# Immunomodulatory Properties of Probiotics and Their Derived Bioactive Compounds
## 6 Challenges and Future Perspectives
Additionally, clarification of the exact molecular mechanisms of probiotic action, selection of the appropriate bacterial strain for targeted therapy, and determination of the most efficient dose remain necessary [173]. It is hypothesized that specific probiotic-derived compounds, mainly bacterioxins and SCFAs, are involved in the improvement of gut immunity without causing the undesirable side effects related to living microorganisms in high-risk patients [51]. Bacterioxins could be a promising therapeutic approach for the eradication of harmful pathogens showing resistance to conventional antibiotics [88; 174]. Nevertheless, there is limited in vitro and in vivo evidence regarding their efficacy and safety as a treatment option for various human diseases [90]. In order to determine their efficiency, pharmacokinetic parameters, including solubility, bioavailability, and biodegradation, must be evaluated [175]. A few studies have indicated that bacterioxins may aggregate in vivo due to disturbance of their physical stability or alterations of their biochemical properties during their exposure in the GI tract. This could lead to low bioactivity and a subsequent immune response [91]. In this context, the encapsulation of purified bacterioxins into a protective matrix or the application of bioengineering methods [176] could be efficient approaches in order to improve their stability when orally administrated [175]. Further studies should be conducted to determine the action of purified bacterioxins since the presence of other postbiotics may mask their actual effects, as well as the combined action of their mixtures, since probiotics produce a variety of them. Thus, it is still challenging to develop more advanced, efficient, and affordable methods for the purification of bacterioxins from cell free supernatants to ideal concentrations for therapeutic applications [176] or the discovery of new bacterioxins [177]. In general, SCFAs comprise the largest group of bioactive compounds residing in the gut lumen, thus participating vigorously in the stimulation of gut immunity synergistically with other endogenous metabolites or exogenously administered drugs [1; 146]. Additionally, SCFAs are endowed with the capacity to promote immune response, not only locally, but also systemically, due to their dissemination to distant tissues and organs [18; 178]. To date, there are limited in vivo studies focusing mostly on the impact of SCFAs administration in chemically induced colitis animal models. The design of randomized controlled trials is required to fully understand the clinical impact of SCFAs in host health [116]. Several factors could affect the efficacy of SCFAs, including their route of administration, their dynamic interactions with commensal bacteria or other metabolites, and the variability in gut microbiota composition between individuals. It still remains a major challenge to determine the in vivo production of SCFAs in tissues other than the gut [179]. In this context, further investigations are necessary to extrapolate conclusions regarding the optimal dose and composition of SCFAs, and comparative analysis experiments could configure the criteria of SCFAs utilization based on the distinctiveness of each individual. Finally, there is lack of evidence regarding the combined action of purified bacterioxins and SCFAs. Further studies are warranted to ascertain their optimal ratio as therapeutics. Conceptualization, MT. ; writing--original draft preparation, C.T. ; writing--review and editing, M.T. and C.T. ; figure editing, C.T. and M.T. ; supervision, M.T. All authors have read and agreed to the published version of the manuscript. This research received no external funding. Not applicable. The authors express their gratitude to [PERSON] for the critical reading of the MS. This study was partially financially supported by the Graduate Program \"Applications of Biology\" of the School of Biology, Aristotle University of Thessaloniki. The authors declare no conflict of interest.