Prosopis nigra, a sucrose-rich crop, was used to enzymatically synthesize fructo-oligosaccharides (FOS). The obtained products were used as stabilizing matrices during freeze-drying and storage of Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333. The centesimal composition of P. nigra flour was firstly determined. FOS were synthesized using Viscozyme L as biocatalyst. The progress of the enzymatic reaction was monitored by HPLC and compared with a reaction carried out using equivalent concentrations of pure sucrose as substrate (control). Then, P. nigra containing or not the obtained FOS (P. nigra + FOS or P. nigra) were used as matrices for freeze-drying and storage of L. delbrueckii subsp. bulgaricus CIDCA 333. P. nigra flour was rich in simple sugars (sucrose and fructose), total dietary fiber, and polyphenols. The main products of synthesis were FOS with degrees of polymerization (DP) within 3 and 5, and these results were comparable with those of the controls. DP3 was the first product obtained, attaining the maximal production after 1.29 hours of synthesis. The maximal production of total FOS (DP3 + DP4 + DP5) was achieved after 2.57 hours, indicating that larger FOS (DP4, DP5) were produced from DP3. Glucose was obtained as secondary product, but with significantly lower Vmax and Kf (maximal velocity for the production and constant for the formation) than DP3. Both P. nigra + FOS or P. nigra matrices stabilized the highly sensitive L. delbrueckii subsp. bulgaricus CIDCA 333 strain during freeze-drying and storage for up to 140 days at 4 °C, and were significantly better protectants than the controls of sucrose (p <0.05). The concomitant presence of prebiotics (FOS), antioxidants (polypyhenols) and lactic acid bacteria in the matrices provides a smart strategy to increase the value of this underutilized regional crop, turning it in an interesting ingredient potentially useful in the food industry.