Bruno Pontoire

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.

Structural features and potential texturising properties of lemon and maize cellulose microfibrils

Abstract Cellulose microfibrils extracted from lemon and maize were studied by solid state NMR, Wide Angle X-ray Scattering, Fourier Transform Infrared Spectroscopy and Transmission Electron Microscopy. Some structural characteristics such as the allomorphic composition, the degree of crystallinity and the lateral or longitudinal crystal size were determined. These depended on the processing conditions, especially grinding, and determined the texturising properties of the microfibrils for liquid food product. Hydrolysed tunicin and Avicel were also studied as reference systems in terms of crystallinity and texturising properties in water. Lemon cellulose was shown to have a higher crystallinity or crystal size and a better texturising behaviour than maize microfibrils. Nevertheless, lemon microfibrils are more sensitive to grinding which decreases systematically the crystallinity and increases the amount of Iβ allomorph. Solid state NMR was also used to probe the mobility of the different regions of microfibrils by measuring T 1ρ which was well correlated to crystallinity. The presence of both surface and internal amorphous areas was discussed with respect to the NMR and X-ray scattering data. Some possible hypotheses for discrepancies observed between lemon and maize microfibrils in terms of texturising properties are expressed. Finally, these various properties were studied in the presence of polysaccharides such as carboxymethyl cellulose, scleroglucan or xanthan.

Comparison of X-ray diffraction patterns and sorption properties of the hydrolyzed starches of potato, wrinkled and smooth pea, broad bean and wheat

Abstract The hydrolysis of native starches by hydrochloric acid (the so-called lintnerization process) yields a more crystalline material with moderately decreased water sorption capacities. X-ray diffraction may be used to rank native or lintnered starches from B to A type with intermediate type C mixtures and this change in order is parallelled by decreasing water affinities. The dependence of the intensity of the X-ray diffractogram on the water contents required for the A and B lattice spacings confirms the intimate role of water in the amylose crystalline structure. Both structural and hydration hysteresis remain important in crystalline rich materials, suggesting that the mechanisms involved are not limited to long range interactions or mechanical tensions.

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.

Coupling lipophilization and amylose complexation to encapsulate chlorogenic acid

Chlorogenic acid (5-caffeoylquinic acid) is a hydrophilic phenolic compound with antioxidant properties. Because of its high polarity, these properties may be altered when formulated in oil-based food. There is therefore an interest in trying to protect the natural antioxidant by molecular encapsulation. Amylose, the linear fraction of starch with essentially α(1-4) linkages, is well known for its ability to form semi-crystalline complexes with a variety of small ligands. Monoacyl lipids, as well as smaller ligands such as alcohols or flavor compounds, are able to induce the formation of left-handed amylose single helices. In contrast, chlorogenic acid is a bulky molecule whose topology requires the amylose helix to be distorted, which could prevent amylose complexation. An innovative strategy has been developed to overcome this problem by grafting an aliphatic chain onto chlorogenic acid then trapping this chain in the helical cavity. The lipophilization reaction was used to obtain a palmitoyl chlorogenic acid derivative and the amylose-palmitoyl chlorogenic acid assemblies were studied by X-ray diffraction, differential scanning calorimetry and NMR to elucidate the interaction. The results showed that such interactions between amylose and palmitoyl chlorogenic acid are effective.

Monitoring the crystallization of starch and lipid components of the cake crumb during staling

Cake staling is a complex problem which has still not been fully understood. Starch polymers retrogradation, which is linked to biopolymers recrystallisation, is the most important factor affecting cake firmness in addition to water migration and fat crystallization. In this study, the effect of storage temperatures of 4°C and 20°C on starch retrogradation and fat recrystallization was investigated. Starch retrogradation can be tracked through changes in crystalline structure via X-rays diffraction as well as through melting of crystals via calorimetry. These techniques have been coupled to study the different phenomena occurring during staling. The results revealed that the storage of cakes at 20°C for 25 days showed more starch polymer retrogradation and more intense fat recrystallization in the β form than at 4°C. Consequently, the staling was delayed when a low storage temperature like 4°C was used, which is recommended to retain high quality cakes during storage.

Brachypodium Cell Wall Mutant with Enhanced Saccharification Potential Despite Increased Lignin Content

Plant lignocellulosic biomass, mostly composed of cell walls, is one of the largest, mostly untapped, reserves of renewable carbon feedstock on the planet. Energy-rich polysaccharide polymers of plant cell walls can be broken down to produce fermentable sugars used to produce bioethanol. However, the complex structure of plant cell walls, and in particular, the presence of lignin, makes them recalcitrant to enzymatic degradation. Reducing this recalcitrance represents a major technological challenge. Brachypodium distachyon is an excellent model to identify parameters underlying biomass quality of energy grasses. In this work, we identified a mutant line spa1 with a so far undescribed phenotype combining brittleness with increased elasticity of the internodes. Mutant cell walls contain less crystalline cellulose and changes in hemicellulose and lignin quality and quantity. Using a dedicated reactor to follow in real-time, the evolution of straw particle size and sugar release during enzymatic digestion, we show that, despite the increased lignin content, the spa1 mutant has a dramatic reduced recalcitrance to saccharification compared to the WT. These observations demonstrate that other parameters besides lignin content are relevant for the improvement of biomass recalcitrance in energy grasses.

Trapping by amylose of the aliphatic chain grafted onto chlorogenic acid: importance of the graft position.

5-Caffeoylquinic acid (chlorogenic acid), is classified in acid-phenols family and as polyphenolic compounds it possesses antioxidant activity. The oxydative modification of chlorogenic acid in foods may lead to alteration of their qualities; to counteract these degradation effects, molecular encapsulation was used to protect chlorogenic acid. Amylose can interact strongly with a number of small molecules, including lipids. In order to enable chlorogenic acid complexation by amylose, a C16 aliphatic chain was previously grafted onto the cycle of quinic acid. This work showed that for the two lipophilic derivatives of chlorogenic acid: hexadecyl chlorogenate obtained by alkylation and 3-O-palmitoyl chlorogenic acid obtained by acylation; only the 3-O-palmitoyl chlorogenic acid complexed amylose. The chlorogenic acid derivatives were studied by X-ray diffraction, differential scanning calorimetry and NMR to elucidate the interaction. By comparing the results with previous work on the complexation of amylose by 4-O-palmitoyl chlorogenic acid, the importance of the aliphatic chain position on the cycle of the quinic acid is clearly highlighted. A study in molecular modeling helped to understand the difference in behavior relative to amylose of these three derivatives of chlorogenic acid.

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.