Lb medium

The strain of E. coli K88 (O8: K87, K88ac) was obtained from China Veterinary Culture Collection Center (Beijing, China). The E. coli K88 strain was grown in LB medium (Beijing Land Bridge Technology Co., Beijing, China) aerobically at 37 °C overnight. The techniques for culturing this strain were based on the descriptions of Marquardt et al. [26 (link)]. Two commercial additives Globigen^® Jump Start and Activo^® (IgY and PM), were provided by EW Nutrition GmbH (Visbek, Germany) as alternatives. According to the manufacturer, IgY is an egg powder containing natural immunoglobulins to support the intestinal health of young animals, while PM is a microencapsulated feed additive, with a mixture of phytomolecules that promote intestinal health through antibacterial, digestive and antioxidant activities. The AGPs premix consisting of 75 g chlortetracycline, 50 g oxytetracycline calcium and 40 g zinc bacitracin per kilogram was provided by Inner Mongolia Xingdaye Agriculture and Animal Husbandry Company Limited (Wulanchabu, Inner Mongolia, China).

SE CICC21513 was cultured on LB medium (Beijing Land Bridge Technology, Co., Ltd., Beijing, China). Fresh SE cultures were washed with PBS, counted, and then adjusted with DMEM basal medium (HyClone Laboratories, Logan, UT, USA) without FBS to 10⁷ CFU/mL for the next experiments.
After changing the culture medium, Caco-2 cells were pre-treated with either sophy β-glucan or vehicle (MEM medium with 10% FBS without antibiotics). After inoculation for 24 h, half of the glucan-treated wells and half of the untreated wells were infected with SE (MOI = 100:1) and further incubated for another 3 h, while the remaining wells were only treated with antibiotics-free DMEM basal medium. Cells were removed using a cell scraper and collected in 1.5 mL tubes. Thereafter, after centrifugation at 4000× g for 5 min at 4 °C, the culture supernatant was collected for the detection of antioxidant and cytokines content, and cell precipitate was collected for the assay of cytokines’ mRNA, protein expression, and intracellular antioxidant enzymes activities. All samples were assessed in triplicate.