Reassessing the safety of dietary emulsifiers through the lens of gut microbiota

Hydrophilic dietary emulsifiers-fed C57BL/6J mouse model.Male C57BL/6 J mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). After acclimation, fifteen-week-old mice were randomly divided into four experimental groups (n=3-4/cage): (i) control [CON], (ii) lecithin [LEC, 7523.3 mg/kg bw/day], (iii) sucrose fatty acid esters [SUC, 1110 mg/kg bw/day], and (iv) carboxymethylcellulose [CMC, 1%]. Mice in each experimental group were allowed free access to food and water for 17 weeks.Lipophilic dietary emulsifiers-fed C57BL/6J mouse model.Six-week-old male C57BL/6J mice were employed for the experiment after an adaptation period of 2 weeks. Eight-week-old male C57BL/6J mice were divided into two groups based on their diet: (i) control diet (CON) group, and (ii) monoglycerides and diglycerides diet (MDG) [5.5%] group. The mice were sacrificed after 14 weeks of the experiment.Cecal samples DNA were extracted according to the manufacturer's instructions using the QIAamp PowerFecal DNA Kit (Qiagen, Netherlands). PCR amplification of 16S rRNA V4 regions was performed using the 515F/806R bacterial primer pair (515F: 5-GTGCCAGCMGCCGCGGTAA-3 and 806R: 5-GGACTACHVGGGTWTCTAAT-3), with a 50 uL reaction volume containing 25 uL 2X Taq Master Mix (Thermo Scientific, USA), 0.2 M of each forward and reverse primer, and 20 ng DNA template. Thermal cycling consisted of initial denaturation at 95 C for 5 min, followed by 25 cycles of denaturation at 98 C for 20 s, annealing at 57 C for 15 s, and elongation at 72 C for 30 s, with a final hold at 72 C for 10 min. Next, amplified products were subjected to 2% agarose gel electrophoresis. The amplified PCR product was attached with Illumina sequencing adapters with a Nextera XT Index kit and then purified using AMPure XP beads. Library quantification was achieved employing the DNA 1000 kit and 2100 Bioanalyzer instrument (Agilent Technologies, USA), and sequencing (paired-end reads) was completed using Illumina NextSeq or Illumina MiSeq (Illumina, USA).