## 4 Discussion
However, through this study, we confirmed that even the same species can have different activities, and the activity is related to the structural differences of LTAs. Similar studies have been reported. When comparing the effect of a lactobacillus genus on immune action, the degree of induced inflammatory response was dependent on the level of MAPK activity [4]. Strain-specific structural differences in LTA led to differences in binding capacity with its receptors, which led to continuity or strength of the signal pathway; a strong signal eventually produced immune activation. Regarding the activity and association of the two grouped _L. plantarum_ strains, there are likely several structural differences in the binding capacity of LTA and its receptor. However, it is expected that the number of fatty acids in the glycolipid and the number of double bonds in the lipid chain had the largest effect. The double bonds of the lipid chain are expected to be involved in generation of immune substances, as well as with signal activity, as they act on strong binding to receptors such as TLRs [24; 25; 26]. It is difficult to see the LTA component of gram-positive bacteria as a single substance. However, the composition and structural differences of the active material act directly. Analysis of SPR using LTAs and cell surface receptors showed faster and stronger association with ligands (TLR2, TLR4, and CD14) with the same pattern as that of the signal activity [11; 27]. In addition, the affinity also was revealed to be high in the same pattern through the analyzed KD index. Through this analysis, we determined that the differences in binding ability with receptors of LTA, a component of the gram-positive bacterial cell wall, directly affect signal activity, which leads to a difference in the expression of immune activity downstream. Indeed, SPR analysis showed that LTAs isolated from K5-5 and K55-5 induced stronger signaling than LTAs isolated from K8 and K88 through association with TLR2 and CD14. The various affinity results of LTAs for TLR2 and CD14 appear to be caused by structural differences in LTAs. Therefore, we separated the glycolipids of the four LTAs, analyzed the distribution of the stroma composing the LTA through GC-MS and LC-MS analyses, and predicted the structures of the four LTAs. All four LTAs contained palmitic acid. In addition to palmitic acid, the K8 and K88 LTAs were composed of 9-octadecenoic acid, 9,10-methylene-octadecanoate, and trans-13-octadecenoic acid. On the other hand, the K5-5 and K55-5 LTAs contained a large amount of oleic acid, in addition to palmitic acid. In this way, not only did the strains contain both common and differing substances, but their retention rates were also different depending on LTAs. This shows that the structure depends on the constituents and their amounts affecting the binding ability with TLR2 and CD14. This is attributed to the difference in the initiation and outcome of signal transduction. In conclusion, through this study, the immune differences of the same bacterial species with different strain number were analyzed using their LTAs. The different binding abilities with the host cell surface receptors, along with the differences in signal pathway, were due to the structural differences in LTAs. LTA is proposed as a major material with multiple functions in lactic acid bacteria. In addition, the structure of a new substance (glycolipid) that can be used in various industrial methods is presented. If this glycolipid can be synthesized as a substitute for lactic acid bacteria, many formulation limitations can be overcome, and the material will be industrially applicable in a variety of fields. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12030954/s1](https://www.mdpi.com/article/10.3390/app12030954/s1)]([https://www.mdpi.com/article/10.3390/app12030954/s1](https://www.mdpi.com/article/10.3390/app12030954/s1)).