Food-grade vanillin, propylene glycol (PG) and triacetin (TA) were supplied by Aromco Ltd. (Nuthampstead, UK). Two simple vanilla flavours were made by mixing vanillin (10 % w/w) with PG or TA as the flavour solvent. Both flavourings were made on the day of application, the standard application dosage for both flavourings was 0.2 % w/w in the biscuit dough (i.e. 200 ppm of vanillin was added initially).
Acetovanillone (≥98 %, SAFC Supply Solutions, St. Louis, USA) was used as the internal standard (IS) for HPLC detection. Methanol (HPLC Grade ≥99.9 %) was ordered from Fisher Scientific UK Ltd, Loughborough, UK. Acetic acid (≥99.85 %) and 5-hydroxymethyl-furfural (HMF, ≥99 %) were purchased from Sigma Aldrich, UK.
Preparation of standard biscuits
Standard dough was prepared from the ingredients listed below: (1) shortening 15 g/100 g; (2) icing sugar, 15 g/100 g; (3) invert sugar, 2 g/100 g; (4) skimmed milk powder, 0.3 g/100 g; (5) salt, 0.3 g/100 g; (6) lecithin, 0.3 g/100 g; (7) sodium bicarbonate, 0.14 g/100 g; (8) ammonium bicarbonate 0.03 g/100 g; (9) water 11 g/100 g; (10) biscuit flour 56 g/100 g; (11) flavouring, 0.02 g/100 g. Shortening was supplied by Cardowan Creameries Ltd (Glasgow, UK), and the biscuit flour was sourced from Rank Hovis (High Wycombe, UK). All other ingredients were supplied by C Holland & Sons Ltd. (Royston, UK).
The ingredients from No. 1 to No. 6 were weighed and blended by a spade blender (Hobart, Windsor, UK). A fat base was then formed after 2 min of continuous mixing of these ingredients. A water base was prepared by dissolving the Ingredients No. 7 and No. 8 in cold tap water. Standard dough was made by gradually mixing flour into the fat base then added the water base into the fat base using a spade blender. The flavouring (PG or TA) was then added and blended to homogeneity by spade blender. Each dough preparation was then rolled to 40 mm thickness using a Pastry Brake (Seewer Rondon, Burgdorf, Switzerland) and shaped by a model cutter (36 mm diameter, round with fluted edge) to produce individual biscuits.
The biscuits with PG and TA flavouring were positioned in alternating rows, with equal separating distances on the same tray to reduce baking variation. The tray of biscuits was placed on the top layer of a Deck Oven (Sveba-dahlen, Fristad, Sweden) and baked at 230 °C for 8 min on the top layer and were then dried on the bottom layer of the oven for 3 min at 100 °C. Finally, the baking tray was removed from the oven to allow the biscuits to cool for 10 min at room temperature (25 °C). Biscuits at the edge of the tray were discarded to minimise baking variation that was known to occur in these positions . The biscuits were then carefully packed and stored in sealed aluminium bags with a minimum headspace within the bag, moisture content was analysed as per Fisk .
X-ray μCT analysis
The microstructure of three PG biscuits and three TA biscuits was analysed by X-ray μCT using a Pheonix Nanotom NF180 X-ray CT System (GE Sensing & Inspection Technologies GmbH, Wunstorf, Germany). Triplicate biscuits were fixed together in the position shown in Fig. 1a, in order to be rotated and scanned as one stack. The scan consisted of 1,440 projection images collected over a 360° rotation using an electron acceleration energy of 80 kV, a current of 180 μA and a scan resolution of 22.5 μm. A 3D rendered model of the biscuit after reconstruction is illustrated in Fig. 1b.
All images were analysed by ‘Image J’ processing software version 1.44 (public domain Java analysis programme, developed by National Institute of Health, Maryland, US) .
Without any physical destruction, a cross-sectioned view of the top (XY slice) and the front slice (ZX slice) for both PG and TA biscuit were extracted from the CT scan, illustrated in Fig. 2. The white areas (high X-ray attenuation) show the biscuit matrix, and the black areas (low X-ray attenuation) inside the biscuits indicate pores (air space).
A ‘remove dark outliers’ filter with a radius of 0.5 pixels was applied to reduce binary noise in the images. Image J was then used to analyse the average size of all the pores in a defined region of the biscuit and area fraction (also known as the porosity) using the ‘Analyze Particles’ function in Image J.
Hundred reprehensive XY slice images were selected per biscuit. The central region (area = 25 × 25 mm) was used in every biscuit layer and the exterior was discarded. The image analysis settings as described were applied for all six biscuit samples. A total of 300 images were generated for either PG or TA biscuits based on triplicate samples.
Additional investigations were carried out at four different blocks within one biscuit (13.5 × 9 mm) as demonstrated in Fig. 3. Similar image analysis was carried out for each block per biscuit with 100 XY image slices, therefore generating 1,200 slices in total.
Aroma distribution analysis
Replicates of standard biscuits were cut across with a sharp knife from left to right into seven pieces (A1–A7, as shown in Fig. 4). Three PG biscuits and three TA biscuits were used to assess the average vanillin and HMF concentration in each section. A further three reps of TA biscuits and PG biscuits were sectioned into four layers from top to bottom (L1–L4, as shown in Fig. 4). Similarly, each layer was weighed and vanillin and HMF concentration quantified in each layer.
Each biscuit piece (~0.11 g) was weighed and extracted with 1 ml methanol, 10 μl of the internal standard (IS) was added prior to extraction. The IS consisted of acetovanillone (100 mg) in 100 ml methanol. All samples were then placed on a roller mixer (Thermo Scientific, Tube roller Spiramix 10) to roll side by side for 30 min and then centrifuged at 1,300×g for 20 min at 5 °C (Thermo CR3i Multifunction Centrifuge, KeyWrite-DTM). The upper solvent layer was isolated, and 0.5 ml of the extract was filtered (nylon syringe filter 4 mm 0.4 μm) into 2-ml amber vials, capped with Teflon-coated lids and analysed by HPLC.
The HPLC (Alliance® Waters 2095, Waters Corporation, Massachusetts, USA) fitted with a photodiode array detector (PDA, Waters 996) was used. Compounds were measured at an absorption wavelength of 270 nm and separated by a C18 column (C18 Techsphere, 5 μm, 250 × 4.6 mm, Thermo Scientific, Manchester, UK). The instrumental settings were as follows: injection volume 10 μl; flow rate 1 ml per min; gradient elution with (1) water (1 % acetic acid) and (2) methanol (ramped from 20 % B v/v to 50 % B v/v over 30 min then to 100 % B v/v over 1 min and held for 2 min). The chromatography data were analysed by Millennium32 software (Waters, USA). The retention time for authentic standard of HMF, vanillin and acetovanillone was 5.1, 15.0 and 17.9 min, respectively. Concentration (ppm, μg/g) was calculated from the ratio of the peak area of compound of interest to the peak area of internal standard.
All results were analysed by SPSS 16.0 (SPSS Inc., Chicago, USA) to calculate the average (AV) and standard deviation (STD) of pore size and porosity for both PG and TA biscuits. ANOVA followed by Tukey’s multiple comparison tests was applied where appropriate (p < 0.05). Pearson’s correlations with two-tailed test were applied to the distribution of vanillin and pores (porosity and average pore size) in the defined areas for both PG and TA biscuit, p < 0.05.