Resistant starch supplementation enhances gut barrier integrity in estrogen deficient mice by modulating the gut microbiota

To evaluate the effects of resistant starch supplementation and estrogen deficiency on gut microbiota composition, mice were divided into six experimental groups: RS3-SES (5% RS3 type resistant starch-supplemented diet with sesame oil), RS3-VCD (5% RS3 type resistant starch-supplemented diet with VCD-induced estrogen deficiency), RS4-SES (5% RS4 type resistant starch-supplemented diet with sesame oil), and RS4-VCD (5% RS4 type resistant starch-supplemented diet with VCD-induced estrogen deficiency), with n = 8 per group. Cecal contents were collected at the end of the study and subjected to microbial DNA extraction using the Qiagen PowerFecal Kit (Qiagen, Maryland) following the manufacturer's protocol. The bacterial 16S rRNA gene V3-V4 region was amplified using primers F341 (5'-CCTACGGGNGGCWGCAG-3') and R806 (5'-GGACTACNVGGGTWTCTAAT-3'), which included Illumina adapter sequences and sample-specific barcodes. Amplicons were sequenced on an Illumina MiSeq platform using paired-end 250 bp reads, generating raw FASTQ files for downstream microbiome analysis.