**Background:** In vitro simulated colon fermentations are increasingly used to study the effects of foods on the gut microbiota. A key challenge is eliminating contaminating microbes from test materials without damaging heat-labile components such as resistant starch. Autoclaving, the standard sterilization method, can gelatinize starch and alter its physicochemical properties, converting Type 2 resistant starch (granular) to Type 3 (retrograded), which has different effects on the gut microbiota. Non-thermal alternatives are needed, but their effectiveness for complex food matrices in microbiome research contexts has not been systematically evaluated.
**Methods:** Four pulse flours (green lentil, sprouted green lentil, chickpea, and field pea) were treated with three liquid chemicals: bleach (1.0% and 1.5% sodium hypochlorite), hydrogen peroxide (2.0%), and reagent alcohol (70% v/v). Treatments involved suspending flours at 4% w/v in chemical solutions, incubating for 24 hours at room temperature (~21°C) with orbital shaking at 218 RPM, followed by ten washes with type 1 DI water. Controls included untreated flours, water-washed flours, and dry autoclaved flours (121°C, 25 minutes). Microbial load was assessed by viable plate counts on anaerobic RUM media (limit of detection: 2.5×10³ CFU/g). Simulated in vitro colonic fermentations (24 hours at 37°C) were performed in deep 96-well plates with and without fecal microbes from a cryogenically stored human fecal sample. Microbial community composition was analyzed by 16S V4 region sequencing using Illumina MiSeq 250×250 paired-end sequencing, with OTU clustering via Mothur and differential abundance analysis using DESeq2, ANCOM-II, and LEfSe. Short-chain fatty acids (acetate and butyrate) were measured by HPLC-UV. Starch granule structure was evaluated by scanning electron microscopy at 5 kV.
**Key Results:** Autoclaving, 1.0% bleach, 1.5% bleach, and 2.0% hydrogen peroxide all reduced microbial loads to below the limit of detection (<2.5×10³ CFU/g) in all pulse flours. Seventy percent alcohol reduced loads but left detectable microbes in green lentil flour (4.9×10⁴ CFU/g). Untreated flours had microbial loads ranging from <2.5×10³ CFU/g (field pea) to 2.5×10⁵ CFU/g (green lentil). Water washing achieved approximately 1-log reduction, but overnight incubation in water resulted in up to 5-log microbial expansion. The dominant endogenous microbe was Erwinia (OTU0003), comprising 65.5% of chickpea, 75.3% of green lentil, and 95.5% of sprouted green lentil endogenous communities; Clostridium (OTU0001) dominated field pea (57.1%). During endogenous fermentation (without fecal addition), all pulses produced significant acetate (23.7–31.9 mM) but no significant butyrate. In simulated colonic fermentations with fecal addition, all sterilization treatments significantly reduced Erwinia abundance (DESeq2: 90- to 500-fold reduction, all corrected p < 0.05). Alpha diversity (Shannon index) showed no significant differences between sterilized and unsterilized samples; Inverse Simpson index showed a significant decrease only for 1.0% bleach (p = 0.0107). Beta diversity (Aitchison and weighted UniFrac) showed significant differences between all sterilized and unsterilized samples (PERMANOVA adjusted p < 0.05 for all comparisons), while Bray-Curtis showed trending but non-significant differences (adjusted p = 0.075–0.099). No significant differences in acetate or butyrate production were found between sterilized and unsterilized fermentations. Scanning electron microscopy showed intact starch granules across all treatments, with only dry autoclaving showing minor signs of starch damage.
**Clinical Implications:** This study provides validated, accessible non-thermal sterilization methods for preparing starchy food substrates for in vitro gut microbiome research. Bleach (1.5%) and hydrogen peroxide (2.0%) are recommended as effective treatments that eliminate contaminating microbes without significantly altering starch structure or fermentation outcomes. These methods enable researchers to study heat-labile food components (e.g., resistant starch) in their native form, avoiding the confounding effects of thermal gelatinization. The findings are particularly relevant for investigating raw or minimally processed foods and supplements. However, the study used a single fecal donor, and further testing is needed to confirm generalizability across different microbiota compositions and other starchy substrates.