Workpackage 3

The objective was to explore new descriptors that have potential impact on grain processing/product quality.
The focus was on grain water status, cell wall polymers, lipids and protein aggregation. Beyond their global content, the mobility of water and the aggregation and modification status of proteins was addressed. AX (arabinoxylans) composing the cell walls were studied as well, as they interact strongly with water and have recognized impact on cereal processing. The WP3 was led by BIA and involved IATE and SAYFOOD units.

Pentosans and lipids distribution in flours (BIA)
The total amount of AX, water-extractable AX (WE-AX) and water-unextractable AX (WU-AX) was determined in the flour after acid hydrolysis of polysaccharides and measurement on individual neutral sugars (Arabinose, Xylose, Galactose, Glucose) by gas chromatography. Specific viscosity and intrinsic viscosity from WE-AX were also established  using high-performance size exclusion chromatography equipped with a differential refractometer, a multi angle laser light scattering detector and a differential pressure viscometer. 
Starch and non-starch lipids were extracted and quantified by gas chromatography (as fatty acid methyl esters equivalent). There are also plans to look into the analysis of the composition of the main lipid fractions, i.e. non polar lipids (triglycerides, mono- and diglycerides, free fatty acids), glycolipids (monoglactosyldiglycerides, digalactosyldiglycerides and their lysoderivatives) and phospholipids (n-acyl phosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, their lysoderivatives…) by normal phase HPLC with a light scattering detection.

Water status in grain and flour (BIA)
Water status of grain was assessed by NMR relaxometry. This is a non-destructive approach, which is commonly used to investigate water contents, mobility, and interactions in matrix systems, such as plant organs (see here). Deconvolution of the multi-exponential curves obtained from T₂ relaxation allows identifying groups and proportions of water protons that share environments, interactions or molecular exchange rates. 
Here is a link to a poster that summarises the initial characterization plan for minor compounds in the evagrain project :

Protein characterization (IATE)
In addition to the classical SE-HPLC analysis of flour proteins (gliadin/glutenin ratio, glutenin polymers size distribution) the objective is also to assess for the occurrence protein free protein thiols contents, in quantity and nature. We made the assumption that the wheat protein free thiols (carried by gliadin or glutenin subunits with odd number of cysteine) could modify the extent of protein thiol/disulfide interchanges occurring during dough mixing and baking, thereby impacting the final quality of bread. We will use a high quantum yield specific fluorescent thiol probe (ATTO-maleimide) to block the total free thiols during flour protein extraction.
The flour protein extracts will then be analyzed by SEHPLC using dual detection (UV and fluorescence) in order to quantify the thiol/protein ratio, as previously described. In addition IATE will carry out, on selected samples, protein pattern analysis by reverse phase chromatography.

Dough rheological properties (BIA)
Dough rheology was characterized for a subset of samples (68) selected based on their composition and their behaviour at Mixolab and Alveograph tests. The rheology of the dough was characterised at large deformations by Lubricated Squeezing Flow test (LSF) as previously described. This test allows determining the extensional viscosity of the dough submitted to compression, whilst cancelling shear deformation thanks to constant lubrication of the sample surfaces during testing. It results in equibiaxial extension, and from the elongational flow curves, 2 indices are extracted: the consistency index and the flow behaviour index. Dough rheology was also characterised at small deformation by oscillatory measurements and its changes under temperature were assessed by dynamic thermomechanical analysis. 
The knowledge of dough rheological properties allowed to relate macroscopic observations to its microstructure.

Fast screening methods  
The project uses a front Face Fluorescence analyser developed by Spectralys to rapidly screen samples of grains and flours. Embedded software allows automatic and real time decomposition of the optical fingerprint that can be further correlated to the technological behaviour of the product. Near-infrared spectroscopy (NIRS) is widely used by wheat breeders in their programs to estimate some grain traits (hectoliter weight, grain hardness, W class, Protein content). NIRS is used to calculate prediction equations of classical parameters and possibly of new parameters obtained from the fine characterization of protein, cell wall components and water distribution.