Cholesterol peg 600

The selection of strains with lowering-cholesterol ability was performed by in vitro tests using water-soluble cholesterol, Cholesterol-PEG 600—polyoxyethylene cholesteryl sebacate (Sigma Aldrich)—and resorted to colorimetry, with a commercial kit for cholesterol quantification (Sigma Aldrich). According to Benítez-Cabello et al. [46 (link)], overnight cultures of LAB were centrifuged (3211× g, 10 min, 5 °C), washed twice with a 0.85% NaCl solution, and re-suspended in PBS. After 2 h at room temperature (to simulate starvation), 20 μL of suspension was inoculated in 200 μL of MRS broth and supplemented with 3 g/L of oxgall (Sigma Aldrich) and 0.100 g/L of cholesterol. After incubation at 37 °C for 24 h, samples were centrifuged (9600× g, 3 min, 5 °C) and pellet was discarded. The cholesterol was quantified according to manufacturer’s instructions, using a standard curve of absorbance at 500 nm vs. cholesterol concentration. MRS and oxgall without cholesterol solution were used as controls. The experiments were repeated for all strains as two biological replicates, each with two technical replicates. After knowing cholesterol concentration, according to said kit, cholesterol assimilation CA(%) was determined via Equation (6): $$\mathrm{CA}(\%)=\left(1-\frac{C_f}{C_i}\right)\times 100$$
where C_i = initial cholesterol concentration and C_f = final cholesterol concentration in the samples.

The capacity of probiotic Lactobacillus strains to assimilate cholesterol in MRS broth was investigated as previously described [18 (link)]. Cholesterol-PEG 600 (Sigma Aldrich, St. Louis, MI, USA) was added to MRS broth at a final concentration of 100 μg/mL. The 1% (v/v) inoculum of each overnight probiotic culture was added and incubated at 37 °C for 24 h. The bacterial cultures were centrifuged at 4000 rpm for 10 min at 4 °C, and the supernatants with non- assimilated cholesterol were collected [24 (link)]. Cholesterol assimilation was calculated by the formula:
where A is the cholesterol that remained with the pellet (as a percentage), B is the absorbance of the sample containing the cells, and C is the absorbance of the sample without bacterial cells. The isolates with the highest cholesterol assimilating potential were selected for in vivo trials. The Lactobacillus plantarum ATCC 14917 (KWIK-STIK, UK) was used as a reference strain only for cholesterol assimilation activity.