# Oxidation in Low Moisture Foods as a Function of Surface Lipids and Fat Content
## 2 Materials and Methods
### Methods  
Cracker preparation (Figure 1) was adapted from [PERSON] et al. (2015) [10]. A mixture of flour (50.6 wt. %), salt (1.2 wt. %), and baking soda (0.5 wt. %) was added to interesterifiedoil (8.1 wt. %), as shown in Table 1. Ingredients were mixed using a Kitchen Aid mixer (model #KSM95, Mississauga, ON, Canada) for 2 min. Water (31.6 wt. %) was added, and the resulting dough was kneaded by hand with the incorporation of additional flour (8.1 wt. %) to minimize sticking. The dough was flattened by a pasta roller (Kitchen Aid KPSA attachment, Mississauga, ON, Canada; thickness setting 2, approximately 3 mm). For the surface oil analysis, the opening of a disposable test tube (13 \(\times\) 75 mm, Fisher Scientific, Hampton, NH, USA) was used to cut the dough into small round pieces that could be used to extract surface lipids. To evaluate the effect of lipid concentration on lipid oxidation and microstructure, the dough was cut into 2.5 \(\times\) 2.5 cm pieces before baking. All crackers were baked at 163 \({}^{\circ}\)C for 21 min. In the latter experiments, the fat content of the crackers was changed by altering the fat and water content while keeping all other ingredient concentrations the same. Table 1 shows the fat and water concentrations. For confocal imaging, a Rhodamine B stock solution was prepared by dissolving 1 g Rhodamine B in 15 mL ethanol. This hydrophilic fluorescent dye solution (40 \(\mu\)L) was added to the water prior to mixing it with other cracker ingredients. BODIPY 493/503 stock \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Ingredient** & **0.1\% Fat** & **1.0\% Fat** & **8.0\% Fat** & **20\% Fat** \\ \hline Flour & 62.5 (50.6\%) & 62.5 (50.6\%) & 62.5 (50.6\%) & 62.5 (50.6\%) \\ \hline DI Water & 48.82 (39.5\%) & 47.82 (38.7\%) & 39 (31.6\%) & 24.30 (19.7\%) \\ \hline Interesterified Soybean Oil & 0.18 (0.1\%) & 1.18 (1.0\%) & 10 (8.1\%) & 24.70 (20.0\%) \\ \hline Salt & 1.5 (1.2\%) & 1.5 (1.2\%) & 1.5 (1.2\%) & 1.5 (1.2\%) \\ \hline Baking Soda & 0.58 (0.5\%) & 0.58 (0.5\%) & 0.58 (0.5\%) & 0.58 (0.5\%) \\ \hline Flour for sprinkling & & & & \\ on surface & 10 (8.1\%) & 10 (8.1\%) & 10 (8.1\%) & 10 (8.1\%) \\ \hline TOTAL & 123.58 g & 123.58 g & 123.58 g & 123.58 g \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Ingredients used for cracker baking (g). Figure 1: Flow diagram of cracker baking. Insert: whole crackers. solution was prepared by dissolving 10 mg dye in 15 mL ethanol:chloroform solution (2:1). Lipophilic BODIPY (2.7 ug BODIPY 493/503/g lipid) was added to the fat prior to mixing. After baking, all crackers were allowed to cool to room temperature before handling. For the surface oil analysis, whole (round) crackers were directly placed in 10 mL glass gas chromatography (GC) vials (Supelco Analytical; Bellefonte, PA, USA). For the evaluation of the effect of lipid concentration on lipid oxidation, the 2.5 \(\times\) 2.5 cm square crackers were crumbled using a mortar and pestle, distributed (0.5 g) in 10 mL volume glass headspace vials, and sealed with silicone/polytetrafluoroethylene (PTFE) septa screw caps for the storage studies. Vials were incubated at 55 \({}^{\circ}\)C in corrugated cardboard containers. The use of light-protected carriers was crucial to avoid the increased effects of ultraviolet-visible (UV/vis) radiation on lipid oxidation rates. Incubation was a maximum of 45 days, with testing occurring every few days. _Confocal Microscopy Analysis_: A Nikon confocal microscope (C1 Digital Eclipse, Tokyo, Japan) with a PL FLUOTAR ELWD 20.0 \(\times\) 0.45 objective lens was used to capture the confocal images in intact 2.5 \(\times\) 2.5 cm crackers. Crackers containing both BODIPY and Rhodamine B were imaged sequentially, and the resulting images were overlaid. An air-cooled argon-ion laser (Model 376 IMA1010 BOS; [PERSON]; Carlsbad, CA, USA) was used to excite BODIPY at 488 nm, and emission spectra were collected at 515 \(\pm\) 30 nm.