Model u 2001

The pre-cultured cells were washed three times with saline and resuspended in saline, and the bacterial cell suspension was prepared in saline at an optical density (OD) value at 660 nm of 1.0 with a spectrophotometer (Model U-2001, (Hitachi, Ltd., Tokyo, Japan)). The cell suspension was inoculated into tested broth at a ratio of 0.1% (v/v) and incubated for 15 h at 37 °C under the anaerobic conditions. The tested broth was composed of (l⁻¹) 2.8 g of Difco Thioglycollate Medium Without Dextrose or Indicator (Becton, Dickinson and Co.), 1.0 g of Oxoid Lab-Lemco Powder (Thermo Fisher Scientific Inc., Waltham, MA, USA), 0.2 g of ammonium citrate, 1.0 g of sodium acetate, 0.05 g of Tween 80, 0.04 g of MgSO₄–7H₂O, 0.002 g of MnSO₄–4H₂O, 0.002 g of FeSO₄–7H₂O, 0.002 g of NaCl, and 2.0 g of carbohydrate (pH 5.8). Carbohydrates included were glucose (98% purity, Wako Pure Chemical Industries, Ltd., Osaka, Japan), kestose (99% purity, B Food Science Co., Ltd., Aichi, Japan), and raffinose (98% purity, Wako Pure Chemical Industries, Ltd.), and sugar-free medium served as a negative control. raffinose, a common prebiotic, was used for a reference. Growth was monitored using the OD value at 660 nm with a spectrophotometer.

A focused ultrasonic extractor (20k Hz, 1400 W, Ever Great Ultrasonic Co., New Taipei City, Taiwan), spectrophotometer (Model U-2001, Hitachi Co., Tokyo, Japan), benchtop centrifuge (HERMLE Z300, Gosheim, Germany), mini-protein system (Bio-Rad, CA, USA), RF with hot air equipment (40.68 MHz, 10 kW, Yh-Da Biotech Co., LTD., Yilan, Taiwan), oven (Channel DCM-45, Yilan, Taiwan), digital pocket refractometer (Pocket, 3810, PAL-1, ATAGO Corp., Tokyo, Japan), and multifunctional infrared thermometer (Testo104-IR, Hot Instruments Co., LTD., New Taipei, Taiwan) were employed.

The method of Arnon (39 (link)) was followed for the determination of photosynthetic pigments. Leaf samples were collected separately from each pot in triplicates. In total 0.2 g of fresh leaves of each sample were taken and ground well separately. Then the grounded samples were mixed up with 80% of acetone. Ten milliliter of each plant triplicates were made by mixing 80% of acetone and were placed in a dark place in the laboratory for 48 hours. Then the samples were run on a centrifuge machine to collect the supernatant which was then analyzed in a spectrophotometer (Hitachi, Model U2001, Tokyo, Japan). The absorbance of solution was measured at 480, 645, and 663 nm for carotenoids, chlorophyll a, and chlorophyll b, respectively. The following formulae were used: