Chemical Analyses

At the end of the collection period, pooled individual feces were thawed, homogenized, and freeze-dried. Feces samples were freeze-dried using a laboratory freeze dryer (5Pascal, Trezzano sul Naviglio, Italy). The process of lyophilization consisted of dry sublimation with water evaporation under low pressure (0.200 mbar) until the samples reached room temperature (25°C). Both the foods and freeze-dried feces were ground to pass through a 1-mm sieve and stored in airtight plastic containers for laboratory tests. The dry matter (DM) of the foods was determined by drying the samples at 103°C to constant weight. The foods and feces were analyzed according to the AOAC (29) standard procedures; thus, ash was determined by muffle furnace incineration (section 942.05), crude protein (CP) was ascertained using the Kjeldahl method (section 954.01), and ether extract (EE) was analyzed following acid hydrolysis (section 954.02). In addition, diet crude fiber (CF) was determined using the method described in section 962.09 (29), and amino acid content by HPLC (Waters Alliance System with a Waters 1525 Binary HPLC Pump, Waters 2707 Autosampler, and Waters 2475 Multi λ Fluorescence Detector, Milford, USA) after pre-column derivatization (30) in samples ground to pass a 0.5-mm sieve. The detection limit ranged from 2.9 to 20.1 pmol/μl depending on the amino acid. Tryptophan was not analyzed.

Samples of foods and feces were burnt to ashes and acid-digested in the microwave (31), prior to the determination of chromium concentrate by inductively coupled plasma optical emission spectrometry (ICP-OES). Calcium and phosphorus were also determined by ICP-OES in the absence of the previous incineration.

Hydroxyproline and the related collagen content were assessed according to the colorimetric method adapted by Kolar (32) and described in the AOAC (29) section 990.26. The acid hydrolysis of the sample was performed under heat; an oxidizing agent was added to the sample, and oxidized hydroxyproline was measured photometrically.