Sustainable probiotic production via AI: medium optimization and metabolic mechanisms in Bifidobacterium animalis ssp. lactis BB-12 using agricultural waste

Bifidobacterium animalis ssp. lactis BB-12 (BB-12) is a well-established probiotic with notable health benefits and broad applications. However, its conventional MRS medium is expensive and poses safety and religious concerns. Agricultural wastes represent sustainable alternatives for microbial cultivation. This study aimed to optimize the BB-12 culture medium using agricultural waste with artificial intelligence (AI) and to investigate their metabolic impact through metabolomic analysis. AI approaches, including RSM (Response Surface Methodology), machine learning, deep learning, and evolutionary optimization methods, were employed to model and optimize the effects of medium composition on OD600, growth rate (μ max), and cost. The optimized media were further evaluated through organic acids analysis and metabolomic profiling to elucidate how variations in nitrogen and carbon sources affect the metabolic responses of BB-12. The O1 medium optimized by Ridge-NSGAII (Non-dominated Sorting Genetic Algorithms II) significantly (p Artificial intelligence was efficient in optimizing medium composition.Sustainable medium for BB-12 (Bifidobacterium animalis ssp. lactis BB-12) was developed using agricultural by-products.Six model-optimizer combinations yielded prediction errors below 10% for both OD600 and μ max.Optimized medium (O1) boosts BB-12 lactic, acetic, and propionic acids production.O1 may promote energy-saving in BB-12 through nucleotide salvage and pentose phosphate pathways.O1 may enhance BB-12 redox and stress responses via glutathione and pentose phosphate pathways. Artificial intelligence was efficient in optimizing medium composition. Sustainable medium for BB-12 (Bifidobacterium animalis ssp. lactis BB-12) was developed using agricultural by-products. Six model-optimizer combinations yielded prediction errors below 10% for both OD600 and μ max. Optimized medium (O1) boosts BB-12 lactic, acetic, and propionic acids production. O1 may promote energy-saving in BB-12 through nucleotide salvage and pentose phosphate pathways. O1 may enhance BB-12 redox and stress responses via glutathione and pentose phosphate pathways.