6n hcl

Defatted milk (250 mL) was added to an equal volume of distilled water (DW), and after pre-warming at 37°C for 10 min, the pH was adjusted to 4.6 with 6N HCl (Wako, Osaka, Japan) to promote casein aggregation [30 (link)]. Casein was separated by centrifugation at 5,000×g at 25°C for 20 min using an R14A rotor (Hitachi Koki) in a Himac CR20GII centrifuge (Hitachi Koki). The supernatant was filtered as described above in order to obtain whey.

Total collagen was estimated by measuring the hydroxyproline, which is an amino acid characteristic of collagen. A portion of the sample was excised with a knife. The weight of the excised tissue was measured, the organ fragment was transferred to a biosmasher tube (Nippi), and four volumes of tissue weight 6 N HCl (Wako) was added and homogenized. The liver samples were hydrolyzed for 12–15 h in 5 mL of 6 N HCl at 96 °C. After removing from the heater and cooling at room temperature, these samples were centrifuged at 15,000 rpm for 5 min. Then, we collected an appropriate amount of the sample and added 0.5 volume of H₂O. After preparing 20 μL of the sample, we added 75 μL of 50% 2-opropanol in citrate-acetate-buffered chloramine T. The mixture was vortex and mixed. Subsequently, we added 75 μL of a mixture of Ehrlich’s reagent (2-propanol (Wako), Dimethylaminobenzaldehyde (Sigma) and perchloric acid (Sigma). Tubes were incubated for 10 min in a water bath at 60 °C. The samples were placed on the range of 560 nm absorbance band in Hitachi spectrophotometer (U-2000). Results were expressed as μmol hydroxyproline/g of liver tissue.