Bile salts

Pancreatin (4 × USP specification) and zein (≥95%) and were bought from Sigma-Aldrich (Missouri, USA). 7,8-dihydroxyflavone (≥98%) was purchased from TCI Co., Ltd. (Tokyo, Japan). Sophorolipid was purchased from the Boliante Chemical Company (Xian, China). Sodium alginate (>90%) was obtained from Macklin (Shanghai, China). Sodium carboxymethyl cellulose was purchased from Aladdin (Shanghai, China). Bile salts and pepsin (activity 3000~3500 U mg⁻¹) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Other utilized reagents and chemicals were of analytical grade.

The bile salt and acid tolerance assay were performed by adopting the method described by Li X. Y. et al. (2021) (link). Briefly, for bile salt tolerance tests, different concentrations (0.3, 0.6, and 0.9% (w/v)) of bile salts (Sangon Biotech, Shanghai, China) were added to MRS broth medium; for acid resistance tests, MRS broth medium was adjusted to different pH values (pH 2.0, 3.0 and 4.0) using 2 mol/mL HCl. Subsequently, L. salivarius CGMCC20700 cultures (10⁷ CFU/mL) were added to MRS broth medium and cultured at 37°C for 4 h and 5 h, respectively. After incubation, 100 μL of each bacterial suspension was separately coated on MRS solid plates by the serial dilution method and inverted incubation at 37°C for 24 h. The viable counts of 30 ~ 300 colonies were counted, and bile salt tolerance was determined by calculating the ratio (%) of viable cells compared to the control without bile salt survival rates (%).

Intestinal and gastric fluid tolerance determinations were performed by adopting the method described by Li X. Y. et al. [17 (link)]. Briefly, for bile salt tolerance tests, different concentrations (0.3, 0.6, and 0.9% (w/v) of bile salts (Sangon Biotech, Shanghai, China) were added to MRS broth medium; for acid-resistance tests, MRS broth medium was adjusted to different pH values (pH 2.0, 3.0, 4.0, and 6.8) using 2 mol/mL HCl. Subsequently, L. paracasei XLK401 cultures (10⁷ CFU/mL) were added to the MRS broth medium and cultured at 37 °C for 4 and 5 h, respectively. After incubation, 100 μL of each bacterial suspension was separately coated on MRS solid plates by the serial dilution method and inverted incubation at 37 °C for 24 h. The viable cell counts of 30 ~ 300 colonies were determined, and tolerance was determined by calculating the ratio (%) of viable cells compared to the control without additives, that is, the survival rates.