## 4 Discussion  
Probiotics, mainly comprised of lactic acid bacteria, are known to provide specific health benefits to the host through the colonization of the gastrointestinal tract [38]. In order to exert such biotherapeutic effects, probiotic strains should survive the passage through the gastrointestinal tract and colonize the small intestine and colon for a sufficient period of time [18; 38]. Strains such as KAU001 and KAU003 were the most acid and bile tolerant, and their adhesion to HT-29 cells were significantly higher. Some LAB isolates from chicken caecum show a limited survival of 60% to 80% after being exposed to an acid environment with pH 2-2.5 for 3 h [2; 4; 39; 40; 41]. In contrast, a survival rate as high as 92.75-97.26% was observed in this study. The natural human defense mechanism against pathogens into the intestinal tract is through the production of hydrochloric acid which lowers the stomach pH. Therefore, strains with acid tolerance are believed to survive the transit through the upper gastrointestinal tract. Whereas in the small intestine and colon, high abundance of bile salt is also a protective barrier to prevent attachment of pathogenic bacteria, hence survival in bile is considered a crucial factor for probiotics to colonize the gut [17]. The mechanism of probiotics in counteracting the  
Figure 4: Antioxidant effect of LAB isolated. (**a**) ABTS scavenging activity assay. (**b**) FRAP assay. (**c**) DPPH scavenging activity assay. Data are expressed as mean \(\pm\) S.D. from three independent experiments. Figure 5: Effect of LAB isolates on (**a**) \(\alpha\)-glucosidase inhibition activity and (**b**) cholesterol lowering activity. Data are expressed as mean \(\pm\) S.D. from three independent experiments. damaging effect of bile salt is through the production of enzyme bile salt hydrolase (BSH), which can break down conjugated bile salts, thereby reducing the toxicity [23; 40]. Another important requirement for probiotic strains is the ability to adhere to intestinal epithelial cells, which enables them to colonize the gut, compete with pathogens, and confer benefits to the host [42; 43]. Among all the strains tested, KAU001, KAU002 and KAU003 depicted a high ability to survive the gastrointestinal tract and adherence to colon cells, conferring them as potential probiotic candidates. KAU001 and KAU002 were found to have significant antioxidant potential, particularly KAU001. These results underpin that both LAB strains are highly effective in scavenging peroxyl radicals. Probiotics with high antioxidant potential may help to reduce the oxidative damage caused by free radicals, thereby promoting health benefits such as slowing down cells aging as aging process are highly associated with accumulated oxidative stress. Additionally, KAU001 and KAU002 inhibited \(\alpha\)-glucosidase activity, which is one of the important determinant factors in metabolic disease such as diabetes and obesity. Reduction of \(\alpha\)-glucosidase could indicate the ability of these strains in reducing intestinal sugar absorption, therefore potentially reducing the risk of diabetes. Besides, both strains also significantly lowered cholesterol levels, which may help to reduce the risk of high-cholesterol levels and cardiovascular disease. It was postulated that BSH produced by LAB strains are responsible for the anti-cholesterol properties through the deconjugation of bile salts. Deconjugated bile salts are less reabsorbed by the intestines, and they are excreted through feces thus the replacement of new bile salts from cholesterol will lower the serum cholesterol level [30]. In response to LPS stimulation in RAW 264.7 macrophages, the cells produced NO as a toxic defense molecule to fight off pathogens, and these led to inflammation. Treatment with _Lactobacillus_ strains reduced the NO production in antigen-stimulated macrophages, in parallel with increasing _Lactobacillus_ concentrations.