A Mixture of Organic Acids and Botanicals Improves the Intestinal Barrier Functionality in Vitro

Organic acids and botanicals are widely used in animal nutrition as antibiotic replacers or adjuvant and recent studies have highlighted their possible role as “intestinal health enhancers” via improvement of gut barrier function. The aim of this study was to assess the impact of a mixture of organic acids and botanicals on the intestinal barrier function using a cell culture model.

Caco-2 cells were seeded on transwell inserts, cultured in a high glucose DMEM+10% FBS (basal medium, BM) in 5% CO₂at 37°C and allowed to grow until stable. Then cells were cultured for 15 days in BM (control group), or BM added with a mixture of citric acid, sorbic acid, thymol and vanillin at 0.2 or 1 g/L. Trans-epithelial electrical resistance (TER) was measured every other day using a voltohmmeter. At d7 and d15 since the addition of the experimental media, the paracellular permeability was measured with FITC-dextran flux and at d15 cells were harvested to assess mRNA expression of tight junction markers by qPCR. Data were analyzed with 1-way ANOVA (FITC-dextran flux and mRNA expression) or ANOVA repeated measures (TER), and the treatments had 5 independent replicates (n = 5).

Compared to control, the blend of organic acids and botanicals at 0.2 g/L significantly improved the TER starting at 4 days since the beginning of the experiment (+13%, P<0.001) and the increase remained significant throughout the experiment (on average +12%, P<0.05). At d15 both treatments improved the TER in a dose-dependent way compared to control (+16% for 0.2 g/L, P<0.05; +27% for 1 g/L, P<0.001). FITC-dextran flux was not affected by the treatments. Tight junction mRNA expression was generally improved by the treatments: while claudin-1 was not affected, occludin tended to be improved at 1 g/L (P<0.2) and zonula occludens-1 was increased in a dose-dependent manner (P<0.01), as shown in Table 1.

In conclusion, the mixture of organic acids and botanicals improved Caco-2 epithelial barrier integrity by increasing the TER and improving the tight junction expression and this could validate the “gut barrier-improving” mechanism of action of these additives in animal nutrition.

Table 1 – Tight junction markers mRNA relative expression*

	CTR	0.2 g/L	1 g/L	Pooled SEM	P-value
Claudin-1	1.02	1.17	1.12	0.10	0.53
Occludin	1.02	1.07	1.31	0.12	0.19
Zonula occludens-1	1.02	1.35	1.61	0.10	0.004

* Normalized on 2 housekeeping genes (GAPDH and RPLP0) and calculated relative to the control group.