Microalgal protein-alginate cryogels for enhanced lyoprotection, gastrointestinal stability, and controlled release of Lacticaseibacillus rhamnosus GG

This study elucidated the role of microalgal proteins, specifically spirulina (SPI), chlorella (CPI), and their equal parts blend, in structuring sodium alginate-based (NaAlg) cryogels and modulating the survivability and adhesion properties of Lacticaseibacillus rhamnosus GG (LGG). All matrices ensured near-complete preservation of LGG during freeze-drying (~10 log CFU g −1 ), reflecting synergistic stabilisation via cryo-concentration, hydrogen bonding, and vitrification within the protein–polysaccharide network. Storage stability was primarily governed by water activity and temperature but was strongly coupled with thermophysical properties: higher glass transition temperatures (T g ) and glassy-state conditions significantly reduced inactivation kinetics, with SPI-based cryogels exhibiting the longest shelf-life (up to 320 days at a w 0.11, 20 °C). During semi-dynamic digestion, protein-dependent colloidal transitions dictated LGG protection and release. SPI systems formed compact gastric aggregates that limited enzyme diffusion and enabled controlled intestinal release, resulting in superior post-digestion viability (~9.6 log CFU g −1 ), whereas CPI and SPI:CPI matrices showed greater losses. These functional differences were mechanistically linked to microstructural features of the cryogels, including highly porous (76–78%) architecture, the protein-specific pore-wall morphology, and aggregation behaviour, despite similar bulk mechanical properties e.g., stiffness and hardness. SPI cryogels preserved LGG cell adhesion to the mucus layer of co-culture model of the intestinal epithelium (4.8 log CFU cm −2 ), showing a strong correlation with post-digestion viability. Overall, microalgal proteins, particularly SPI, govern the interplay between cryogel structure, physical state, and colloidal behaviour. This allows for high lyoprotection, prolonged shelf-life, and improved gastrointestinal survivability and cell adhesion properties, matching or exceeding those of conventional lyophilised microcarriers.