No 1 filter paper

Soft-shelled turtle fat obtained from Sanhe Biotech (Ligang, Pingtung, Taiwan) was used to prepare SSTO. Briefly, 100 g of fat were placed in a steel pan, stirred and heated to 100, 200, 300, or 400°C in water. An infrared thermometer was used to determine the temperature of the material. After the target temperature had been reached, the fat was continually stirred for an additional 10 min to obtain SSTO. After cooling, the oil was centrifuged at 10,000 × g and filtered through Advantec No. 1 filter paper to remove solid impurities or precipitation from the oil. The purified turtle oil was stored at 4°C.

Adding 50 mL TSB medium (Tryptone Soya Broth, HIMEDIA^®, Shenzhen, China) and 3% feathers into a 250 mL flat-bottomed Erlenmeyer flask. After sterilization (121 °C, 20 min), inoculate 0%, 5% Bacillus subtilis var. natto N21 (N21) and mixed strains culture (N21, B. subtilis Da2 (Da2) and Da15 (Da15), B. amyloliquefaciens Da6 (Da6), Da16 (Da16)) (10⁹ cfu/mL), and incubate at 37 °C, 100 rpm, for 0, 24, 48 and 72 h. The hydrolysate was passed through a filter paper (No. 1 filter paper, ADVANTEC^®, Tokyo, Japan) to remove unhydrolyzed feathers at different times. The feather degradability was calculated using the following formula:
where A is the dry weight of the feathers before degradation, B is the dry weight of the feathers and filter after degradation, and C is the dry weight of the filter.

Valerian powder (15 g) was added to 150 mL of water or various concentrations (25%, 50%, 75%, and 95%) of ethanol in a shaking water bath at 150 rpm for 30 min at 25–75 °C, then centrifuged at 6932× g (10,000 rpm, 20 min), and filtered through Advantec No.1 filter paper. The residue was re-extracted with 150 mL portions of the solvent, as described above. The combined filtrate was concentrated under reduced pressure at a 50 °C water bath and freeze-dried. The lyophilized extracts obtained by extracting the samples with water, and 25%, 50%, 75%, and 95% ethanol in a 25 °C water bath were labeled 25C0E, 25C25E, 25C50E, 25C75E, and 25C95E, respectively. The first set of numbers and letters indicates the bath temperature in Celsius, and the second set of numbers and letters indicates the percentage of ethanol. All treatments were randomly generated and performed in quadruplicate.

Decayed P. densiflora blocks were removed from the incubation bottle. Blocks surface were carefully cleaned from fungal mycelium. The woods were freeze-dried overnight, cut into smaller pieces, and ground using a mortar and pestle to obtain decayed wood powder. Five grams of the decayed wood powder was inserted into a 200 mL conical flask and subjected to extraction with 100 mL of n-hexane at 18–25 °C overnight (10–12 h). The mixture was filtered using filter paper (No. 1, Advantec Toyo Roshi, Tokyo, Japan) and concentrated using a rotary evaporator into a 10 mL solution.
For extract preparation from fungal cultures cultivated on growth media, the mycelium-covered medium surface was carefully scraped using a spatula; the collected material was ground with a glass stirrer, and then added to a 300 mL conical flask. The ground medium (100 g) was extracted with 100 mL of n-hexane overnight or up to 24 h. The mixture was filtered using No. 1 filter paper (Advantec Toyo Roshi) and concentrated to 5 mL in a rotary evaporator at 10 °C, then refrigerated (2–3 °C) for a maximum of 14 days after extraction.

The antioxidant activity of the sample was determined using a modified 2, 2-diphenyl-1- picrylhydrazyl (DPPH) free radical scavenging assay [30 (link)]. A quantity of 5 g of chicken meatball after each F-T cycle was homogenized (BagMixer^® 400W, Interscience, St. Nom la Bretêche, Île-de-France, France) with 45 mL of 0.1 M sodium phosphate buffer (pH 7.0) for 2 min. Then, the homogenate was filtered using No.1 filter paper (ADVANTEC, Bunkyo, Tokyo, Japan) to remove impurities and then centrifuged at 12,000× g for 30 min. A volume of 1 mL of 0.2 mM DPPH dissolved in methanol (Sigma-Aldrich Co., St. Louis, MO, USA) was added to 200 μL of the supernatant and 800 μL of distilled water in 15 mL of the conical tube. The mixture was vortexed and placed in a darkroom for 30 min. The conical tube containing 1 mL of methanol and 1 mL of 0.2 mM DPPH dissolved in methanol was used as the control, whereas 2 mL of methanol alone was used as a blank. The absorbance of the sample solution was measured using a spectrometer-based ELISA reader at 517 nm. The scavenging activity of the sample against the DPPH radical was calculated using the following equation: $\%\mathrm{inhibition}\mathrm{of}\mathrm{DPPH}=\frac{\left[\left(\mathrm{Abs}\mathrm{control}-\mathrm{Abs}\mathrm{blank}\right)-\left(\mathrm{Abs}\mathrm{sample}-\mathrm{Abs}\mathrm{blank}\right)\right]}{\mathrm{Abs}\mathrm{control}}\times 100$