Whatman 541 filter paper

Soluble and insoluble collagen were extracted and quantified using a protocol adapted from [11 (link),12 ] as outlined by Ebarb et al. [13 (link)]. Briefly, 3 g of ground muscle tissue was incubated in Ringer’s solution at 77 °C for 80 min. After centrifugation at 2250× g for 12 min at 20 °C, the supernatant containing the soluble collagen fraction was separated from the insoluble collagen containing precipitate. Sulfuric acid was added (3 mL concentrated sulfuric acid to soluble portion; 30 mL 3.5 M sulfuric acid to the insoluble portion) and fractions were incubated at 105 °C for 16 h to hydrolyze the collagen. Extracts were diluted, filtered with Whatman 541 filter paper (Fisher Scientific; Waltham, MA, USA), and hydroxyproline determination was carried out using a BioTek Eon spectrophotometer [14 (link)] (Biotek Instruments Inc., Winooski, VT, USA) reading absorbance at 558 nm. Collagen content was determined by multiplying hydroxyproline content of the soluble fraction by 7.25 and the insoluble fraction by 7.52 [15 (link)]. Total collagen content was determined by adding the collagen fractions together.

The concentration of NH₃-N was determined using the phenol-hypochlorite colorimetric method according to the study of Broderick and Kang (16 (link)). The VFA concentrations were measured by using GC-2010 (Shimadzu Laboratory Supplies Co., Ltd, Shanghai, China) equipped with a flame-ionization detector and a capillary column (HP-Innowax, 19091N-133, Agilent Technologies, Santa Clara, CA), as described by the study of Zhang et al. (3 (link)).
The MCP synthesis was estimated according to the study of Hall and Herejk (17 (link)). Briefly, 300 mg of freeze-dried fermentation residue was weighed, transferred into a 50 ml flask containing 10 ml of trichloroacetic acid (TCA; 19.4%), and the mixtures were then placed on ice for 45 min. Following centrifugation at 7,719 × g for 20 min, the whole flask content was filtered through Whatman 541 filter paper (Fisher Scientific, Atlanta, GA). Subsequently, the filtrate was filtered again through a Whatman GF/A glass fiber filter (Fisher Scientific, Atlanta, GA) using 2% TCA to rinse the flask. Both Whatman 541 and GF/A filters were dried at 55°C overnight and then subjected to Kjeldahl analysis.