Melt processing of paramylon using a water:ionic liquid mixture as plasticizer.

Paramylon is a linear ß-1,3-glucan produced by the microalgae Euglena Gracilis. Due to its native crystalline structure, involving hexagonally packed triple helices, paramylon is neither water soluble nor thermoplastic. While such properties are generally obtained by chemical modification of paramylon, the present work demonstrates that using ionic liquid/water mixtures as solvents or plasticizers may be an alternative: A mixture of water with cholinium glycinate (40:60) allowed: i) obtaining paramylon solutions at 80 °C, that form reversible ionogels upon cooling at 20 °C, when used as a solvent, and ii) the thermomechanical processing of paramylon below 100 °C by extrusion and hot-press into transparent films, when used as a plasticizer. The thermoplastic paramylon obtained consists of an amorphous matrix, self-reinforced by oriented triple helices packed as nanofibers. This results in a storage modulus ranging from 300 to 450 MPa at 25 °C, depending on the plasticizer content, and in a tensile strain at break of 27 %. For storage times larger than 1 month, a recrystallization of paramylon is observed, with an unidentified crystalline structure different from the native one. Recrystallized samples can be reprocessed into amorphous films by hot pressing.

Effects of Sucrose Replacement by Polyols on the Dough-Biscuit Transition: Understanding by Model Systems.

This study investigated the impacts of the complete substitution of sucrose by maltitol and/or sorbitol on the dough-crumb transition in biscuits. To this end, the phenomena of starch gelatinization/melting were studied at different moisture contents, both in the biscuit dough and model systems, by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and by environmental scanning electron microscopy (ESEM). Observation of doughs in ESEM revealed sorbitol had a structure very different from sucrose and maltitol crystals. After forming the dough pieces, it could be seen that at least some sugar and maltitol crystals were still present while sorbitol flakes were solubilized. At a limiting real water content (~20% dry basis), adding sweeteners to the mixture increased the gelatinization temperature, more markedly for sucrose and maltitol, as well as increasing the enthalpy. These results were confirmed by the model systems analyses. The calorimetric study with mixing batch cells revealed that sorbitol dissolved completely while maltitol and sucrose competed with the flour constituents to capture water. The proportion of water available for the sorption of the starch grain and its gelatinization was therefore different according to the affinity of the sweetener for water, and might influence the degree and temperature of starch gelatinization/melting.

Influence of the Presence of Choline Chloride on the Classical Mechanism of "Gelatinization" of Starch.

The aim of this research is to contribute to a better understanding the destructuration of three native starches and a wheat flour in mixtures of water and choline chloride. Model systems have thus been defined to allow a better approach to hydrothermic transformations related to the interactions between choline chloride and starch. We have observed that choline chloride has an impact on the gelatinization of starch which corresponds to the stabilizing salts phenomenon. The depolymerization and dissolution of the starch have also been demonstrated and can there dominate the gelatinization. However, the results obtained in X-ray diffraction by heating cell have shown that the exotherm which appeared was not only related to the depolymerization of the starch, but that a stage of crystalline rearrangement of the starch coexisted with this phenomenon.

Evolution of flax cell wall ultrastructure and mechanical properties during the retting step.

Flax retting is a major bioprocess in the cultivation and extraction cycle of flax fibres. The aim of the present study is to improve the understanding of the evolution of fibre properties and ultrastructure caused by this process at the plant cell wall scale. Initially, investigations of the mechanical performances of the flax cell walls by Atomic Force Microscopy (AFM) in Peak Force mode revealed a significant increase (+33%) in the cell wall indentation modulus with retting time. Two complementary structural studies are presented here, namely using X-Ray Diffraction (XRD) and solid state Nuclear Magnetic Resonance (NMR). An estimation of the cellulose crystallinity index by XRD measurements, confirmed by NMR, shows an increase of 8% in crystallinity with retting mainly due to the disappearance of amorphous polymer. In addition, NMR investigations show a compaction of inaccessible cell wall polymers, combined with an increase in the relaxation times of the C4 carbon. This densification provides a structural explanation for the observed improvement in mechanical performance of the secondary wall of flax fibres during the field retting process.

Whole Proteome Analyses on Ruminiclostridium cellulolyticum Show a Modulation of the Cellulolysis Machinery in Response to Cellulosic Materials with Subtle Differences in Chemical and Structural Properties.

Lignocellulosic materials from municipal solid waste emerge as attractive resources for anaerobic digestion biorefinery. To increase the knowledge required for establishing efficient bioprocesses, dynamics of batch fermentation by the cellulolytic bacterium Ruminiclostridium cellulolyticum were compared using three cellulosic materials, paper handkerchief, cotton discs and Whatman filter paper. Fermentation of paper handkerchief occurred the fastest and resulted in a specific metabolic profile: it resulted in the lowest acetate-to-lactate and acetate-to-ethanol ratios. By shotgun proteomic analyses of paper handkerchief and Whatman paper incubations, 151 proteins with significantly different levels were detected, including 20 of the 65 cellulosomal components, 8 non-cellulosomal CAZymes and 44 distinct extracytoplasmic proteins. Consistent with the specific metabolic profile observed, many enzymes from the central carbon catabolic pathways had higher levels in paper handkerchief incubations. Among the quantified CAZymes and cellulosomal components, 10 endoglucanases mainly from the GH9 families and 7 other cellulosomal subunits had lower levels in paper handkerchief incubations. An in-depth characterization of the materials used showed that the lower levels of endoglucanases in paper handkerchief incubations could hypothetically result from its lower crystallinity index (50%) and degree of polymerization (970). By contrast, the higher hemicellulose rate in paper handkerchief (13.87%) did not result in the enhanced expression of enzyme with xylanase as primary activity, including enzymes from the "xyl-doc" cluster. It suggests the absence, in this material, of molecular structures that specifically lead to xylanase induction. The integrated approach developed in this work shows that subtle differences among cellulosic materials regarding chemical and structural characteristics have significant effects on expressed bacterial functions, in particular the cellulolysis machinery, resulting in different metabolic patterns and degradation dynamics.

Expression of Escherichia coli glycogen branching enzyme in an Arabidopsis mutant devoid of endogenous starch branching enzymes induces the synthesis of starch-like polyglucans.

Starch synthesis requires several enzymatic activities including branching enzymes (BEs) responsible for the formation of α(1 → 6) linkages. Distribution and number of these linkages are further controlled by debranching enzymes that cleave some of them, rendering the polyglucan water-insoluble and semi-crystalline. Although the activity of BEs and debranching enzymes is mandatory to sustain normal starch synthesis, the relative importance of each in the establishment of the plant storage polyglucan (i.e. water insolubility, crystallinity and presence of amylose) is still debated. Here, we have substituted the activity of BEs in Arabidopsis with that of the Escherichia coli glycogen BE (GlgB). The latter is the BE counterpart in the metabolism of glycogen, a highly branched water-soluble and amorphous storage polyglucan. GlgB was expressed in the be2 be3 double mutant of Arabidopsis, which is devoid of BE activity and consequently free of starch. The synthesis of a water-insoluble, partly crystalline, amylose-containing starch-like polyglucan was restored in GlgB-expressing plants, suggesting that BEs' origin only has a limited impact on establishing essential characteristics of starch. Moreover, the balance between branching and debranching is crucial for the synthesis of starch, as an excess of branching activity results in the formation of highly branched, water-soluble, poorly crystalline polyglucan.

Shape memory starch-clay bionanocomposites.

1-10% starch/clay bionanocomposites with shape memory properties were obtained by melt processing. X-ray diffraction (XRD) and TEM evidenced the presence of a major fraction of clay tactoids, consisting of 4-5 stacked crystalline layers, with a thickness of 6.8 nm. A significant orientation of the nanoparticles induced by extrusion was also observed. Tensile tests performed above the glass transition of the materials showed that the presence of clay nanoparticles leads to higher elastic modulus and maximum stress, without significant loss in elongation at break which typically reached 100%. Samples submitted to a 50% elongation and cooled below the glass transition showed shape memory behavior. Like unreinforced starch, the bionanocomposites showed complete shape recovery in unconstrained conditions. In mechanically constrained conditions, the maximum recovered stress was significantly improved for the bionanocomposites compared to unreinforced starch, opening promising perspectives for the design of sensors and actuators.

Isolated starches from yams (Dioscorea sp) grown at the Venezuelan Amazons: structure and functional properties.

This work aimed to characterize the molecular structure and functional properties of starches isolated from wild Dioscorea yams grown at the Amazons, using conventional and up-to-date methodologies. Among the high purity starches isolated (≥99%), the chain lengths were similar, whereas variations in gelatinization profile were observed. Starches have shown varied-shaped granules with monomodal distribution, and B-type crystallinity. Variations in amylose contents found by three analyses were hypothesized being related to intermediate material. Linear chain lengths were similar, and their amylopectins showed a dense, spherical conformation and similar molecular characteristics. The average molar mass and the radius of gyration of the chromatograms of the yam amylopectin, M¯W and R¯G were ranging between 174×10(6) g mol(-1) and 237×10(6) g mol(-1), and 201 nm and 233 nm, respectively. The white yams starches were more sensible to enzymes than the other two. All starches have shown a wide range of functional and nutritional properties.

Physicochemical, functional, and macromolecular properties of waxy yam starches discovered from "Mapuey" (Dioscorea trifida) genotypes in the Venezuelan Amazon.

"Mapuey" tubers in Venezuela are staple food for indigenous peoples from the Caribbean coast and Amazon regions. Noticeable differences between genotypes of yam starches were observed. Granules were large, triangular, or shell-shaped with monomodal particle size distribution between 24.5 and 35.5 µm. Differential scanning calorimetry (DSC) analyses revealed onset gelatinization temperatures from 69.1 to 73.4 °C with high gelatinization enthalpy changes from 22.4 to 25.3 J g(-1). All X-ray diffractograms of starches exhibit B-type crystallinity. Crystallinity degrees varied from 24% to 40%. The highest crystallinity was found for the genotype having the highest amylose content. Iodo-colorimetric, amperometric, and DSC amylose determinations varied from 1.4 to 8.7%, 2.2 to 5.9%, and 1.4 to 3.5% for Amazonian genotypes, in comparison with commercial Mapuey starches: 12.0, 9.5, and 8.7%, respectively. Solubility and swelling power at 90 °C varied from 2.1 to 4.4% and 20.5 to 37.0%, respectively. Gel clarity fluctuated from 22.4 to 79.2%, and high rapid visco analyzer (RVA) viscosity was developed at 5% starch suspension (between 1430 and 2250 cP). Amylopectin weight average molar mass M(w), radius of gyration R(G), hydrodynamic coefficient ν(G), and apparent molecular density d(Gapp) were determined using high-performance size exclusion chromatography (HPSEC) and asymmetrical flow field flow fractionation (A4F) techniques coupled with multiangle laser light scattering (MALLS) on the Dioscorea trifida genotypes exhibiting the lowest and highest amylose contents. Amylopectins showed very similar molecular conformations. M(w) values were 1.15 × 10(8) and 9.06 × 10(7) g mol(-1) using HPSEC and A4F, respectively, thus, 3-5 times lower than those reported with the same techniques for other yam species, and very close to those of potato and cassava amylopectins. This discovery of a new natural amylose-free starch in the neglected yam "Mapuey" could present some potential for the food industry.

Further evidence for the mandatory nature of polysaccharide debranching for the aggregation of semicrystalline starch and for overlapping functions of debranching enzymes in Arabidopsis leaves.

Four isoforms of debranching enzymes are found in the genome of Arabidopsis (Arabidopsis thaliana): three isoamylases (ISA1, ISA2, and ISA3) and a pullulanase (PU1). Each isoform has a specific function in the starch pathway: synthesis and/or degradation. In this work we have determined the levels of functional redundancy existing between these isoforms by producing and analyzing different combinations of mutations: isa3-1 pu1-1, isa1-1 isa3-1, and isa1-1 isa3-1 pu1-1. While the starch content strongly increased in the isa3-1 pu1-1 double mutant, the latter decreased by over 98% in the isa1-1 isa3-1 genotype and almost vanished in triple mutant combination. In addition, whereas the isa3-1 pu1-1 double mutant synthesizes starch very similar to that of the wild type, the structure of the residual starch present either in isa1-1 isa3-1 or in isa1-1 isa3-1 pu1-1 combination is deeply affected. In the same way, water-soluble polysaccharides that accumulate in the isa1-1 isa3-1 and isa1-1 isa3-1 pu1-1 genotypes display strongly modified structure compared to those found in isa1-1. Taken together, these results show that in addition to its established function in polysaccharide degradation, the activity of ISA3 is partially redundant to that of ISA1 for starch synthesis. Our results also reveal the dual function of pullulanase since it is partially redundant to ISA3 for degradation and to ISA1 for synthesis. Finally, x-ray diffraction analyses suggest that the crystallinity and the presence of the 9- to 10-nm repetition pattern in starch precisely depend on the level of debranching enzyme activity.

Structure and mechanical properties of hydroxypropylated starch films.

Films of acid-hydrolyzed hydroxypropylated pea starch with average molecular weight M w ranging from 3.3 x 10 (4) g/mol to 1.6 x 10 (6) g/mol were prepared from 25% (w/w) solution by casting. The structure of the films was investigated by means X-ray diffraction and calorimetry, evidencing a B-type crystalline structure. In similar drying conditions, 25 degrees C and 40% of relative humidity, the crystallinity varied from 24% for the low molecular weight (A5) to almost none for the highest molecular weight (A160). The influence of the drying temperature was also investigated. A reduction of the crystallinity from 16% to almost none was found when increasing temperature from 25 to 65 degrees C. The glass transition temperature ( T g) at different water contents was determined. The difference of T g between the first and the second scan was interpreted by changes in the water distribution between phases into the B-type crystalline structure. Mechanical properties of the films determined by tensile tests and by DMTA in the glassy state showed no effect of the average molecular weight or of crystallinity. In contrast, thermomechanical experiments by DMTA showed that the average molecular weight of the sample influenced the mechanical relaxation and the moduli in the rubbery state.

Assessment of in vitro binding of isolated pectic domains to cellulose by adsorption isotherms, electron microscopy, and X-ray diffraction methods.

Isolated pectic domains representative of the pectic backbone and the neutral sugar side chains were tested for their ability to interact with cellulose in comparison to the well-known binding of xyloglucan. Pectic side chains displayed a significant in vitro binding capacity to cellulose, whereas pectic backbone domains exhibited only slight adsorption to cellulose microfibrils. To support the binding results, electron microscopy and X-ray diffraction were applied. Celluloses from bacteria and sugar beet cell walls were used as substrates for the precipitation of isolated pectic domains or xyloglucan by acetone vapor diffusion. Pectic side chains grew attached to the cellulose surfaces, whereas pectic backbone domains were observed separately from cellulose microfibrils. Xyloglucan seeded with cellulose provoked a decrease of microfibrils entanglement, but no clear cross-links between neighboring microfibrils were observed. These results led to the elucidation of the pectic domains responsible for binding with cellulose microfibrils.