Probiotics must be safe for human consumption, have the ability to endure the gastrointestinal environment, adhere to intestinal epithelial cells, and perform various biological functions in the hosts [24]. Through a series of probiotic experiments, it was identified that L. brevis KU15159 can survive in the gastrointestinal environment and attach to intestinal cells. Additionally, L. brevis KU15159 can produce useful enzymes without the risk of transferring antibiotic resistance to harmful bacteria or producing harmful enzymes. Leucine arylamidase and valine arylamidase produced by Lactobacillus influence the decomposition of proteins and peptides, contributing to the flavor of the final product [25]. β-Galactosidase helps alleviate lactose intolerance in the gut and is important for probiotics [26, 27]. In addition, β-glucosidase contributes to taste by hydrolyzing bitter compounds and converting flavorless glucosides into aromatic compounds [28]. L. brevis KU15159 showed a higher production of β-glucosidase than L. rhamnosus GG.
The immune-improving effect of heat-treated L. brevis KU15159 was confirmed using NO and phagocytosis assays. Treatment of RAW 264.7 cells with 7 log CFU/mL of heat-treated L. brevis KU15159 increased NO production more effectively than the L. rhamnosus GG treatment. When macrophages are activated, they secrete NO to counteract free radicals; NO production is the important molecules in immune response. In addition, it is fatal to intracellular parasites and bacteria [29]. The induction of phagocytic activity by heat-treated L. brevis KU15159 was similar to that by L. rhamnosus GG. The phagocytic activity of macrophages is important to defend their host against pathogens, which is crucial for immunity [30].
The increased expression of iNOS in RAW 264.7 cells treated with heat-treated L. brevis KU15159, compared to that in the LPS or L. rhamnosus GG treatment, further implicates the immune-enhancing effect of L. brevis KU15159. iNOS adjusts the function and differentiation of immune cells through nitration of principal molecules related to signaling or transcriptional pathways [31, 32]. In addition, heat-treated L. brevis KU15159 upregulated the expression of TNF-α, IL-6, and IL-1β. The upregulation of TNF-α by L. brevis KU15159 was higher than that in the other treatments. Macrophages secrete cytokines, as well as TNF-α, IL-6, and IL-1β, and have considerable effects on immune responses [33]. They can bind to particular receptors of other cells and activate them immunologically [24]. TNF-α protects the host against infectious pathogens [3], initiates the expression of adhesion molecules in endothelial cells and neutrophils, and activates leukocytes [34]. The amounts of iNOS, TNF-α, IL-6, and IL-1β in the heat-treated L. brevis KU15159 treatment were similar to or higher than that in the L. rhamnosus GG treatment.
Western blot analysis showed that heat-treated L. brevis KU15159 activated the MAPK pathway (Fig. 5). The MAPK signaling pathway is related to regulating mRNA expression associated with the expression and release of inflammatory cytokines for modulating immune responses [35]. MAPKs are regulators of immune responses; their activity is regulated by reversible phosphorylation of threonine and tyrosine residues [36]. There are trinity specific MAPK pathways, the p38, ERK, and JNK pathways [36]. Heat-treated L. brevis KU15159 phosphorylated p38, JNK, and ERK 1/2 more than LGG did. Therefore, elevated cytokine production in macrophage handled with heat-treated L. brevis KU15159 was associated with the activity of MAPK pathway.
L. plantarum KU15149 exhibited immune-enhancing potential. In the RT-PCR and ELISA data, L. plantarum KU15149 treatment had similar or slightly lower values than L. brevis KU15159. Unlike L. brevis KU15159, which affected cell viability at 8 log CFU/mL in the MTT assay, L. plantarum KU15149 did not affect viability. Therefore, it is necessary to evaluate the use of higher concentrations and longer treatment times. In addition, it is important to assess inflammatory reactions that could occur with increased bacterial concentrations. This applies to the immune-enhancing potential of L. brevis KU15159 as well, and in vivo tests are necessary to evaluate the effectiveness and the possibility of an inflammatory reaction.
In summary, L. brevis KU15159 was characterized for its probiotic properties, and heat-treated L. brevis KU15159 was investigated to detect its immune-enhancing effects in macrophage. Heat-treated L. brevis KU15159 augmented the phagocytic activity and the NO production in macrophage by regulating iNOS. The TNF-α, IL-1β, and IL-6 expression was upregulated. At the protein level, L. brevis KU15159 showed an effect similar to that of LGG. Therefore, L. brevis KU15159 has immune-enhancing potential in vitro.