# The Functional Properties of _Lactobacillus casei_ HY2782 Are Affected by the Fermentation Time
###### Abstract  
Maintaining probioticic effectiveness represents the most important task for the development of functional foods. Gastrointestinal stability and intestinal adhesion properties comprise one criterion for probiotictic selection. Here, we investigated the benefits of milk fermented with _Lactobacillus casei_ HY2782 at different fermentation times. The probiotictic strain used was _L. casei_ HY2782 and the reference strain was _L. casei_ ATCC393 for comparisons. The samples were fermented for 7 days at 30 \({}^{\circ}\)C. We determined the pH, CFU/mL, survival rate during simulated gastrointestinal digestion, adhesion ability to HT-29 cells, and gene expression of tight-junction proteins known to regulate intestinal permeability in Caco-2 cells. _L. casei_ HY2782 exhibited significantly higher survival rates in simulated gastrointestinal digestion during long-term fermentation than _L. casei_ ATCC393. The adhesion ability to HT-29 cells was significantly increased with _L. casei_ HY2782 (3.3% to 8.7%) after 7 days of fermentation; however, only a slight increase was observed for _L. casei_ ATCC393 (3.1% to 4.7%). In addition, _L. casei_ HY2782 can significantly increase the expression of genes encoding tight-junction proteins during long-term fermentation of milk. In conclusion, we confirmed that long-term fermentation could be a novel manufacturing process for fermented milk containing _L. casei_ HY2782 and showed the beneficial effects.  
Lactobacillus casei, fermentation; adhesion ability; simulated gastrointestinal digestion; tight-junction proteins +
Footnote †: journal: Medical  
as the most efficient product to deliver probiotics or lactobacilli to the small and large intestines without resulting in a significant loss of activity [16]. Fermented milk is a suitable carrier for probiotics and its consumption might be beneficial for improving health. In the development of probiotic-fermented milk, probiotic viability during fermentation and storage time and the interaction with the starter culture of the product and its impact on the sensory properties of the product should be considered. Yakult is prepared by adding glucose to skimmed milk and then inoculating the mixture with the lactobacillus and incubating it for 6 to 7 days at 37 \({}^{\circ}\)C [17]. _L. casei_ HY2782 is used as a starter strain for Korean Yakult fermented milk products. Previous studies have shown that _L. casei_ HY2782 has various functions such as increasing the expression of interleukin 12 in dendritic cells [18] and preventing the toxic effect induced by particulate matter [19,20]. Previous studies found that growth and metabolism mostly cease after 24 h of incubation. However, some _Lactobacillus_ strains' metabolism and growth can be unconnected. It is important to adjust the fermentation time since probiotic strains produce different amounts of metabolic products throughout that time [21]. Previous studies have shown that lactic acid bacteria exposed to an acidic environment for a certain period of time increase their adaptability to acids and bile [22,23]. In addition, it has been suggested that acidic stress can improve the ability of lactobacilli to adhere to intestinal cells [24]. According to a previous study, the products of secondary metabolism and peptide components produced by lactic acid bacteria during the fermentation process can increase the expression of tight-junction proteins [25].  
In this study, we aimed to improve the functional properties of fermented milk with _L. casei_ HY2782 using long-term fermentation. We measured the growth characteristics, decreasing pH, titratable acidity (TA), and viable cells in fermented milk at different fermentation times in comparison to those with _L. casei_ ATCC393. We also confirmed the gastrointestinal tolerance, intestinal cell adhesion ability, and tight-junction protein-related gene expression in intestinal cells.