Materials and Methods

The ingredients used were potato starch (Emsland group, Germany), white rice flour (Doves Farm Foods Ltd, UK), dry yeast (Puratos, Belgium), margarine (Storck Unilever, Ireland), whey protein isolate (Isolac®, Carbery group, Ireland), sugar (Siucra, Ireland), salt (Glacia British Salt Limited, UK), Xanthan gum (SUMA Whole Foods, UK), HPMC (Dow Wolff Cellulosics, Germany), bakers flour from hard wheat (protein content 11.5 %; moisture 13.8 %) (Odlums, Ireland) and tap water. Beta-glucan powder extracted from oats was purchased from Bioatlantis (Ireland) and inulin Raftilose P95 from Beneo – Orafti (Belgium).

5.3.2 Rheology on gluten free batters and wheat dough

For the rheological measurements a Physica MCR 301 rheometer (Anton Paar Germany GesmbH, Germany) equipped with parallel plate geometry, consisting of a 50 mm diameter corrugated probe and plate, was used. A circulating water bath and a controlled pelletier system were utilised to set the temperature to 30 °C. Batter or dough samples, prepared as described for the bread making procedure (without the addition of yeast) were loaded onto the serrated plate. The probe was lowered to a gap width of 1 mm and the exposed edges of the samples were trimmed off. Samples rested for 5 min to allow equilibration and recovery. The edges of the samples were covered with mineral oil and the test was run in a humidified chamber to reduce water loss during testing. Storage modulus (G’), loss modulus (G’’), complex modulus (G*) and phase angle (δ) were calculated using the manufacturers software (Rheo- plus, AntonPaar, Germany).

The linear viscoelastic range and the target strain were determined using amplitude sweeps applying strain (γ) increasing from 0.001 to 100 % at a constant frequency (ω) of 10 Hz. Frequency sweep tests were performed at frequencies between 0.1 and 50 Hz with a target strain of (0.01 %). All results are the average of two measurements.

5.3.3 Determination of the water addition level

The water level was determined as previously described by Nunes et al. (2009). Briefly, a single frequency test was performed at an angular frequency of 10 Hz and a strain of 0.01 %. Firstly, the complex modulus of the control formulations was determined. Then the complex moduli of the fiber containing samples were determined and the water level was altered until the complex modulus equalled that of the control formulation. Ten measuring points of G*, were recorded during each measurement and the calculated average value was used. Results of the single frequency test are the average of three measurements.

5.3.4 Bread production

Gluten free breads were prepared as described by Nunes et al. [2008]. The wheat dough was prepared, using a standard recipe (Ryan et al. 2008). Water and functional ingredients were incorporated at the levels shown in Table 5.1. Yeast and sugar were suspended in water (35 °C) and regenerated for a period of 10 min in a proofer (KOMA sunriser, Roermond, The Netherlands) set to 30 °C at a relative humidity (RH) of 85 %. This was added to the premixed dry ingredients. For the gluten free batter margarine was also added. Mixing was then carried out at low disk speed with a Kenwood chef classic equipped with a batter attachment (3.5 min for wheat dough and 1 min for the gluten free formulation). The bowl containing the gluten free batter was scraped down and a further mixing of 2 min at a higher disk speed was carried out. Bulk fermentation for the wheat dough was carried out for 15 min at 30 °C, 85 % RH. Then wheat doughs and gluten free batters were scaled to 90 g into 9 baking tins of 10 x 5.5 x 3.5 mm and placed in a proofer for 45 min and 25 min, respectively (30 °C, 85 % RH). The breads were baked for 25 min at 230 °C top and 240 °C bottom heat in a deck oven (MIWE condo, Arnstein, Germany), previously steamed with 0.3 L of water. Wheat and gluten free bread loaves were removed from the tins and cooled down at room temperature. The loaves were subsequently analysed or packaged in containers (polystyrol-ethylene vinyl alcohol-

polyethylene) under modified atmosphere (60 % N2 and 40 % CO2). Three batch replicas were

prepared.

5.3.5 Bread analysis

Loaf specific volume were analysed upon cooling using rapeseed displacement method (AACC Method 10-05.01). Bake loss was determined by substracting loaf weight from dough weight. Moisture was determined using the AACC approved air-oven method (44-15A). Crumb texture and crust colour were determined after cooling at 2 and 5 days of storage. The two bread slices (20 mm thickness) taken from the centre of each loaf were used to evaluate the crumb texture. Texture profile analysis (TPA) was performed using a TA-XT2i texture analyser (Stable Micro Systems, Surrey, UK) equipped with a 25 kg load cell and a 20 mm aluminium cylindrical probe. The settings used were a test speed of 5 mm/s with a force of 0.98 N to compress the middle of the breadcrumb to 50 % of its original height. Crust colour was analysed with a Chroma Meter (Minolta CR-300, Japan) and expressed according to the CIE L*a*b* colour system. Three loaves per batch were analysed.

5.3.6 Beta-glucan degradation and molecular weight distribution

The level of β-glucan after baking was assessed with an enzyme test kit (Megazyme, Ireland) for mixed-linkage β-glucan. The molecular weight of β-glucan was estimated using a high performance size-exclusion chromatography system as described by Galle et al (2010). Beta- glucan was extracted from the bread sample with 1 N NaOH for 16 hrs on a shaker at room temperature, neutralized with 1 N HCl and precipitated by adding 2 volumes of 96 % ethanol and overnight storage at 4 °C (Tieking et al. 2003). Beta-glucan was collected by centrifugation; the pellet was redissolved in MES-TRIS blend buffer (pH 8.2) and treated with heat-stable α- amylase and protease (Megazyme, Ireland). Beta-glucan in solution were precipitated again with ethanol and freeze dried. The freeze dried samples were dissolved in Millipore H2O [2 mg

mL-1] at 60 °C and analysed using a Superdex 200 Column (GE Healthcare, Canada). Water was

The standards of known molecular weight used were inulin (104 Da) (Sigma, Canada) as well as

high and low molecular weight dextran (5x106-4x107 and 105 Da) (Sigma, Canada).

5.3.7 Confocal Laser Scanning Microscopy

Methyl blue (Sigma, Ireland) 0.2 % in 150 mM KH2PO4 was used to selectively stain β-glucan.

Bread samples were soaked in the dye solution over night and destained in 150 mM KH2PO4

for 15 min. Proteins and starch were stained for 1 min with 1 % solution of fluorescence isothiocyanate (FITC) (Sigma, Ireland) in N,N-Dimethylformamide (Sigma, Ireland) and rinsed with N,N-Dimethylformamide for approximately 30 min. An MRC-1024 confocal laser-scanning system (Biorad, Herts, UK) mounted on an upright microscope (Axioskop, Zeis, Germany) with 10x and 20x objectives was used. Fluorescence images of a number of optical sections were acquired by scanning the sample along the optical axis in 1.83 µm steps. For methyl blue and isothiocyanate, respectively, the 405 nm and 488 nm excitation line was used and signals were collected through a BA430-460 and a BA510IF emission filter.

5.3.8 Statistical analysis

Multiple samples were compared with PASW Statistics 18 (SPSS Inc., Chicago, Illinois). A One- way ANOVA and Tukey’s Honesty significant differences post hoc test were used to describe means at a significance level of p<0.05. For loaf specific volume, values are the average of nine measurements. All other values are the average of triplicate measurements.