## 4 Discussion  
Postbiotics have beneficial effects such as anti-pathogenicity, anti-diabetic, anti-inflammatory, anticancer, anti-allergic, and angiogenic activities. However, to achieve these beneficial effects, postbiotics must be able to survive and adapt under intestinal conditions such as acidic pH, enzymes, biliary salts, and heat. The term postbiotics, the so-called fourth-generation LAB (lactic acid bacteria), indicates the cell metabolites rather than living cells. Therefore, they are not toxic to the human body and not affected by intestinal conditions [6, 13]. In addition, postbiotics must have various beneficial effects and nutritional components for human health. Recently, postbiotics have been spotlighted as next-generation compounds that could improve human health. This study was performed to investigate the effects of postbiotics against colorectal cancer cells. Two probiotic strains (_Lactobacillus_ sp. La1 and La2) were prepared by the sonication cell-death method. The WST-1 assay was performed to confirm the effect of Pobt-La1 and La2 on the proliferation of HT-29 cells. [PERSON] reported that 200 ug/ml of _Lactobacillus casei_ postbiotics (heat-killed and sonicated) have a 40% or less inhibition rate against HT-29 cells [14]. In the present study, we observed an inhibition rate of 41% for Post-La1 and 46% for Post-La2 when treated at 150 uL/mL. Thus, the postbiotics used in this research gave the very same effective levels of inhibitory activity compared to the previous report which used postbiotics prepared from _Lactobacillus casei_. Moreover, it was verified that low concentrations of Pobt-La1 and La2 effectively inhibit the HT-29 cells. Morphological changes in HT-29 cells were observed under a microscope depending on the times of treatment with Pobt-La1 and La2. In the control group, the number of cells was increased by the incubation time, showing that normal cell proliferation was clearly observed, and the cell nuclei showed a normal shape. However, in the case of the test groups, there was no change in the number of cells with the incubation time, the morphology of the cells was irregular, and cell nuclei were not observed. In addition, suspended and condensed cells were observed which were not properly attached to the culture plate. Apoptosis causes chromatin condensation in cells, blisters on the plasma membrane, and cell division in the membrane-encapsulated body. In addition, lamellipodia  
Figure 4: Expression of caspase-3 and 8 proteins in HT-29 cells treated with Pobt-La. and filopodia--which play important roles in cell migration--are lost, resulting in a loss of adhesion [15]. For this reason, irregular morphology, condensation, and suspended cells appeared to be observed in the cells treated with Pobt-La1 and La2. The gene expression associated with apoptosis was analyzed through RT-PCR. Apoptosis occurs by two different major pathways. One pathway is the induced release of cytochrome c from the mitochondrial transmembrane, in which caspase-9 is involved. This is called the intrinsic pathway. The other pathway is death-receptor ligation, which involves caspase-8. This is called the extrinsic pathway. Both caspase-9 and caspase-8 are initiator caspases capable of activating caspase-3, an initiator of apoptosis [16]. As a result of the present study, the expression of box was also increased in the test group. From these results, it seems that Pobt-La suppresses the apoptosis of HT-29 cells, which is a case of the extrinsic pathway. The expression level of caspase factors, which plays a key role in the occurrence of apoptosis, was confirmed by Western blot analysis. Caspase exists in an inactive form in the outer membrane of the nucleus and mitochondria, and is activated by stimuli that induce apoptosis [17]. That is, the activation of caspase can act as further evidence of the occurrence of apoptosis. In caspase-3 and -8, the inactive form of pro-caspase is cleaved and converted into the activated protein form (cleaved-caspase).