of the suspension viscosity and a solubilization that released <30 % of the wheat bran NSPs. Upon longer exposure, the fragmentation of particles continued at a very slow rate without any further solubilization. Contrary to this cocktail, xylanase C alone caused a moderate 25% drop of viscosity and a very weak fragmentation. However, the amount of xylose and arabinose from solubilized sugars after 6 h treatment with this enzyme was similar to that obtained after 2 h with Rovabio®. Altogether, this multi-scale analysis supported the synergistic action of enzymes mixture to readily solubilize complex polysaccharides, and revealed that in spite of the richness and diversity of hydrolytic enzymes in the cocktail, the deconstruction of NSPs in wheat bran was largely incomplete.

Impacts of environmental conditions on product formation and morphology of Yarrowia lipolytica.

The yeast Yarrowia lipolytica is an industrially important microorganism with distinctive physiological and metabolic characteristics. A variety of external factors (e.g., pH, temperature, and nutrient availability) influences the behavior of the yeast and may act as stress conditions which the cells must withstand and adapt. In this mini review, the impacts of environmental factors on the morphology and metabolite production by Y. lipolytica are summarized. In this regard, detailed insights into the effectors involved in the dimorphic transition of Y. lipolytica, the cultivation conditions employed, as well as the methods applied for the morphological characterization are highlighted. Concerning the metabolism products, a special focus is addressed on lipid and citric acid metabolites which have attracted significant attention in recent years. The dependence of lipid and citric acid productivity on key process parameters, such as media composition and physico-chemical variables, is thoroughly discussed. This review attempts to provide a recent update on the topic and will serve as a meaningful resource for researchers working in the field.

Influence of oxygen availability on the metabolism and morphology of Yarrowia lipolytica: insights into the impact of glucose levels on dimorphism.

Dynamic behavior of Yarrowia lipolytica W29 strain under conditions of fluctuating, low, and limited oxygen supply was characterized in batch and glucose-limited chemostat cultures. In batch cultures, transient oscillations between oxygen-rich and -deprived environments induced a slight citric acid accumulation (lower than 29 mg L-1). By contrast, no citric acid was detected in continuous fermentations for all stress conditions: full anoxia (zero pO2 value, 100% N2), limited (zero pO2 value, 75% of cell needs), and low (pO2 close to 2%) dissolved oxygen (DO) levels. The macroscopic behavior (kinetic parameters, yields, viability) of Y. lipolytica was not significantly affected by the exposure to DO fluctuations under both modes of culture. Nevertheless, conditions of oxygen limitation resulted in the destabilization of the glucose-limited growth during the continuous cultivations. Morphological responses of Y. lipolytica to DO oscillations were different between batch and chemostat runs. Indeed, a yeast-to-mycelium transition was induced and progressively intensified during the batch fermentations (filamentous subpopulation reaching 74% (v/v)). While, in chemostat bioreactors, the culture consisted mainly of yeast-like cells (mean diameter not exceeding 5.7 µm) with a normal size distribution. During the continuous cultures, growth at low DO concentration did not induce any changes in Y. lipolytica morphology. Dimorphism (up to 80.5% (v/v) of filaments) was only detected under conditions of oxygen limitation in the presence of a residual glucose excess (more than 0.75 g L-1). These data suggest an impact of glucose levels on the signaling pathways regulating dimorphic responses in Y. lipolytica.

Dynamic behavior of Yarrowia lipolytica in response to pH perturbations: dependence of the stress response on the culture mode.

Yarrowia lipolytica, a non-conventional yeast with a promising biotechnological potential, is able to undergo metabolic and morphological changes in response to environmental conditions. The effect of pH perturbations of different types (pulses, Heaviside) on the dynamic behavior of Y. lipolytica W29 strain was characterized under two modes of culture: batch and continuous. In batch cultures, different pH (4.5, 5.6 (optimal condition), and 7) were investigated in order to identify the pH inducing a stress response (metabolic and/or morphologic) in Y. lipolytica. Macroscopic behavior (kinetic parameters, yields, viability) of the yeast was slightly affected by pH. However, contrary to the culture at pH 5.6, a filamentous growth was induced in batch experiments at pH 4.5 and 7. Proportions of the filamentous subpopulation reached 84 and 93 % (v/v) under acidic and neutral conditions, respectively. Given the significant impact of neutral pH on morphology, pH perturbations from 5.6 to 7 were subsequently assayed in batch and continuous bioreactors. For both process modes, the growth dynamics remained fundamentally unaltered during exposure to stress. Nevertheless, morphological behavior of the yeast was dependent on the culture mode. Specifically, in batch bioreactors where cells proliferated at their maximum growth rate, mycelia were mainly formed. Whereas, in continuous cultures at controlled growth rates (from 0.03 to 0.20 h-1) even closed to the maximum growth rate of the stain (0.24 h-1), yeast-like forms predominated. This pointed out differences in the kinetic behavior of filamentous and yeast subpopulations, cell age distribution, and pH adaptive mechanisms between both modes of culture.

Rheology of Lignocellulose Suspensions and Impact of Hydrolysis: A Review.

White biotechnologies have several challenges to overcome in order to become a viable industrial process. Achieving highly concentrated lignocellulose materials and releasing fermentable substrates, with controlled kinetics in order to regulate micro-organism activity, present major technical and scientific bottlenecks. The degradation of the main polymeric fractions of lignocellulose into simpler molecules is a prerequisite for an integrated utilisation of this resource in a biorefinery concept. The characterisation methods and the observations developed for rheology, morphology, etc., that are reviewed here are strongly dependent on the fibrous nature of lignocellulose, are thus similar or constitute a good approach to filamentous culture broths. This review focuses on scientific works related to the study of the rheological behaviour of lignocellulose suspensions and their evolution during biocatalysis. In order to produce the targeted molecules (synthon), the lignocellulose substrates are converted by enzymatic degradation and are then metabolised by micro-organisms. The dynamics of the mechanisms is limited by coupled phenomena between flow, heat and mass transfers in regard to diffusion (within solid and liquid phases), convection (mixing, transfer coefficients, homogeneity) and specific inhibitors (concentration gradients). As lignocellulose suspensions consist of long entangled fibres for the matrix of industrial interest, they exhibit diverse and complex properties linked to this fibrous character (rheological, morphological, thermal, mechanical and biochemical parameters). Among the main variables to be studied, the rheological behaviour of such suspensions appears to be determinant for process efficiency. It is this behaviour that will determine the equipment to be used and the strategies applied (substrate and biocatalysis feed, mixing, etc.). This review provides an overview of (i) the rheological behaviour of fibrous materials in suspension, (ii) the methods and experimental conditions for their measurements, (iii) the main models used and (iv) their evolution during biocatalytic reactions with a focus on enzymatic hydrolysis.

Carbon accumulation in Rhodotorula glutinis induced by nitrogen limitation.

BACKGROUND: Oleaginous microorganisms, such as bacterium, yeast and algal species, can represent an alternative oil source for biodiesel production. The composition of their accumulated lipid is similar to the lipid of an oleaginous plant with a predominance of unsaturated fatty acid. Moreover this alternative to conventional biodiesel production does not create competition for land use between food and oleo-chemical industry supplies. Despite this promising potential, development of microbial production processes are at an early stage. Nutritional limited conditions, such as nitrogen limitation, with an excess of carbon substrate is commonly used to induce lipid accumulation metabolism. Nitrogen limitation implies modification of the carbon-to-nitrogen ratio in culture medium, which impacts on carbon flow distribution in the metabolic network. RESULTS: The goal of the present study is to improve our knowledge of carbon flow distribution in oleaginous yeast metabolism by focusing carbon distribution between carbohydrate and lipid pools in order to optimize microbial lipid production. The dynamic effects of limiting nitrogen consumption flux according to carbon flow were studied to trigger lipid accumulation in the oleaginous yeast Rhodotorula glutinis. With a decrease of the specific nitrogen consumption rate from 0.052 Nmol.CmolX (-1).h(-1) to 0.003 Nmol.CmolX (-1).h(-1), a short and transitory intracellular carbohydrate accumulation occurred before the lipid accumulation phase. This phenomenon was studied in fed-batch culture under optimal operating conditions, with a mineral medium and using glucose as carbon source. Two different strategies of decreasing nitrogen flow on carbohydrate accumulation were investigated: an instantaneous decrease and a progressive decrease of nitrogen flow. CONCLUSIONS: Lipid production performance in these fed-batch culture strategies with R. glutinis were higher than those reported in the previous literature; the catalytic specific lipid production rate was 0.07 Cmollip.CmolX* (-1).h(-1). Experimental results suggested that carbohydrate accumulation was an intrinsic phenomenon connected to the limitation of growth by nitrogen when the nitrogen-to-carbon ratio in the feed flow was lower than 0.045 Nmol.Cmol(-1). Carbohydrate accumulation corresponded to a 440% increase of carbohydrate content. These results suggest that microbial lipid production can be optimized by culture strategy and that carbohydrate accumulation must be taken account for process design.

In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis.

This work combines physical and biochemical analyses to scrutinize liquefaction and saccharification of complex lignocellulose materials. A multilevel analysis (macroscopic: rheology, microscopic: particle size and morphology and molecular: sugar product) was conducted at the lab-scale with three matrices: microcrystalline cellulose (MCC), Whatman paper (WP) and extruded paper-pulp (PP). A methodology to determine on-line viscosity is proposed and validated using the concept of Metzner and Otto (1957) and Rieger and Novak's (1973). The substrate suspensions exhibited a shear-thinning behaviour with respect to the power law. A structured rheological model was established to account for the suspension viscosity as a function of shear rate and substrate concentration. The critical volume fractions indicate the transition between diluted, semi-diluted and concentrated regimes. The enzymatic hydrolysis was performed with various solid contents: MCC 273.6 gdm/L, WP 56.0 gdm/L, PP 35.1 gdm/L. During hydrolysis, the suspension viscosity decreased rapidly. The fibre diameter decreased two fold within 2 h of starting hydrolysis whereas limited bioconversion was obtained (10-15%).