Organic acids (OA) are increasingly used as feed additives in aquaculture due to their potential benefits in improving performance and health. Previous research has shown that OAs, such as citric acid, can improve growth, feed intake, and feed conversion ratio in various aquaculture species. Sorbic acid, another OA, exhibits strong antimicrobial properties. Nature-identical compounds (NICs), like thymol and vanillin, also show promise as feed additives due to their potential benefits on feed palatability, immune system, and pathogen reduction. Blending OAs and NICs is hypothesized to create synergistic effects on growth and health. This study aimed to evaluate the effects of a microencapsulated blend of citric acid, sorbic acid, thymol, and vanillin on the growth, feed utilization, intestinal cytokine gene expression, and gut bacterial community of European sea bass under normal and suboptimal rearing conditions. The suboptimal conditions, simulating stressful summer conditions in the Mediterranean, were designed to test the resilience of the additive's benefits under pressure.

Extensive literature supports the use of organic acids as growth promoters and health enhancers in terrestrial livestock. However, research on their application in aquaculture is still relatively limited. Several studies have reported positive effects of citric acid on growth and feed utilization in various fish species, including red drum, rainbow trout, beluga sturgeon, yellowtail, tilapia, and red sea bream. Sorbic acid has been shown to improve weight gain in rainbow trout when used in blends with other organic acids. The use of botanicals and nature-identical compounds (NICs) as feed additives in aquaculture has also gained attention. Thymol and vanillin, two promising NICs, have demonstrated antimicrobial and anti-inflammatory properties in mammals. However, their effects on marine fish have not been extensively studied. The combination of microencapsulated OA and NIC blends has shown promising results in terrestrial livestock and some aquaculture species (Pacific white shrimp and rainbow trout). This study aimed to investigate the effects of such a blend on European sea bass, a species not previously studied with this specific combination.

A 71-day feeding trial was conducted using European sea bass juveniles (initial weight 13.23 ± 0.18 g). Fish were randomly distributed into twelve 500 L tanks (60 fish per tank), with triplicate groups for each of four experimental diets. The diets consisted of a commercial diet coated with increasing levels (0, 250, 500, 1000 ppm) of a microencapsulated blend of citric acid (25%), sorbic acid (16.7%), thymol (1.7%), and vanillin (1%) in a hydrogenated fat matrix. Fish were fed to visual satiation twice daily. After 71 days, growth parameters (final body weight, SGR, FI, FCR, survival) were recorded. Biometric indices (VSI, HSI, CF) and whole-body composition were determined. Intestinal samples were collected to assess the gene expression of six cytokines (IL-1β, IL-6, IL-8, IL-10, TNF-α, TGF-β) involved in immune and inflammatory responses. Distal intestine content was collected for gut microbiota characterization using 16S rRNA gene sequencing. To evaluate the effects under suboptimal conditions, fish from the control (D0) and highest dose (D1000) groups were subjected to high temperature (30.0 ± 0.4 °C) and low oxygen (4.6 ± 0.6 mg L⁻¹) for 8 days. RNA extraction, cDNA synthesis, and real-time PCR were performed to quantify cytokine gene expression. DNA extraction, 16S rRNA gene amplification, and sequencing were performed for gut microbiota analysis. Data analysis involved linear regression, two-way ANOVA, Wilcoxon rank-sum test, and principal component analysis (PCA).

No significant differences in growth performance (final body weight, SGR, FI, FCR, survival) were observed among the dietary treatments over the entire 71-day trial or during intermediate periods. Similarly, biometric indices (VSI, HSI, CF) and whole-body composition showed no significant differences. However, the analysis of cytokine gene expression revealed a significant upregulation of IL-8 (pro-inflammatory), IL-10, and TGFβ (anti-inflammatory) with increasing dietary levels of the OA and NIC blend. 16S rRNA gene sequencing showed that the blend had prebiotic effects, increasing the abundance of beneficial bacteria such as *Lactobacillus*, *Leuconostoc*, and *Bacillus* sp. Picrust analysis indicated a functional reconfiguration of the gut microbiota, reducing inflammation-promoting functions at higher blend concentrations. Exposure to suboptimal rearing conditions significantly altered the gut microbiota composition, reducing the abundance of LAB and increasing Proteobacteria, particularly *Enterobacteriaceae*. This was consistent with the observed upregulation of pro-inflammatory cytokines (IL-1β, IL-6, IL-8) and downregulation of TGFβ. The OA and NIC blend seemed to mitigate some negative effects of suboptimal conditions on the gut microbiota, maintaining diversity levels comparable to those under normal conditions.

The lack of growth promotion despite the observed immunomodulatory effects suggests that the tested blend may primarily function as an immunostimulant and gut microbiota modulator rather than a growth promoter, at least at the concentrations tested. The upregulation of both pro- and anti-inflammatory cytokines could reflect a complex interplay of immune responses aimed at maintaining gut homeostasis. The prebiotic effects observed on the gut microbiota corroborate the blend's potential to improve gut health. The significant changes in gut microbiota composition and diversity under suboptimal conditions highlight the sensitivity of the gut microbiome to environmental stressors. The mitigating effect of the blend on these changes suggests its potential to enhance resilience to environmental stress. Future research should explore higher concentrations of the blend and test its efficacy in combating specific pathogens.

The study demonstrated that the microencapsulated blend of organic acids and nature-identical compounds did not enhance growth performance in European sea bass but exhibited immunomodulatory and prebiotic properties. The blend effectively stimulated beneficial bacteria and potentially reduced inflammation. Exposure to suboptimal conditions negatively impacted gut microbiota; however, the blend showed some potential in mitigating these effects. Further research is necessary to evaluate higher inclusion levels and the blend's effectiveness against specific pathogens.

The study focused on a specific blend of organic acids and nature-identical compounds; therefore, the results may not be generalizable to other blends. The suboptimal rearing conditions were simulated in a controlled environment and may not completely replicate the complexity of natural environmental variations. The study did not investigate the long-term effects of the blend on growth or health.