Bert Lagrain

Reductive lignocellulose fractionation into soluble lignin-derived phenolic monomers and dimers and processable carbohydrate pulps

A catalytic lignocellulose biorefinery process is presented, valorizing both polysaccharide and lignin components into a handful of chemicals. To that end, birch sawdust is efficiently delignified through simultaneous solvolysis and catalytic hydrogenolysis in the presence of a Ru on carbon catalyst (Ru/C) in methanol under a H2 atmosphere at elevated temperature, resulting in a carbohydrate pulp and a lignin oil. The lignin oil yields above 50% of phenolic monomers (mainly 4-n-propylguaiacol and 4-n-propylsyringol) and about 20% of a set of phenolic dimers, relative to the original lignin content, next to phenolic oligomers. The structural features of the lignin monomers, dimers and oligomers were identified by a combination of GC/MS, GPC and 2D HSQC NMR techniques, showing interesting functionalities for forthcoming polymer applications. The effect of several key parameters like temperature, reaction time, wood particle size, reactor loading, catalyst reusability and the influence of solvent and gas were examined in view of the phenolic product yield, the degree of delignification and the sugar retention as a first assessment of the techno-economic feasibility of this biorefinery process. The separated carbohydrate pulp contains up to 92% of the initial polysaccharides, with a nearly quantitative retention of cellulose. Pulp valorization was demonstrated by its chemocatalytic conversion to sugar polyols, showing the multiple use of Ru/C, initially applied in the hydrogenolysis process. Various lignocellulosic substrates, including genetically modified lines of Arabidopsis thaliana, were finally processed in the hydrogenolytic biorefinery, indicating lignocellulose rich in syringyl-type lignin, as found in hardwoods, as the ideal feedstock for the production of chemicals.

Molecular Basis of Processing Wheat Gluten toward Biobased Materials

The unique properties of the wheat grain reside primarily in the gluten-forming storage proteins of its endosperm. Wheat glutens structural and functional properties have led to an expanding diversity of applications in food products. However, its viscoelastic properties and low water solubility also are very interesting features for nonfood applications. Moreover, gluten is annually renewable and perfectly biodegradable. In the processing and setting of gluten containing products, temperature plays a very important role. In this review, the structure and reactivity of gluten are discussed and the importance of sulfhydryl (SH) and disulfide (SS) groups is demonstrated. Wheat gluten aggregation upon thermosetting proceeds through direct covalent cross-linking in and between its protein groups, glutenin and gliadin. Predominant reactions include SH oxidation and SH/SS interchange reactions leading to the formation of SS cross-links. Additionally, thermal treatment of gluten can result in the formation of other than SS covalent bonds. We here review two main technological approaches to make gluten-based materials: wet processes resulting in thin films and dry processes, such as extrusion or compression molding, exploiting the thermoplastic properties of proteins under low moisture conditions and potentially resulting in very useful materials. Gluten bioplastics can also be reinforced with natural fibers, resulting in biocomposites. Although a lot of progress has been made the past decade, the current gluten materials are still outperformed by their synthetic polymer counterparts.

Wheat Gluten Functionality as a Quality Determinant in Cereal-Based Food Products

The unique properties of wheat reside primarily in its gluten-forming storage proteins. Their intrinsic viscoelastic behavior is responsible for the characteristics of different wheat-based foods and for the use of wheat gluten proteins in different food products. Wheat-based food processing generally develops and sets the gluten protein network. Heat-induced gluten aggregation proceeds through cross-linking within and between its protein fractions. Prominent reactions include sulfhydryl (SH) oxidation and SH-disulfide (SS) interchange, which lead to SS cross-links. Other covalent bonds are also formed. Gluten functionality can be (bio-) chemically impacted. We focus on bread making, in which gluten proteins contribute to dough properties, bread loaf volume, and structure, and on pasta production, in which gluten proteins generate the desired cooking quality. Furthermore, it is speculated that the structure and texture of soft wheat products are also, at least to some degree, shaped by the heat-induced changes in the gluten protein fraction.

Wheat gluten amino acid composition analysis by high-performance anion-exchange chromatography with integrated pulsed amperometric detection

A simple accurate method for determining amino acid composition of wheat gluten proteins and their gliadin and glutenin fractions using high-performance anion-exchange chromatography with integrated pulsed amperometric detection is described. In contrast to most conventional methods, the analysis requires neither pre- or post-column derivatization, nor oxidation of the sample. It consists of hydrolysis (6.0M hydrochloric acid solution at 110 degrees C for 24h), evaporation of hydrolyzates (110 degrees C), and chromatographic separation of the liberated amino acids. Correction factors (f) accounted for incomplete cleavage of peptide bonds involving Val (f=1.07) and Ile (f=1.13) after hydrolysis for 24h and for Ser (f=1.32) losses during evaporation. Gradient conditions including an extra eluent (0.1M acetic acid solution) allowed multiple sequential sample analyses without risk of Glu contamination on the anion-exchange column. While gluten amino acid compositions by the present method were mostly comparable to those obtained by a conventional method involving oxidation, acid hydrolysis and post-column ninhydrin derivatization, the latter method underestimated Tyr, Val and Ile levels. Results for the other amino acids obtained by the different methods were linearly correlated (r>0.99, slope=1.03).

Assignments of proton populations in dough and bread using NMR relaxometry of starch, gluten, and flour model systems.

Starch-water, gluten-water, and flour-water model systems as well as straight-dough bread were investigated with (1)H NMR relaxometry using free induction decay and Carr-Purcell-Meiboom-Gill pulse sequences. Depending on the degree of interaction between polymers and water, different proton populations could be distinguished. The starch protons in the starch-water model gain mobility owing to amylopectin crystal melting, granule swelling, and amylose leaching, whereas water protons lose mobility due to increased interaction with starch polymers. Heating of the gluten-water sample induces no pronounced changes in proton distributions. Heating changes the proton distributions of the flour-water and starch-water models in a similar way, implying that the changes are primarily attributable to starch gelatinization. Proton distributions of the heated flour-water model system and those of fresh bread crumb are very similar. This allows identifying the different proton populations in bread on the basis of the results from the model systems.

Biopolymer interactions, water dynamics, and bread crumb firming.

To establish the relationship between biopolymer interactions, water dynamics, and crumb texture evolution in time, proton mobilities in starch and gluten model systems and bread were investigated with NMR relaxometry. Amylopectin recrystallization was observed as an increased amount of fast-relaxing protons, while network strengthening and changes in water levels were noted as a reduced mobility and amount, respectively, of slowly relaxing protons. Amylopectin recrystallization strengthened the starch network with concomitant inclusion of water and increased crumb firmness, especially at the beginning of storage. The inclusion of water and the thermodynamic immiscibility of starch and gluten resulted in local gluten dehydration during bread storage. Moisture migration from crumb to crust further reduced the level of plasticizing water of the biopolymer networks and contributed to crumb firmness at longer storage times. Finally, we noted a negative relationship between the mobility of slowly relaxing protons of crumb polymers and crumb firmness.

Mapping of Saccharomyces cerevisiae metabolites in fermenting wheat straight-dough reveals succinic acid as pH-determining factor

Fermenting yeast does not merely cause dough leavening, but also contributes to the bread aroma and might alter dough rheology. Here, the yeast carbon metabolism was mapped during bread straight-dough fermentation. The concentration of most metabolites changed quasi linearly as a function of fermentation time. Ethanol and carbon dioxide concentrations reached up to 60 mmol/100g flour. Interestingly, high levels of glycerol (up to 10 mmol/100g flour) and succinic acid (up to 1.6 mmol/100g flour) were produced during dough fermentation. Further tests showed that, contrary to current belief, the pH decrease in fermenting dough is primarily caused by the production of succinic acid by the yeast instead of carbon dioxide dissolution or bacterial organic acids. Together, our results provide a comprehensive overview of metabolite production during dough fermentation and yield insight into the importance of some of these metabolites for dough properties.

Reaction kinetics of gliadin-glutenin cross-linking in model systems and in bread making.

The gluten proteins gliadin and glutenin are important for wheat flour functionality in bread making, where, during baking, they polymerize through a heat-induced sulfhydryl-disulfide exchange mechanism. A model system was used to study the kinetics of this reaction. Thus, gluten was subjected to hydrothermal treatment with the rapid visco analyzer (RVA) with holding temperatures of 80, 90, and 95 degrees C. At these temperatures, omega-gliadin solubility did not change, but the solubilities of alpha- and gamma-gliadin in 60% ethanol decreased according to first-order reaction kinetics. All reaction rate constants increased with temperature. The activation energies for the heat-induced exchange reaction were 110 and 147 kJ/mol for alpha- and gamma-gliadin, respectively. Starch did not influence the reaction rates of the association of alpha- and gamma-gliadin with glutenin. During gluten-starch model bread baking, glutenin oxidized first, and when the internal crumb temperature reached 100 degrees C, alpha- and gamma-gliadin cross-linked to glutenin, again following first-order reaction kinetics. The experimental findings and similarities in temperature conditions and reaction kinetics suggest that the RVA system can be instrumental in understanding gluten behavior in concentrated food systems, such as bread making.

Identification of Intact High Molecular Weight Glutenin Subunits from the Wheat Proteome Using Combined Liquid Chromatography-Electrospray Ionization Mass Spectrometry

The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for further top-down proteomics of individual HMW-GS and the entire wheat glutenin fraction.

Identification of Isopeptide Bonds in Heat-Treated Wheat Gluten Peptides

Results in this paper confirm heat-induced isopeptide bond formation in wheat gluten. Heating (24 h, 130 °C) of wheat gluten [moisture content 7.4%] decreased its extractability in sodium dodecyl sulfate containing buffer (pH 6.8), even after reduction of disulfide (SS) bonds. Thus, both SS bonds and non-SS bonds were responsible for the extractability loss. Cross-links of the lysinoalanine and lanthionine type were not present in the heated samples, but heat treatment reduced levels of available amino groups. Heating of purified and alkylated high molecular weight glutenin subunits (HMW-GS) under similar conditions also resulted in extractability loss, demonstrating that cross-linking did not solely depend on the availability of cysteine or cystine. These observations indicated that heat treatment had induced isopeptide bond formation, resulting in larger and unextractable molecules. Heating HMW-GS lysine- and glutamine-containing peptides induced the formation of isopeptide bonds, thereby supporting the above hypothesis. The level of isopeptide bond formation increased with heating time.