Spray‐Dried Microencapsulation of Probiotics With Genipin‐Crosslinked Whey Protein Isolate for Enhanced Stability in Fortified Instant Cereal Drinks

Probiotics face significant challenges during processing, storage, and gastrointestinal transit, leading to reduced viability and diminished health benefits in functional foods. This study investigated the effects of genipin crosslinking (2.5, 5, and 10 mM) on the structural and functional properties of whey protein isolate (WPI) as an encapsulant for Limosilactobacillus reuteri KUB-AC5 in probiotic-fortified cereal instant drinks. Fourier transform infrared spectroscopy (FTIR) confirmed covalent bonding between genipin and WPI, forming a reinforced protein matrix with reduced surface hydrophobicity, increased surface tension, and enhanced turbidity. Differential scanning calorimetry (DSC) further demonstrated that crosslinking initially improved thermal stability, with WPI-2.5G exhibiting the highest denaturation temperature (78.97°C), and enthalpy values (0.54 J/g), before decreasing at higher genipin concentrations due to potential protein aggregation. WPI-2.5G significantly improved probiotic survival after spray drying (8.13%), with flow cytometry confirming a higher proportion of intact cells and fewer injured cells, indicating enhanced protective functionality. After 4 months of storage at 4°C and 25°C, WPI-2.5G maintained superior probiotic viability, 2.69 × 10⁹ CFU/serving (12.44% survival) and 3.47 × 10⁶ CFU/serving (0.02% survival), respectively, compared to other formulations. Furthermore, WPI-2.5G exhibited the highest survival rates after thermal reconstitution (2.98 × 10⁸ CFU/serving) and dynamic gastrointestinal digestion (5.69 × 10⁸ CFU/serving), exceeding the recommended threshold for probiotic efficacy. These findings demonstrate that genipin crosslinking, particularly at 2.5 mM, offers a promising strategy for enhancing probiotic viability and developing heat-stable, nondairy functional beverages.