Sl 40r

From a 30 ml log phase SGL culture (OD₆₀₀ ≈ 1.5–2.5) of each indicated strain, 6 ml aliquot were transferred to separate tubes containing proline (10 mM final concentration) or ddH₂O. Cultures were incubated for 2 h at 30°C with aeration and then a 100 μl aliquot was transferred to each well of a round bottom 96-well microplate placed on ice. Exactly 100 μl of 10% TCA was added to each well and were allowed to incubate for 15 min protected from light. The direct extraction of P5C in TCA is crucial as P5C is stable in acidic condition [38 (link)]. Using a multichannel pipette, a 50 μl volume of 2-aminobenzaldehyde (2-ABZ) solution in 99.5% ethanol was added to each well and then incubated on ice for another 5 min. Plates were centrifuged at room temperature for 5 min at 4,000 rpm (SL 40R, Thermo Scientific). Supernatants (100 μl) were transferred to a fresh 96-well microplate (clear, flat bottom) and then immediately analyzed for absorbance at 444 nm using the Enspire microplate reader (Bio-Rad). All readings were normalized to cell density (OD₆₀₀) and were presented as fold change relative to wildtype strain grown without proline.

On day 42, two randomly selected birds per pen were euthanized by intracardiac injection with T61 (0.1 ml/kg BW; Intervet Nederland BV), and jejunal digesta samples were taken for viscosity measurement and gizzards were collected. Jejunal digesta samples of two birds per pen were pooled and mixed thoroughly. Samples were centrifuged for 10 min (3500×g, 4°C, centrifuge model SL40R; Thermo Scientific, Thermo Fisher Scientific, Langenselbold, Germany). Viscosity of the filtered supernatant (0.5 ml) was measured at 6 r.p.m. at 20°C using a viscometer (Model LVCP; Brookfield Eng Labs Inc., Stroughton, MA, USA). The results of the viscosity measurements are reported in Cps. Full and empty gizzard weight were determined.

Bean flour of the 18 accessions was obtained by milling (A11 mill, IKA, Staufen, Germany) and passing through MESH 60. Later, maceration was carried out at room temperature from 16 h by weighing 15 g of bean flour (dry weight, dw) and 150 mL of acetone solvent mixture (acetone, water, and acetic acid, 70:29.5:05 v/v/v). Then, the extracts were centrifuged at 3488× g for 15 min (SL 40R, Thermo Scientific, Waltham, MA, USA), washed with the solvent mixture once, and the supernatant was retained. Finally, the extract was concentrated using a rotary evaporator at 45 °C and 450 mbar (R-210, BUCHI, Flawil, Switzerland). The extracts were frozen at −20 °C until analysis [13 (link),20 (link)].