## 4 Discussion  
### Isolation and Characterization of Probiotic Candidate Lactic Acid Bacteria from Dadih  
The presence of Ca-lactate is shown by the formation of a clearly defined zone surrounding the colony. This zone is formed as a result of the combination of primary metabolites produced by lactic acid bacteria, namely lactic acid and organic acids, along with CaCO\({}_{3}\). Figure 6 displays an isolate that exhibited a distinct zone encompassing the colony. Lactic acid bacteria can be classified as Gram-positive bacteria that are negative for catalase activity and lack motility [19]. The qualitative tests conducted on the isolates DK1, DK2, and DK3 indicate the presence of lactic acid bacteria. Furthermore, the metabolites derived from the three probable lactic acid bacteria (LAB) isolates were extracted. The extraction of bacterial liquid culture involved a 24-h incubation period. Subsequently, the bacterial culture underwent centrifugation in order to achieve the separation of the bacteria from their byproducts, which are often referred to as cell-free supernatant (CFS). The antimicrobial effectiveness of the cell-free supernatant from each colony of lactic acid bacteria (LAB) was subsequently assessed against the test pathogens often found in beef, namely _E. coli_ and _Salmonella_ sp. The metabolic byproducts produced by lactic acid bacteria encompass a range of substances such as organic acids, diacetyl, hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)), reuterin, antifungal peptides, and bacterioxins [26]. These compounds exhibit potent antibacterial activity. The outcomes of the inhibitory zone's diameter for each isolate were found to be diverse. Several variables influence the outcome, including the sensitivity of the test microorganisms, the rate at which antimicrobial chemicals diffuse, and the concentration of antimicrobial substances. The outcomes of antimicrobial activity can be classified according to the size of the inhibition zone diameter observed. The inhibitory zones were classified into three categories based on their sizes: faint (+), moderate ++ (3-6 mm), and strong +++ (more than 6 mm) [27]. The diameter of the inhibitory zone was determined by dividing the total diameter by the diameter of the paper disc. The disc paper employed in this study possesses a diameter of 6 mm. Consequently, the inhibitory potency may be classified into three distinct groups, namely weak (+), medium (+), and strong (+), for diameters ranging from 6 to 12 mm. Due to the observed strong and moderate inhibition of _E. coli_ and _Salmonella_ species by the CFS generated by the DK1 isolate, it was deemed necessary to evaluate the DK1 isolate as a potential probiotic candidate bacterium. Additionally, the effects of the CFS produced by the DK1 isolate on the quality of beef during storage were investigated (see Table 2). The probiotic bacteria exhibit the ability to withstand an acidic environment characterized by a pH range of 2-4, as well as the presence of bile salts at a minimum concentration of 0.3%. The results also indicated that the bacteria exhibited a yield of 6 log CFU/mL at temperatures of 25 \({}^{\circ}\)C, 37 \({}^{\circ}\)C, and 45 \({}^{\circ}\)C. In order to exert an influence on the gut environment, a concentration of probiotic bacterial strains ranging from six to eight logarithmic  
Figure 6: Lactic acid bacteria isolate from dadih showing clear zone on MRSA + CaCO\({}_{3}\) 1% (\(\omega/\upsilon\)) media, 180 \(\times\) 155 mm (59 \(\times\) 59 DPI). colony-forming units per milliliter (CFU/mL), or 10\({}^{6}\)-10\({}^{8}\) CFU/mL, is required [28]. The LAB DK1 isolate has been identified as a potential probiotic candidate based on its ability to survive and proliferate across a wide temperature range of 25 \({}^{\circ}\)C to 45 \({}^{\circ}\)C, with acidic pH levels ranging from 2 to 4, and a bile salt concentration of 0.3%.