Abstract
Background: Freeze-dried probiotic dairy products enriched with plant-derived bioactive compounds have attracted growing interest as functional foods for their potential to improve nutritional quality, probiotic stability, and shelf life. However, simultaneously optimizing cryoprotective systems, lyophilization conditions, and formulation components remains a significant technological challenge. Objective: This study aimed to develop and technologically characterize freeze-dried probiotic dairy ingredient prototypes from mare’s and camel’s milk. A sequential strategy was used: first, a cryoprotective system was optimized to preserve probiotic viability; second, the selected system was applied to walnut-shell extract-enriched dairy matrices, and freeze-drying conditions were optimized for residual moisture and reconstitution performance. Methods: A probiotic consortium comprising Lactobacillus acidophilus LSM-7, L. acidophilus LSM-9, and Lactobacillus casei LS-1 was studied. Two three-factor central composite rotatable designs were applied. Stage I optimized sucrose, maltodextrin, and ascorbic acid concentrations, using the viable cell count after freeze-drying as the response. Stage II evaluated walnut shell extract concentration (1–5%), freeze-dryer shelf temperature (−10 to −30°C), and drying time (6–30 h), using residual moisture and solubility index as technological responses. Control and extract-enriched mare’s- and camel’s-milk powders were physicochemically characterized. Results: Probiotic viability varied nonlinearly with the cryoprotective composition. The selected formulation contained 11.8% sucrose, 13.2% maltodextrin, and 0.32% ascorbic acid, yielding a model-predicted viable count of 2.85 × 10¹¹ CFU/g. In Stage II, shelf temperature and drying time primarily governed residual moisture, whereas extract concentration contributed to variation in the solubility index. Extract-enriched powders had higher measured protein, fat, carbohydrate, and total-solids contents than their corresponding controls. These results establish technological feasibility but do not, by themselves, demonstrate a clinical health benefit or an evidence-based effective dose. Conclusion: The two-stage optimization directly links probiotic cryoprotection to the production of stable, reconstitutable mare’s- and camel’s-milk powders containing walnut shell extract. On the evidence available, the outputs should be described as candidate functional dairy ingredients or product prototypes. Confirmation as functional foods requires standardized bioactive-compound profiling, biological-activity testing in the final matrix, storage validation, safety assessment, and dose-response or clinical studies. Novelty of the Study: The novelty is the sequential transfer of an optimized probiotic cryoprotective system into two non-traditional dairy matrices, followed by joint optimization of walnut shell extract concentration and freeze-drying conditions. This integrated workflow links preservation of probiotic viability with powder manufacturing while explicitly defining the evidence still required for functional-food validation. Keywords: Camel milk; freeze-drying; functional dairy products; lyophilization; mare milk; plant extracts; probiotic cultures; response surface methodology; walnut shell extract