# The Functional Properties of _Lactobacillus casei_ HY2782 Are Affected by the Fermentation Time
## 4 Discussion  
In general, each probiotic strain might have different characteristics; therefore, probiotics are used for a selection of products by comprehensively considering strain characteristics such as safety, functionality, and technical aspects [31]. For lactic acid bacteria to be characterized as probiotics, they must pass through the stomach and duodenum, reach the final target site (the intestine), and attach to the intestinal epithelial cells to function [32; 33]. Probiotics can contribute to the improvement of the intestinal mucosal barrier function and have been reported to affect the formation of proteins that help the physiological barrier function of the tight-junction structure by modulating various signaling systems of intestinal epithelial cells [34; 35]. For probiotics to exhibit health-promoting effects through  
Figure 4: Relative mRNA levels of genes encoding the tight-junction proteins tight-junction protein 1 (ZO-1) (**a**), occludin (OCLN) (**b**), claud 1 (CLDN1) (**c**), and CLDN3 (**d**) in Caco-2 cells after 10 h exposure to fermented milk with _Lactobacillus casei_ HY2782 or _L. casei_ ATCC393 versus fermentation time. Different letters above the bars indicate statistically significant differences from each sample on day 1 of fermentation (* \(p<\) 0.05, **_p_\(<\) 0.01). positive interactions with the intestinal environment and intestinal epithelial cells, the ability to colonize the intestine is essential [36]. It has been reported that bifidobacteria, a representative commercial probiotic, have lost the functions necessary to colonize the human intestinal environment [37]. Therefore, when probiotics are taken orally, it is necessary to investigate their ability to survive and reach the intestine and colonize the intestinal environment. Our results suggested that _L. casei_ HY2782 has beneficial effects such as an increased survival rate in the digestive tract, better adhesion to intestinal cells, strengthening the intestinal barrier, and protecting the mucous membrane through long-term fermentation. Previous studies have shown that when lactic acid bacteria are exposed to an acidic environment for a certain period of time, their adaptability to acid and bile increases [22; 23]. It has also been suggested that acidic stress can improve the ability of lactobacilli to adhere to intestinal cells [24]. Previous studies showed that _L. casei_ ATCC 393 possesses significant adherence capacity in vitro [38], and thus, we used it as a reference strain for comparisons, but it did not result in an increase in the intestinal cell adhesion rate and tight-junction genes with long-term fermentation. _L. casei_ produces a large amount of exopolysaccharideacharide (EPS) during growth [39; 40]. Research has shown that EPS components protect lactic acid bacteria from being killed in the human digestive system [41]. With low temperatures and the application of glucose as the carbohydrate source, the highest amounts of EPS and the highest specific EPS production are achieved [42]. _L. casei_ can increase the expression of adhesion junction proteins [43; 44] and might increase the expression of mucin proteins [45]. A previous study showed that the products of secondary metabolism and peptide components produced by lactic acid bacteria during the fermentation process can increase the expression of the tight junction protein and the expression of mucin proteins [25]. In particular, a condition in which the intestinal permeability increases due to the loosening of the tight junction is called intestinal leakage syndrome, which causes diseases such as abdominal bloating, diarrhea, abdominal pain, and irritable bowel syndrome [46; 47; 48]. The long-term fermentation of _L. casei_ HY2782 can prevent intestinal diseases by increasing the expression of genes encoding tight-junction proteins. Recently, coating materials or encapsulation technologies for probiotics have been studied to increase the survival rate during the digestion process [49].