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While the e ff ect of rice NPN on TNF- α is seen to be moderate post-week 4, this trend was in keeping with those of previous studies [62,63] and may be explored in future work through optimization of study parameters such as population size, dose, or duration. It is also worth noting that the Christmas holidays overlapped significantly with this period of the trial and it is likely subjects did not comply with the study protocol as rigorously during this time. It was statistically shown above that a serious confounding factor a ff ectinge ffi cacy was compliance; the higher the reported level of compliance, the greater the e ff ect observed. In accordance with our findings here, Rein et al., (2019), investigated a commercial scale up of rice NPN, and also demonstrated a significant reduction in TNF- α in a 24-h kinetic study in healthy subjects [26]. Finally, in line with other studies [64–66], despite a positive impact on TNF- α , herewe report no observable increase over the test period on other inflammatory markers (IL-6, IL- β andCRP) and, as such, no measurable e ff ect of rice NPN. We further characterized the rice NPN by identifying seven peptides that exhibited anti-inflammatory e ffi cacy against the LPS-induced TNF- α secretion, with some exhibiting comparable e ff ects to the parent rice NPN. We do note that the reported e ff ects of a number of our peptides are more modest versus those of the rice NPN. However, this is to be expected since parent protein ingredients benefit from the synergistic e ff ects of their constituent peptides [67]. Additionally, the bioavailability of bioactive peptides in rice NPN are under further investigation. While the precise mechanism of action of our peptides against TNF- α is not known, it has been shown previously that both endogenous [68] and exogenous [69] peptides can inhibit signal transduction pathways involved in the expression of inflammatory cytokines [20]. Moreover, some of the peptides presented here have been shown (both in silico and in vitro) to bind with high a ffi nity to TNF- α directly [26] potentially causing the disassembly of this cytokine [70], or preventing the binding of TNF- α to its receptors on human tissues [71,72]. Further work will be required to elucidate which of these potential mechanisms alone or in combination may be responsible for the observed e ffi cacy of these peptides, and indeed the rice NPN. 5. Conclusions Ultimately, in this study we have validated a functional ingredient possessing anti-inflammatory effects in vitro and in human, with rice NPN conferring beneficial effects on physical performance, circulating cytokines, cholesterol, and glucose control in a relevant human population. We have also validated the use of an AI approach for predicting constituent bioactive peptides within a functional ingredient. By adopting this innovative framework, we show that targeted, characterized, functional ingredients o ff er potential in the maintenance of health or the prevention and treatment of various conditions. Supplementary Materials: The following are available online at http: // www.mdpi.com / 2304-8158 / 9 / 9 / 1147 / s1, Figure S1: Cell viability following rice NPN treatment Figure S2: Ion spectra for predicted peptides detected in rice NPN. Figure S3: Predicted peptide did not reduce LPS induced TNF- α secretion. Table S1: Subject Anthropometric Data. Figure S4: AUC change from baseline for (a) IL-1 β , (b) IL-6 and (c) C-Reactive Protein. Author Contributions: Conceptualization, T.A.H., A.M.W. and N.K.; methodology, B.M., B.K., K.K., T.A.H. and A.M.W.; validation, K.K., R.C. and A.K.; formal analysis, A.M.W., G.J. and T.A.H.; investigation, K.K., A.K.; data curation, A.A. and C.L.; writing—Original draft preparation, T.A.H., A.M.W., B.K., K.K. and N.K.; writing—Review and editing, Therese Holton, Audrey Wall, Brian Keogh, Kathy Kennedy and Nora Khaldi visualization, T.A.H., A.M.W., A.A. and C.L.; supervision, N.K.; project administration, K.K., B.K. and A.M.W. All authors have read Acknowledgments: The authors thank Wim Calame (StatistiCal BV), Fergus Shanahan (University City Cork; Atlantia Food Clinical Trials), Tim Considine (Nuritas Ltd.), Sanja Trajkovic (Nuritas Ltd.), Sean O’ Callaghan (Nuritias Ltd.) and John Savage (Nuritas Ltd.) for useful discussion. Conflicts of Interest: All authors are employees of Nuritas Limited and declare no conflict of interest. Human trial analysis was carried out by Wim Calame (StatistiCal BV). The design of the study; the collection, all other analyses and interpretation of data; the writing of the manuscript and the decision to publish the results was performed by Nuritas. and agreed to the published version of the manuscript. Funding: This research received no external funding.
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