## 3 Results and Discussion
### Cell Survival during In Vitro Digestion Model  
To investigate the potential protective effect of cell immobilization on the viability of _L. rhamns_ OLXAL-1 cells during GI transit, both wet and freeze-dried immobilized cultures were subjected to a static in vitro digestion model. For comparison purposes, wet and freeze-dried free _L. rhamns_ OLXAL-1 cells were also tested in the same digestion process. According to Table 1, incubation in the simulated salivary phase had no effect on the viable cell counts of wet and freeze-dried free or immobilized _L. rhamns_ OLXAL-1 cultures [7; 32], while incubation in the simulated gastric and intestinal phase led to a significant (_p_ < 0.05) reduction in all cases. Notably, the survival rates (%) of all immobilized _L. rhamns_ OLXAL-1 cultures after in vitro simulated digestion were higher (_p_ < 0.05) compared to free cells. Additionally, higher survival rates were observed in the wet compared to the corresponding freeze-dried cultures for both free and immobilized cells, a result that can be explained by the cellular stress during freeze-drying [33]. Furthermore, statistical analysis showed strong interactions between the state (wet or freeze-dried) and the nature (immobilized or free) of _L. rhamns_ OLXAL-1 cells regarding their survival during in vitro digestion. Figure 3: Electron micrographs showing (in red circles) immobilized _L. rhamns_ OLXAL-1 cells on apple pieces (**left**) and oat flakes (**right**). In detail, after in vitro digestion, the survival rates of wet and freeze-dried free cells were determined at 36.29% and 33.42%, respectively. Among the wet immobilized cultures, the survival rates ranged from 51.01% to 84.76% and the highest (\(p<0.05\)) survival rate was noted in wet immobilized cells on oat flakes, while the lowest (\(p<0.05\)) rate was observed in wet immobilized cells on apple pieces. A similar pattern was also observed in the freeze-dried immobilized cultures, with the highest (\(p<0.05\)) survival rate recorded in freeze-dried immobilized cells on oat flakes (81.22%) and the lowest (\(p<0.05\)) rate in freeze-dried immobilized cells on apple pieces (48.65%). Resistance to the harsh and degradative conditions of the human GI tract is considered an essential prerequisite for probiotic cultures, in order to confer potential health benefits [34; 35]. This requirement is particularly important for LAB strains as they are generally characterized by a poor ability to survive such challenging conditions [36; 37]. Several strategies have been proposed to enhance the survival and viability of bacterial cells in the GI tract [38]. Among these strategies, a notable approach is cell immobilization, which involves physically or biochemically confining probiotic cells, preventing their unrestricted movement and providing them with a protective matrix or support structure [39]. In particular, cell immobilization on food matrices/natural carriers has been reported to offer distinct advantages, mainly due to their food-grade nature, which ensures safety compliance, their low-cost and high availability, and, finally, their ease of use for processing during the immobilization process [13]. Several studies have reported a protective effect when comparing immobilized cells on food matrices to free cells after in vitro exposure to low pH, bile salts and digestive enzymes. For instance, in the study conducted by [PERSON] et al. [34], _L. plantarum_ NCIMB 8826 cells were immobilized on a weanimix (cereal-legume blend) food matrix and then exposed to two separate tolerance assays: one against low pH and another against the presence of bile salts. In both cases, the weanimix food matrix provided a protective effect on _L. plantarum_ cells, as significantly higher survival rates were determined in immobilized cultures compared to free cells after in vitro digestion. In the same manner, [PERSON] et al. [40] reported that cell immobilization on barley and malt fibers contributed to the stability of LAB cells after exposure to simulated gastric and bile juices. Apart from