Lactose standard

Organic acids and lactose were extracted using a method modified from Upreti et al. (2006) . Permeates were analyzed at 10% solids. One hundred microliters of each sample was extracted in 900 μL of 0.013 N H 2 SO 4 (Sigma Aldrich, St. Louis, MO), vortexed, and centrifuged at 8,000 × g at room temperature (21°C) for 5 min. The top layer was removed and filtered using 0.45-μm nylon syringe filters (VWR International, West Chester, PA). Twenty microliters of each sample was injected on the HPLC (Waters 1525 Binary Pump, Waters, Milford, MA) using an autosampler (Waters 2707 Autosampler) onto the column (0.8 mL/min, 300 × 7.8 ion exclusion, 55°C; Bio-Rad Labs, Richmond, CA). Each replicate of each permeate was injected in duplicate. The temperature of the injector was 4°C and a photodiode array detector (Waters 2998) was used for organic acids and a refractive index detector (Waters 2414 Refractive Index Detector, 30°C) was used for lactose. The maxima used for organic acid calculation was 254 and 285 nm due to citric and orotic acids coeluting at 254 nm. Citric acid was not detected at 285 nm. A standard curve was created for each organic acid and lactose. Organic acid standards were obtained from Thermo Fisher Scientific Inc. (Pittsburgh, PA) and lactose standard was obtained from Sigma Aldrich.

Acetonitrile HPLC and LC/MS grade, water LC/MS grade, Formic acid LC/MS grade, Lactose standard, Lactulose standard, Ethanol HPLC grade and D-Lactose-1-13 C monohydrate, Potassium hexacyanoferrate (II) trihydrate, zinc sulphate, trichloroacetic acid were bought from Sigma Aldrich (US-MO).