## 4 Discussion
However, the study protocol did not include intermediate measurements of the biomarkers tested for more accurate investigation of the metabolic outcomes. Extending the experimental period to 8-12 weeks and incorporating midpoint measurements would offer valuable insights and enable a comprehensive assessment of metabolic alterations. Secondly, the trial population consisted of a small number of healthy participants, leading to a lack of power in the reliability of the findings; therefore, the generalization of the metabolic effects remains a challenge. Moreover, it is unclear whether the results reflect the efficacy for individuals who suffer from chronic diseases associated with oxidative stress and chronic inflammation, such as diabetic and cardiovascular patients. In addition, a week-long washout period between the two interventions may be insufficient to fully eliminate the residual effects of the initial intervention, particularly if certain compounds, such as carotenoids, have a prolonged half-life. In addition, despite extensive guidelines, the lifestyle factors were not controlled in this trial protocol, so there is a chance that part of the observed results might be related to healthy lifestyle habits during the study period. Also, data management did not include the adjustment of other dietary variables. Therefore, the results of the clinical study may not include the metabolic responses associated with these confounding parameters, limiting the generalizability of the findings. A stricter control of these factors with daily diet records is in order, and further data analysis should examine the effect of the adjustment on the association between the biofunctional miso-type sauce consumption and the biochemical biomarkers. Another limitation of this clinical study procedure is the absence of a nonenriched miso-type sauce control, leading to unclear conclusions regarding whether our findings are attributed to the miso-type sauce itself, to the presence of bioc carotenoids, or to the synergy of both. So, the addition of one more experimental group where participants would consume a control miso-type sauce would be crucial. Concerning future perspectives, further investigation of the impact of habitual intake of the biofunctional miso-type sauce enhanced with biocarotenoids, in a dose-response manner, could be suggested to explore the exact dose at which the bioactive compounds are bioavailable and effective in altering metabolic parameters. Under this design, the determination of plasma carotenoids could lead to safer conclusions about the effectiveness of the enhancement with valuable biocarotenoids, which was performed during the final formulation of the miso-type sauce. Further research should involve a larger sample of participants, including both healthy individuals and those with metabolic or chronic diseases. Moreover, a possible association between habitual biofunctional miso-type sauce consumption and specific oxidative stress inflammation could yield valuable insights. Measurement of insulin and hemoglobin A1C (HbA1C) could offer more accurate insights into the levels of long-term glycemic control. Additionally, the evaluation of the triglycerides to HDL cholesterol ratio may facilitate a more in-depth investigation of the effectiveness on insulin resistance [17]. Regarding oxidative status, current knowledge should be expanded by the inclusion of novel oxidative stress biomarkers, such as the performance of a Plasma Antioxidant Test (PAT), as well as the evaluation of Reactive Oxygen Metabolites (ROMs) and the Oxidative Stress Index (OSI) [39]. Finally, due to the crucial role of proinflammatory cytokines in the up-regulation of inflammatory reactions, the determination of inflammatory biomarkers, including Interleukin (IL)-6, IL-18, tumor necrosis factor alpha (TNF-\(\alpha\)), and the inflammatory high sensitivity C-reactive protein (CRP), is needed to enhance the understanding of the modulatory effects on inflammation status.