Genotypic profiling of the microbial community of functional ready-to-eat baked products from gluten-free composite flour

There is increasing global interest in developing gluten-free functional foods using composite flours from locally available crops to enhance the agro-economic value of underutilized botanicals in non-wheat-producing countries and to provide gluten-free alternatives for individuals who require or choose gluten-free diets. This study evaluated and genotypically profiled the microbial community associated with three ready-to-eat baked products (bread, biscuits, and cake) prepared from pre-fermented and post-fermented gluten-free composite flours of sweet potato, maize, and pigeon pea. The botanicals were commercially obtained in Etsako-West Local Government Area, Edo State, Nigeria, authenticated, and processed into flours. A ternary composite flour blend of sweet potato: pigeon pea:maize (67.70:20.00:12.31), previously optimized and experimentally validated using a D-optimal mixture design (Expert 13.0), was adopted from our published studies in 2024. Microbiological analyses combined conventional culture-based enumeration with molecular identification of isolates using 16S rRNA (bacteria) and ITS (fungi) sequencing and phylogenetic analysis. Pre-fermented products recorded higher microbial loads than products made from post-fermented flour. Bacterial counts ranged from 7.32–7.60 Log10 CFU/g, while fungal counts ranged from 1.1–4.5 Log10 CFU/g. Bread made from postfermented flour had the lowest bacterial and fungal counts, whereas biscuits and cake made from pre-fermented flour recorded the highest bacterial and fungal counts, respectively. All bacterial isolates belonged to the spore-forming genus Bacillus ( B. velezensis, B. licheniformis, B. tequilensis, B. subtilis ), while fungal isolates included Xylaria feejeensis, Aspergillus niger, Talaromyces islandicus , and Cladosporium oxysporum . These findings indicate that fermentation of the raw botanicals improves the microbiological quality of the resulting gluten-free baked products. Comprehensive consumer safety assessments that would additionally require pathogen-specific testing and shelf-life evaluation, are recommended as follow-up studies.