Faecalibacterium duncaniae A2‐165 growth is strongly promoted by yeast extract and vitamin B5 in cGMP medium

Several gut microbial species within the Faecalibacterium genus have emerged as promising next‐generation probiotics (NGP) due to their multifunctional protective effects against gastrointestinal and systemic disorders. To enable clinical studies and further applications, improved methods for cultivating Faecalibacterium must be developed in compliance with current Good Manufacturing Practice regulations, which is complicated by its oxygen sensitivity and complex nutritional requirements. Different yeast‐based nutrients (YBNs), including yeast extracts (YEs) and yeast peptones (YPs), are ubiquitously used when cultivating microbes to supply a broad range of macro‐ and micronutrients. In this study, we evaluated six experimental YBNs, namely three YEs, two YPs and a yeast cell wall product (YCW), and eight B‐vitamins in the cultivation of Faecalibacterium duncaniae A2‐165, former Faecalibacterium prausnitzii, using growth assays in microtitre plates, dose‐effect studies in Hungate tube fermentations and fully controlled bioreactor experiments. We demonstrated that YEs promote F. duncaniae A2‐165 growth in a nutritionally limited medium, while YPs and YCW lacked essential growth factors for enabling cell propagation. High cell density was obtained in controlled bioreactors using a medium containing 2–4% of a selected YE and 1% casein peptone (3.4 ± 1.7 × 109–5.1 ± 1.3 × 109 cells mL−1). Among all tested B‐vitamins, we identified B5 as a strong growth promoter. Replacing casein peptone with YP and supplementing with vitamin B5 further increased biomass by approximately 50% (6.8 ± 1.7 × 109 cells mL−1). Hence, empirical selection of YE, YP and B5 allowed formulation of a high‐yielding animal allergen‐free nutritive medium to produce F. duncaniae A2‐165. Selecting nutritionally suitable YBNs and combining these with other key nutrients are important steps for optimizing production of NGP with high yields and lower cost. In this study, an animal allergen‐free production medium for the next‐generation probiotic Faecalibacterium duncaniae A2‐165 was designed and evaluated using a stepwise approach. Different yeast‐based nutrients and B‐vitamins were tested, allowing the design of an effective medium formulation containing yeast extract, yeast peptone and vitamin B5 that yielded high F. duncaniae A2‐165 cell numbers (6.8 ± 1.7 × 109 cells mL−1) in bioreactor experiments. Selecting nutritionally suitable yeast‐based nutrients and combining these with other key nutri