# Oxidation in Low Moisture Foods as a Function of Surface Lipids and Fat Content
## 2 Materials and Methods
### Methods
A Melles Griot helium-neon laser (Model 05-381 LGP-193; Carlsbad, CA, USA) was used to excite Rhodamine B at 543 nm, and emission spectra were collected at 605 \(\pm\) 75 nm. Detector pinhole size was always 150 \(\upmu\)m. All resulting images consisted of 512 \(\times\) 512 pixels, with a pixel size of 414 nm and a pixel dwell time of 10.40 \(\upmu\)s. Confocal microscopy images were analyzed using EZ-CS1 (version 3.8) software (Nikon; Melville, NY, USA). _Water activity measurement_: Crushed samples were used to measure water activity using a Decagon Devices AquaLab Series 3 water activity meter (Pullman, WA, USA). _Lipid isolation_: For the evaluation of the effect of lipid concentration on lipid oxidation, the crushed crackers (0.105 \(\pm\) 0.001 g) were extracted with 5 mL chloroform:methanol solvent (2:1 \(\
u\)/\(\
u\)) by vortexing for 30 s, followed by centrifuging (Centrific TMCentrifuge, Thermo Fisher Scientific Inc., Fairlawn, NJ, USA) at 3400\(\times\)\(g\) for 10 min and isolation of the solvent phase. On the other hand, for surface oil analysis, two whole crackers that weighed 0.5 \(\pm\) 0.05 g each were selected and extracted with chloroform:methanol solvent (2:1 \(\
u\)/\(\
u\)) at the ratio of 1:7.5 (cracker:solvent) by gently shaking for 1 min. All lipid concentrations were normalized using actual sample weights. _Primary lipid oxidation products_: The [PERSON] (1994) method was used to monitor lipid hydroperoxide formation [27]. This method uses a modified version of the International Dairy Federation method. After lipid extraction, the solvent phase (200 \(\upmu\)L) was mixed with 16.7 \(\upmu\)L of 1:1 FeSO4 (in water):BaCl2 (in 0.4 N HCl) solution. Then, 16.7 \(\upmu\)L of NH4 SCN in water was added. The absorbance was measured at 500 nm after 20 min using a Genesys 20 spectrophotometer (ThermoSpectronic; Waltham, MA, USA). A standard curve was prepared with cumene hydroperoxide. _Secondary lipid oxidation products_: The formation of a secondary oxidation product, hexanal, was monitored by a GC-2014 Shimadzu gas chromatograph equipped with an AOC-5000 autosampler (Shimadzu; Kyoto, Japan) using the method by [PERSON] et al. (2009) [28]. A 50/30 \(\upmu\)m divinylbenzene/carboxen/polydimethylsiloxane (DVB/Carboxen/PDMS) solid-phase microextraction (SPME) fiber (Supelco, Bellefonte, PA, USA) was inserted through the vial septum and exposed to the sample headspace for 10 min at 55 \({}^{\circ}\)C. The SPME fiber was desorbed at 250 \({}^{\circ}\)C for 3 min in the gas chromatograph detector at a split ratio of 1:7. The temperatures of the oven, injector, and flame ionization detector were 65, 250, and 250 \({}^{\circ}\)C, respectively, and the samples ran for 6 min. Volatile aldehydes were separated on a fused-silica capillary column (30 m \(\times\) 0.32 mm internal diameter \(\times\) 1 \(\upmu\)m) coated with 100% PDMS (Equity-1, Supelco, Bellefonte, PA, USA), and peak integration was calculated using Shimadzu EZstart (version 7.4) software. A standard curve was prepared by using a standard hexanal solution in methanol added to crackers, and concentrations of hexanal in the samples were calculated using this curve. Statistical analysis: All experiments were conducted in triplicate. All calculations compared the means by analysis of variance (ANOVA) and used a significance level of \(p<0.05\). Lag phases for the oxidation studies were identified as Day 1, which had significantly higher (>0.05) concentrations of oxidation products than Day 0. All calculations were performed using IBM SPSS Statistics 20 statistical software.