Pure acetic acid

The DNA concentration in the matrix was estimated using the Dische method (Dische, 1930 ). This method is based on the reaction of desoxyribose with diphenylamine in acetic acid. When heated, levulinic aldehyde is produced, which then condenses with diphenylamine to form a colored compound. Briefly, the diphenylamine solution was prepared by dissolving 1 g of diphenylamine (Sigma) in 100 ml of pure acetic acid (Sigma, United States). Then, 2.75 ml of 96% SA was added. A calibration series of standard DNA solutions (50–500 μg/ml) was then prepared using herring sperm DNA (Sigma), and the matrix samples were prepared for analysis. All samples (2 ml) were distributed into glass tubes. Then, a double amount of Dische reagent (4 ml) was added, and the samples were heated for 15 min at 100°C in a water bath. The OD595 values were measured after cooling. MQ water was used as a control.

Stored rat feces were added to HPLC-grade water (Sigma-Aldrich) and sonicated to prepare tissue homogenate. Following centrifugation of the homogenate at 13,000× g for 3 min, the supernatant was filtered through a 0.2 μm filter (Millipore, Bedford, MA, USA) Prepared samples were analyzed using an Agilent 1220 Infinity LC system (Agilent, Santa Clara, CA, USA) equipped with an Aminex-87H column (150 mm, 4.6 mm, Bio-Rad, Hercules, CA, USA) and a diode array detector. Pure acetic acid, propionic acid, and butyric acid (Sigma-Aldrich) were used as standards. Samples were eluted with 0.008 N sulfuric acid for 30 min at a flow rate of 0.6 mL/min. All chromatograms were obtained using a wavelength of 215 nm and data acquisition and calculation was performed using Agilent chemstation software (Agilent).