Are essential oil nano-emulsions better than essential oils?

This was the finding of a study recently published in Nature. Essential oils (EOs) can be a novel alternative to antibiotics in poultry production as they possess antimicrobial properties that benefit animal health performance. However, essential oils mainly contain lipophilic and aromatic bioactive compounds which have some limiting aspects for use due to their low solubility and stability. Nano-emulsions are one prospective approach for efficiently delivering essential oils in poultry farming. Nano-emulsions of essential oils are carrier systems that can be used to overcome the volatile nature of EOs which limits their application. The encapsulation of EOs by nano-emulsions is an innovative technology that can help overcome the factors limiting EO use and preserve therapeutic efficacy by reducing volatility, increasing stability, and solubility. Due to their increased surface area, nano-emulsions stabilise and improve the antimicrobial effects of oils in aqueous solutions.

Essential oil nano-emulsion study

The study evaluated the growth-promoting effects of thymol and thymol nano-emulsion and their protection against Salmonella Typhimurium infection in broilers. One-day-old 2400 chicks were assigned to either the control groups (negative and positive control groups), thymol group, or thymol nano-emulsion group.

• Negative control birds received a control diet without thymol or thymol nano-emulsion and were not challenged;
• Positive control birds received a control diet without thymol additives but were challenged at d 23 of age with S. Typhimurium strain;
• Birds in the thymol and thymol nano-emulsion groups received a control diet supplemented with either thymol or thymol nano-emulsion at concentrations of 0.25, 0.5 and 1% and were also challenged.

All experimental birds in the positive control group, thymol, and thymol nano-emulsion groups were challenged with the S. Typhimurium inoculum dose (one mL of 3×106 CFU/bird), while only the negative control group was not challenged.

Growth performance

The study showed that over the total growing period, birds fed 1% thymol nano-emulsion had better growth performance (Table 1) even after the S. Typhimurium challenge, which was attributed to reduced infection severity. The possible mechanism of essential oils as it affects growth performance could be attributed to the increased digestibility of feed by stimulation of endogenous enzymes and regulation of the gut microbial fora.

In this study, in line with the increased growth rate and feed utilisation in the nano-emulsion group, the expression of genes encoding digestive enzymes was also upregulated. It was proven that dietary thyme oil increased the digestive enzymes and improved the nutrient utilisation of broilers. The mRNA expression of pancreatic alpha 2A amylase, pancreatic lipase, and cholecystokinin genes was up-regulated in response to dietary inclusion of higher levels of thymol or thymol nano-emulsion when compared with negative and positive control groups. Notably, the most prominent effects were ­reported for the thymol nano-emulsion group.

Gut integrity

Intestinal integrity

The mRNA expression of digestive enzymes (pancreatic alpha 2A amylase (AMY2A), pancreatic lipase (PNLIP), and cholecystokinin (CCK)) genes was up-regulated in response to dietary inclusion of higher levels of thymol or thymol nano-emulsion when compared with negative group pre-infection and positive group post-infection, with the most prominent effects reported for the thymol nano-emulsion group. In addition, the group supplemented with 1% thymol nano-emulsion showed the most significant upregulation of mRNA expression of the tight junction protein genes. In the current study, a higher concentration (1%) of thymol nano-emulsion greatly upregulated genes encoding occludin, zona occludens-1, claudins -1, JAM, MUC-2, and FABP2, controlling the barrier functions even after experimental infection, and it was better than thymol. Dietary supplementation with thymol nano-­emulsion was therefore shown to enhance intestinal integrity and strengthen the mucosal barrier function.

Modulation of cytokine gene expression

A distinct array of cytokines that are released in response to Salmonella infection contributes to the development of inflammatory reactions in the intestine. The current results showed that dietary thymol or thymol nano-emulsion supplementation downregulated IL-2 and IL-6 genes expression levels with a more remarkable effect for thymol nano-emulsion than for thymol. The higher reduction of pro-inflammatory cytokines in the thymol nano-emulsion groups even after S. Typhimurium infection indicated its strong anti-inflammatory efficacy. It was suggested this could be due to the uniformly dispersed nano-droplets of thymol nano-emulsion, which easily penetrate and disrupt the microbial membrane.

Microbiota modulation

The microbiota contributes to the development and maintenance of the intestinal epithelial barrier as well as the development of the immune system and competition with pathogenic microorganisms. In the current study, dietary thymol and thymol nano-emulsion altered the microbiological profile of total aerobic and anaerobic bacteria, Lactobacillus and Enterobacteriaceae in the caecum of birds at d 22 (pre-infection) and d 30 and 42 (post-infection). In comparison to the control groups, the caecal Enterobacteriaceae and anaerobic bacterial populations were reduced while the number of Lactobacillus loads was increased in groups fed different concentrations of thymol and thymol nano-emulsion in a dose-dependent manner even after infection with S. Typhimurium. Again, the thymol nano-emulsion group had stronger effects than thymol.

Protection against salmonella infection

Salmonella Typhimurium is a common enteric pathogenic bacterium that causes serious economic losses to the poultry sector. Besides economic losses, reducing Salmonella populations in chickens can potentially reduce the contamination of poultry meat and its products and protect against food-borne salmonellosis in humans. In this study, at 14 days post-challenge (dpi), the supplementation of 1% thymol, and 0.5% and 1% thymol nano-emulsion significantly reduced Salmonella counts compared to the positive control group. The most striking result was reported for 1% thymol nano-emulsion, where it greatly reduced Salmonella counts by 2.27 log10 CFU/g. In addition, the relative mRNA expression levels of the S. Typhimurium invA gene (a marker of Salmonella virulence) were downregulated more in thymol nano-emulsion birds than in the thymol group.

Recommended dosage

The researchers concluded that essential oil nano-emulsions are more effective in improving the growth performance and health status of broilers compared to essential oils alone, as observed in this study that saw more beneficial effects of thymol nano-emulsion compared with thymol essential oil. “On the basis of our results we recommend the use of 1% thymol nano-emulsion as a promising candidate with growth-promoting and anti-virulence properties for the control of S. Typhimurium infection, which is an additional benefit for consumers’ health,” they said.