In-Silico Rational Design of Proline-Containing Peptides (PCPs) for Efficient Utilization by Bifidobacterium bifidum and Identification of Their Key Transported Sequences.

Industrial production of Bifidobacterium bifidum (B. bifidum) is constrained by its poor use of conventional nitrogen sources. Previous studies have demonstrated that supplementation with proline-containing peptides (PCPs) enhances both growth density and freeze-drying survival rate of B. bifidum. Thus, we developed an in silico-guided strategy to prepare proline-containing peptide-rich hydrolyzates tailored to its nitrogen preference. Casein, glutenin, and whey protein hydrolyzate were selected as substrates, and six enzymatic hydrolysis routes were designed using simulated digestion and molecular docking. The optimal nitrogen source, WA-CAF, a nitrogen source rich in PCPs, enabled B. bifidum CCFM16 to achieve higher biomass and freeze-drying survival rate than De Man-Rogosa-Sharpe (MRS) medium at a lower dosage. Viable-cell yield per unit nitrogen increased by 178.6%. Peptidomics revealed that efficiently utilized peptides were shorter than 10 residues, enriched in Pro, Leu, and Val, with internal Pro residues. This work provides a practical, sequence-guided nitrogen-source design framework for difficult-to-cultivate probiotics.