Characterization and antibacterial application of peppermint essential oil nanoemulsions in broiler

Highlights

• •Menthol, 32.3 % as a major compound of Peppermint essential oil has been observed by Gas chromatography and Mass spectroscopy.
• •A smaller droplet size of Peppermint essential oil nano-emulsion (PEONE) has been observed by transmission electron microscopy (TEM) micrograph image.
• •Peppermint essential oil nano-emulsion (PEONE) showed positive in silico, in vitro, and in vivo outcomes as compared to traditional antibiotics.
• •100 µl treatment of PEONE showed higher weight gain and lower Feed conversion ratio (FCR) at day 42.
• •Due to efficient antibiotic activity of menthol in peppermint reduction in total coliform with increase in lactobacillus count was observed.
• •Positive effect of PEONE on hematology, serum biochemistry, and internal organ was observed.
• •To access the meat quality characteristic no significant difference in meat pH, drip loss, cooking loss, and colorimetry was analyzed, and it was observed that tenderness and overall acceptability of broiler's meat treated with PEONE was better than control and antibiotics.

Abstract

In order to mitigate the risk of antibiotic resistance in poultry, scientists nowadays consider plant secondary metabolites to be a major organic antibacterial substitute. This study aimed to characterize and investigate the in silico, in vitro, and in vivo antibacterial effects of peppermint essential oil (PEO) in the form of a nanoemulsion (NE), termed PEONE. Menthol as a major compound of PEO has been identified by gas chromatography and mass spectroscopy (GCMS) analysis as 32.3 %, while lower droplet size, polydispersity Index (PDI), and optimum zeta potential values depicted the stability of PEONE have been observed and validated by transmission electron microscopy (TEM) micrograph image. In silico antibacterial activity was studied by molecular docking of menthol and enrofloxacin with Topoisomerase IV protein (PDB: 1s16;) of Escherichia coli K12 MG1655 and this effect was validated by in vitro and in vivo analysis. In vitro analysis, sustained release of PEONE has been observed against Escherichia coli and Staphylococcus aureus. In this study for in vivo experiments (n = 90) day-old broiler chicks were distributed into 6 dietary treatments with 5 replicates of 3 birds per replication. Dietary treatments included 1) Negative control (basal diet), 2) Positive control (basal diet + 200 µl enrofloxacin), 3) 25 µl PEONE + basal diet, 4) 50 µl PEONE + basal diet, 5) 75 µl PEONE + basal diet, and 6) 100 µl PEONE + basal diet. Analyzed data by different statistical tools confirmed that PEONE significantly affected body weight gain (BWG) with an improved feed conversion ratio (FCR) compared to the control group. A significant increase in cecal Lactobacillus count and a decrease in total coliform was observed. Positive effects on physiological parameters, visceral organs, and meat quality characteristics have been observed. In conclusion, our experiments suggest that PEONE can be used in the broiler industry as a substitute for antibiotics to minimize bacterial resistance.

Graphical abstract

Introduction

In recent decades, the poultry industry has gained increasing attention for providing affordable and instant meat and meat products. Broiler farming is critical in the meat industry because it efficiently converts feed into meat. To fulfil consumers' meat requirements, antibiotics are used as growth promoters (Muaz et al., 2018). According to Gadde et al. (2017), these antibiotics were able to enhance growth by improving feed efficiency and preventing poultry diseases. More strains of antibiotic-resistant bacteria are the output of excessive antibiotic use which pose health risks (Serwecinska 2020). In broiler meat antibiotic residues have been reported, which promote health issues (Haque et al., 2020). Keeping these issues in knowledge, scientists are finding alternatives to antibiotics for the broiler industry.

Due to their great eco-friendliness, the immunomodulatory, antioxidant, and antibacterial effects of natural bioactive compounds of plants in the form of EOs have shown great potential as substitutes for growth promoters (Ibrahim et al., 2022). The direct application of EOs faces the challenges of low water solubility, high volatility, and low stability (Liao et al., 2021). These hurdles have encouraged scientists to take advantage of nanotechnology for the efficient delivery of active EO ingredients in the form of nanocarrier systems (El Asbahani et al., 2015). Among these nanocarrier systems, Nanoemulsions (NEs) offer advanced delivery because of their easy production, low cost, high stability, increased surface area, and direct tissue distribution capabilities (Ibrahim et al., 2022; Esfanjani et al., 2018). These NEs encapsulate bioactive compounds by reducing their hydrophobicity (Falleh et al., 2021).

Among these natural antibiotic substitutes, peppermint (Mentha piperita L.) is a traditional medicinal plant, has antibacterial, anti-inflammatory, and antioxidant properties that promote health and well-being (Dawood et al., 2022). Menthol is considered as a major constituent of peppermint with antimicrobial properties that improve the resistance of broilers against heat stress (Abdel-Wareth et al., 2019). Based on this beneficial effect of menthol, it can be assumed that the encapsulation of menthol in the form of NE may increase its bioavailability and effectiveness. This study has been designed to prepare and characterize PEONE for in vitro and in vivo application in broilers as an organic growth promoter and substitute to antibiotics in silico approach to drug characterization and molecular docking.

Materials and methods

Materials

PEO was purchased from the inner Fareed Gate Market of Bahawalpur, Pakistan. Tween 80, De, Man-Rogosa-Sharpe (MRS), and Total Plate count (TPC) agar from Sigma Aldrich, USA. Day-old Chicks and Poultry feed were purchased from Faisal Chick's Hatchery and Feed Pvt. Ltd. Multan, Pakistan. The chemicals used in this study were in the analytical grade.

Preparation of PEONE

The formulations were composed of PEO as the oil phase and Tween 80 as the surfactant. PEONE was prepared using a probe ultrasonicator (UCD-1200, BIOBASE China). The 5-15 % PEO as oil phase was mixed with 2-3 % Tween 80 surfactant for coarse emulsion preparation via a DH-1500 inverted homogenizer at 18000 rpm for 3 min. This coarse emulsion was homogenized using an ultrasonicator for 10 min (Rest: Work 30/30 sec). Prepared NE was further characterized for mean droplet diameter, PDI for particle size distribution, and zeta potential to measure stability by using nano Zeta-Sizer (3000 HS, Malvern Instruments, Malvern, UK).

Morphology visualization of PEONE

The morphology of the PEONE droplet was investigated through digital imaging by TEM (JEOL 2100, Hitachi High-Technologies Corp., Tokyo, Japan). PEONE droplets were placed on a carbon-coated copper grid with a dropper and stained with 2 % (w/v) phosphotungstic acid. Before analysis, the carbon-coated copper grid was dried for 24 h, and the examination was conducted using an accelerating voltage of 100 kV

Gas chromatography and mass spectroscopy (GC-MS) analysis

GC-MS analysis of PEO was performed according to Kelidari et al. (2021), through a set of 7890A Network GC systems coupled with a 5975C VL MSD with Triple-Axis Detector (Agilent Technologies, Santa Clara, CA, USA). HP-5MS silica fused column was used for separation of components, with 40 °C as initial temperature for 1 min with 3 °C/minute increase in temperature of up to 250 °C as the final temperature for 90 min. Split-flow 10 mL/min with 6 mL/min septum purge, and 1mL/min column flow rate. Helium gas was used as a carrier gas with 99.99 % purity.

Drug characterization and molecular docking

The major compound of PEO was compared in silico with enrofloxacin as a synthetic antibacterial compound using Swiss ADME (absorption, distribution, metabolism, and excretion), which has been utilized for drug characterization by notable researchers (Bakchi et al., 2022; Daina et al., 2017). Toxicity analysis was performed by pkCSM server (https://biosig.unimelb.edu.au/pkcsm/theory). The Stitch database (https://stitch.embl.de/) was used for Ligand-Protein and further Protein-Protein Interaction by adding ligand names (menthol and enrofloxacin) in the item search bar while Escherichia coli K12 MG1655 was selected as organism (Carpenter and Altman 2024). Interacting proteins showing 100 % similarity were selected for further analysis, while selected proteins validation was performed by Ramachandran Plot using PROCHECK analysis (Liu et al., 2024). All the chemical structures were retrieved from the Pubchem database (https://pubchem.ncbi.nlm.nih.gov/). The uniport system (https://www.uniprot.org/) was used to collect the function data of the concerned protein (Zhang et al., 2024). Interacting protein structure was obtained from the Protein data bank system (https://www.rcsb.org/). SwissDock tool of the Swiss institute of Bioinformatics (https://www.swissdock.ch/) was used for molecular docking and the ligand with the lowest binding affinity was considered for favorable interaction with the docking receptor. All the docking results were visualized by UCSF chimera version 1.17.3 (3D and solid surface) (Ait et al., 2024), while the 2D structure was obtained by BIOVIA Discovery Studio Visualizer (Sharma et al., 2019).

Minimum inhibitory concentration (MIC) of PEONE

MIC of PEONE was measured by liquid culture test at 600nm optical density (OD) measurement. 80ul, 100ul, 120ul, 150ul, and 200ul of PEONE were mixed with 100 µl of nutrient broth in the 96-well microtiter plate respectively. After that 10 µl bacteria (E. coli and S. aureus) were inoculated in each well of different PEONE concentrations and one well was kept blank only with bacteria and nutrients without any treatment of PEONE and the plate was incubated at 37 °C for 24, 48, and 72 h and the readings were measured at respective times (Hou et al., 2021).

Animals, diets, and formulations

In this experiment, day-old (n = 90) broiler chicks were randomly assigned to 6 treatment groups, each with 5 chicks in 3 replicates, according to the guidelines of the Ethical Review Committee of CUVAS Bahawalpur, Pakistan. All the birds were properly acclimatized and then grouped into a negative control group (only basal diet), positive control (basal diet + 200 µl of enrofloxacin), 25 µl (basal diet + 25 µl of PEONE), 50 µl (basal diet + 50 µl of PEONE), 75 µl (basal diet + 75 µl of PEONE), and 100 µl (basal diet + 100 µl of PEONE) and treatments were applied during growth period (15-28 days). During the project, animal welfare was the main goal of the project which has been strictly followed according to the ethical review committee guidelines from CUVAS. Table 1 illustrates the composition of the basal diets and their nutrient levels.

Table 1.

Basal diet composition (g/kg). d = days.

Item	Starter (0-14 d)	Grower (15-28 d)	Finisher (28-42 d)
Corn (8 % CP)	520	500	530
Soyabean meal (43 % CP)	438.5	429.0	398
Soyabean oil	13	30	35
Calcium carbonate	9.5	8.5	8
Dicalcium phosphate	15	11	12
Wheat Bran	5	10	15
Canola meal	45	45	45
DL-methionine	3.22	2.2	1
L-Lysine	1	-	-
Vitamin premix	3.5	3.5	3.5
Mineral premix	2.5	2.5	2.5
Gluton 30 %	1	-	-
Salt	1	1	1

Growth performance

On day 14, the initial body weight (BW) was recorded, and when the treatments were started, the BW, BWG, feed intake (FI), and FCR recorded at 21, 28, and 42 days of the age.

$$\text{FCR}=\frac{\text{Feed}\text{Intake}}{\text{Weight}\text{Gain}}$$ (1)

The mortality rate was assessed daily, and any deceased bird was promptly sent to the pathology laboratory for further analysis. Then, the feed was adjusted accordingly.

Blood hematology and serum biochemistry

At the day 42 blood was collected from 5 birds per treatment by puncturing the wing vein in two tubes containing EDTA for hematology analysis and without EDTA for serum biochemical analysis. For hematology red blood cell (RBC), white blood cell (WBC) counts, Hemoglobin (HB), lymphocytes (LYM), and platelets (PLT) were measured by a Mindray BC-1800 hematology analyzer. While biochemical parameters Alanine transaminase (ALT), aspartate aminotransferase (AST), High-density lipids (HDL), Low-density lipids (LDL), total protein, and Triglycerides were evaluated by a CONTEC BC300 Semiautomatic Blood Biochemistry Analyzer.

Relative organ %

To determine the relative organ (liver, gizzard, heart, and abdominal fat) weight of birds on day 42, birds that were used for blood collection were slaughtered under the supervision of the ethical review committee of CUVAS Bahawalpur, Pakistan strictly following their guidelines for animal welfare and any pathological lesion was carefully examined by an expert pathologist from CUVAS Bahawalpur, Pakistan. After weighing the other carcasses, the dressed weight was calculated based on the following:

$$\text{Dressing}\%=\frac{\text{Dressed}\text{Weight}}{\text{Live}\text{Weight}}\mathrm{x}100$$ (2)

$$\text{Relative}\text{Organ}\text{Weight}=\frac{\text{Organ}\text{Weight}}{\text{Total}\text{Body}\text{Weight}}\mathrm{x}100$$ (3)

Meat color, pH, and drip loss of broiler meat

The meat pH was recorded by a pH meter (Model PH 211, Hanna Instruments), with the pH after slaughter considered the initial pH and the pH after 24 h considered the final pH. Meat color, such as lightness (L*), redness (A*), and yellowness (B*), was determined by a chroma meter (Konica Minolta, CR-400-Japan) after one hour of slaughter (Lee et al., 2022).

The drip loss of the meat was assessed by the bag method by hanging meat sample in an airtight polythene bag at 4 °C$.$ The initial weight of the sample was recorded before freezing, and the final weight was measured after 24, 72, 120, and 168 h, while the change in sample weight was expressed as a percentage (Holman et al., 2020).

$$\text{Drip}\text{Loss}\%=\frac{\text{Initial}\text{Muscle}\text{Weight}-\text{Final}\text{Muscle}\text{Weight}}{\text{Initial}\text{Muscle}\text{Weight}}\mathrm{X}100$$ (4)

Meat sensory evaluation

Fresh breast meat was used for sensory analysis by an educated panel aged 20-50 years eating chicken once a week. Aluminum foil-wrapped meat samples tagged with 3-digit numbers were cooked in the oven (75 °C), and evaluation was performed after 10 min of cooking. Uninformed panelists were seated in light-controlled individual booths and were asked to evaluate each sample for sensory attributes according to the performa provided in the supplementary information (Amorim et al., 2016).

Microbial analysis

On day 42, cecal contents were collected aseptically and stored at 4 °C in a sterile container. One gram of cecal content was diluted in a 10-fold serial solution of PBS, and 10⁻⁵ g was used for plating. MRS agar was used for Lactobacillus species, while the total coliform count was determined on TPC agar. Lactobacillus plates were counted after 48 h of inoculation, while total coliform plates were counted after 24 h at 37 °C (Islam et al., 2022).

Statistical analysis

Optimization of PEONE was performed by Box–Behnken designs (BBD) concerning Equation 5 by Design expert-13 software. One-way analysis of variance (ANOVA) in IBM SPSS Statistics, version 22, was used for statistical analysis of data collected as in-vivo dataset, and Tukey's test was applied. Data was represented in the form of Means and standard error (P value-0.05).

$$Y={\beta }_0+{\beta }_1{X}_1+{\beta }_2{X}_2+{\beta }_3{X}_3+{\beta }_{12}{X}_1{X}_2+{\beta }_{13}{X}_1{X}_3+{\beta }_{23}{X}_2{X}_3+{\beta }_{11}{X}_{12}+{\beta }_{22}{X}_{22}+{\beta }_{33}{X}_{32}$$ (5)

where Y is response function, X₁, X₂, and X₃ represents Tween 80, EO, and Sonication time respectively while β₀ is intercept, β₁, β₂, and β₃ linear, β₁₂, β₁₃, and β₂₃ interactive, and β₁₁, β₂₂, and β₃₃ represents quadratic coefficients.

Results

GCMS

In GC-MS analysis, the compound with the highest concentration was taken into consideration. According to Fig. 1, the highest peak area was observed for menthol, which constituted 32.3 % of the total composition at a retention time of 21.6 min (Fig. 1)

Fig. 1.

GCMS graph of PEO: Horizontal X-axis showing retention time while Vertical Y-axis showing peak area for determination of major compound % age.

Optimization of PEONEs

The smallest droplet size was approximately 85nm, achieved with 12ml EO and 2.5g Tween 80. Extended sonication time consistently reduced droplet size, with the smallest size occurring at longer sonication durations. The PDI values observed in this study ranged from 0.021 to 0.1, indicating a homogeneous distribution and low aggregation affinity for PEONE. The lowest PDI was observed at 5ml EO and 2g Tween 80, with a notable decrease in PDI at increased sonication times, particularly at 15 min and 2.5g Tween 80. The zeta potential, estimation of surface charge, and indicator of the physical stability of NEs were observed within the acceptable range (either higher than +30 mV or more negative than -30 mV). The lowest zeta potential was observed at 12ml EO and 2.5g Tween 80 (-18.5mV). (Fig. 2)

Fig. 2.

Optimization of PEONEs: Size: (a-c), PDI (d-f), Zeta Potential: (g-i).

Morphology visualization of PEONE by TEM

TEM images (Fig. 3a) revealed that the nano-size of NE exhibited a clear core-shell structure. The core of the particles appeared lighter, while the outer shell was darker, confirming the oil-in-water (O/W) nature of the NEs. The NE droplets were spherical, uniform, and well-dispersed. This morphology indicated that the particles were consistently sized and well-distributed. Fig. 3b shows the normal distribution curve of droplet size. The droplet size ranged from 20-200 nm, with a mean value of 138.77 ± 28.16 nm.

Fig. 3.

(a) TEM micrograph image of PEONE: (b) Normal Distribution Model of Droplet size visualized by TEM micrograph.

Drug characterization

Enrofloxacin was larger and more complex, with a molecular weight of 359.39g/mol and more heavy and aromatic atoms as compared to Menthol, which has a molecular weight of 156.27g/mol. Enrofloxacin showed a lower fraction of sp³ carbons and more rotatable bonds and hydrogen bond acceptors. Menthol was more saturated, with fewer rotatable bonds and hydrogen bond acceptors and a lower molar refractive. Enrofloxacin was more soluble in water than Menthol, with better solubility and a higher topological polar surface area (TPSA). This suggested that Enrofloxacin had more polar functional groups. Both compounds showed similar profiles regarding problematic reactivity indicators (PAINS, Brenk) and were considered lead-like. Menthol was slightly easier to synthesize. Both drugs have high GI absorption and can cross the blood-brain barrier (BBB). Enrofloxacin was a P-glycoprotein substrate. Menthol also inhibits more cytochrome P450 enzymes and has better skin permeability compared to Enrofloxacin. Both compounds have the same bioavailability score. (Table 2)

Table 2.

Bioavailability characterization of Menthol and enrofloxacin by SWISS ADME server-based program.

Model Name	Enrofloxacin	Menthol	Model Name	Enrofloxacin	Menthol
Physicochemical Properties			Lipophilicity
MW (g/mol)	359.39	156.27	Log Po/w (iLOGP)	2.58	2.55
Heavy atoms	26	11	Log Po/w XLOGP3	-0.25	3.40
Aromatic heavy atoms	10	0	Log Po/w WLOGP	1.91	2.44
Fraction Csp3	0.47	1.00	Log Po/w MLOGP	1.75	2.45
Rotatable bonds	4	1	Log Po/w (Silicos-IT)	2.21	2.06
H-bond acceptors	5	1	Consensus Log Po/w	1.64	2.58
MR	104.95	49.23	Medicinal Chemistry
TPSA (Ų)	65.78	20.23	PAINS	0	0
Water Solubility			Brenk	0	1
Log S (ESOL)	-1.93	-2.88	Lead likeness	1	1
Class (ESOL)	Very soluble	Soluble	Synthetic Accessibility	2.73	2.63
Log S (Ali)	-0.67	-3.50	Pharmacokinetics
Class (Ali)	Very soluble	Soluble	GI absorption	High	High
Log S (SILICOS-IT)	-3.56	-1.48	BBB permeant	Yes	Yes
Class (SILICOS-IT)	Soluble	Soluble	P-gp substrate	Yes	No
Drug likeness			CYP1A2 inhibitor	No	Yes
Lipinski	Yes	Yes	CYP2C19 inhibitor	No	No
Ghose	Yes	Yes	CYP2C9 inhibitor	No	No
Veber	Yes	Yes	CYP2D6 inhibitor	Yes	No
Egan	Yes	Yes	CYP3A4 inhibitor	No	No
Muegge	Yes	Yes	Skin Permeability	-8.67	-4.84
Bioavailability Score	0.55	0.55	-	-	-

TPSA= total polar surface area, Consensus Log P= average of all predicted (Log P_o/w), BBB = blood-brain barrier, GI = gastrointestinal, CYP = cytochrome, log Kp = skin permeation, Pgp = P glycoprotein, MW=Molecular weight.

Toxicity analysis

Table 3 presents the toxicological and safety parameters for Enrofloxacin and Menthol. Both compounds were found non-toxic in the Salmonella/microsome mutagenicity assay (AMES) and do not inhibit the human Ether-a-go-go-Related Gene (hERG) I and II channels. The maximum tolerated dose in humans was higher for Menthol (0.94 log mg/kg/day) as compared to Enrofloxacin (0.51 log mg/kg/day), indicating a higher safety margin. However, Enrofloxacin is identified as hepatotoxic, whereas Menthol was safe. Menthol, on the other hand, showed skin sensitization potential. Higher oral rat acute toxicity (LD₅₀ of 2.431 mol/kg) of Enrofloxacin was compared to Menthol (1.946 mol/kg), but Enrofloxacin's oral rat chronic toxicity (LOAEL of 1.891 log mg/kg BW/day) was slightly lower than that of Menthol (2.017 log mg/kg BW/day). In terms of environmental toxicity, Enrofloxacin showed lower toxicity to Tetrahymena pyriformis (0.288 log µg/L) compared to Menthol (0.333 log µg/L) but was more toxic to minnows (2.408log mM) compared to Menthol (1.29 log mM).

Table 3.

pKCSM toxicity analysis.

Parameters	Unit	Enrofloxacin	Menthol
AMES toxicity	Yes/No	No	No
Max. tolerated dose (human)	(log mg/kg/day)	0.51	0.94
hERG I inhibitor	Yes/No	No	No
hERG II inhibitor	Yes/No	No	No
Oral Rat Acute Toxicity (LD50)	mol/kg	2.431	1.946
Oral Rat Chronic Toxicity (LOAEL)	log mg/kg BW/day	1.891	2.017
Hepatotoxicity	Yes/No	Yes	No
Skin Sensitization	Yes/No	No	Yes
T. pyriformis toxicity	log µg/L	0.288	0.333
Minnow toxicity	log mM	2.408	1.29

hERG: the human Ether-a-go-go-Related Gene, AMERS tpxicity: Salmonella/microsome mutagenicity assay

Ligand-protein and protein-protein interaction

Fig. 4 shows that both menthol and enrofloxacin were directly interacting with parE protein (PDB: 1s16; Topoisomerase IV), showed 100 % similarity, and the highest purity by Ramachandran plot was further considered for docking analysis. As Ramachandran plot rule of 90 % residues in the allowed region for good quality protein model. According to the Ramachandran Plot statistics protein structure in current study was considered a good quality structure containing 92.1 % residues in the allowed region.

Fig. 4.

Ligand-protein and protein-protein interaction.

Molecular docking

Comparative molecular docking analysis has been shown in (Fig. 5, Fig. 6). Notably, menthol (-6.39 Kcal/mol) showed slightly higher binding affinity than enrofloxacin (-6.58 Kcal/mol) with interacting proteins. A detail examination revealed by 2D structure of menthol docking results that ASP 1326 and THR 1246 engaged by two Hydrogen bonds with the receptor, characterized by bond lengths of 2.53Å and 2.83Å. While Enrofloxacin showed Hydrogen bonding with GLU and THR by 1.98 and 3.15 bond length, additional alkyl bonding was found by PHE 2173 (2.58 bond length) and THR showed carbon-hydrogen bonding by 2.16 bond length. Exploring alternatives to conventional synthetic antibiotics, menthol was assessed for its binding efficacy. Although its binding energy of -6.39 Kcal/mol was marginally higher than that of enrofloxacin.

Fig. 5.

Docking results of menthol with topoisomerase IV at -6.39 (kcal/mol) as lowest bonding energy.

Fig. 6.

Docking results of enrofloxacin with topoisomerase IV at -6.58 (kcal/mol) as lowest bonding energy.

Time kill dynamics

According to Fig. 7 antibacterial activity of PEONE showed the highest MIC for E. coli at 120 µl, while for S. aureus at 200 µl concentration at 72 h.

Fig. 7.

MIC of E. coli and S. aureus under different concentrations of PEONE.

Growth performance

Table 4 represents BW, BWG, FI, and FCR. On day 21, the groups treated with the antibiotic (259.62 ± 3.70) and 100 µl of PEONE (268.45 ± 17.76) had significantly greater live body weights than the control group (194.33 ± 4.43), indicating early growth-promoting effects (p < 0.05). This trend persisted on day 28 when the antibiotic (548.08 ± 17.71) and 100 µl PEONE (737.97 ± 23.2) groups exhibited sustained positive impacts on broiler growth (p < 0.05). Notably, 100 µl of PEONE showed an efficiency comparable to that antibiotic. At the days 21 and 28, the antibiotic (560.32 ± 8.00 and 1011.71 ± 21.82) and 100 µl of PEONE (569.79 ± 22.29 and 1117.87 ± 5.69) concentrations significantly increased the body weight gain compared to the control treatment (496.49 ± 13.16 and 933.03 ± 18.93) (p < 0.05). Feed intake remained relatively stable across treatment groups during the early and late growth phases. However, a significant increase in feed intake was observed on day 28 in both the antibiotic (766.16 ± 23.89) and 100 µl NE (737.97 ± 23.2) groups compared to the control group (649.12 ± 22.34) (p < 0.05). The FCR showed a concentration-dependent response.

Table 4.

Effects of PEONE on BW (g/week), BWG (g/week), FI (g/week), and FCR (g/g).

	NC	PC			25 µl	50 µl	75 µl	100 µl	P value
Day 14
BW	302.16 ± 10.44	300.70 ± 5.26	311.20 ± 11.09			296.47 ± 5.59	300.17 ± 10.76	301.33 ± 4.53	0.88
Day 21
BW	496.49 ± 13.16b	560.32 ± 8.00a	530.40 ± 9.03ab			509.85 ± 11.20ab	541.46 ± 4.64ab	569.79 ± 22.29a	0.01
BWG	194.33 ± 4.43	259.62 ± 3.70a	219.20 ± 2.14ab			213.38 ± 16.74ab	241.28 ± 15.19ab	268.45 ± 17.76a	0.006
FI	478.17 ± 20.19	471.08 ± 28.78		477.47 ± 5.91		480.62 ± 21.57	463.57 ± 19.45	452.63 ± 22.53	0.92
FCR	1.68 ± 0.05	1.49 ± 0.07	1.49 ± 0.03			1.83 ± 0.26	1.68 ± 0.04	1.80 ± 0.09	0.29
Day 28
BW	933.03 ± 18.93b	1011.71 ± 21.82ab	975.69 ± 25.10ab			970.74 ± 28.11ab	1006.87 ± 60.26ab	1117.87 ± 5.69a	0.02
BWG	436.53 ± 7.71	451.38 ± 14.02	445.28 ± 20.97			460.88 ± 17.88	465.41 ± 55.61	548.08 ± 17.71	0.11
FI	649.12 ± 22.34b	671.27 ± 15.00b	672.09 ± 15.62b			659.17 ± 10.71b	766.16 ± 23.89a	737.97 ± 23.2ab	0.005
FCR	2.15 ± 0.10	1.83 ± 0.11	1.76 ± 0.13			1.63 ± 0.25	1.81 ± 0.29	1.86 ± 0.09	0.53
Day 42
BW	1246.92 ± 18.59b	1354.28 ± 22.11a	1297.21 ± 28.20ab			1261.02 ± 30.24ab	1353.68 ± 7.13a	1352.65 ± 15.11a	0.009
BWG	313.89 ± 1.29	342.57 ± 10.62	321.51 ± 49.50			290.28 ± 8.34	346.81 ± 67.38	234.78 ± 9.43	0.28
FI	933.86 ± 33.07	966.09 ± 11.91	944.70 ± 15.98			970.79 ± 22.17	950.02 ± 47.81	953.22 ± 24.50	0.94
FCR	2.52 ± 0.24	2.29 ± 0.16	2.21 ± 0.14			2.37 ± 0.16	1.70 ± 0.22	1.79 ± 0.07	0.03
Overall 14-42
BWG	1011.37 ± 59.62b	1158.56 ± 25.02ab	1181.11 ± 19.75a			1161.94 ± 40.21ab	1307.40 ± 19.45a	1186.55 ± 15.32a	0.002
FI	2150.75 ± 35.81	2192.17 ± 38.55	2183.72 ± 7.12			2201.84 ± 40.55	2201.28 ± 22.94	2146.53 ± 3.29	0.61
FCR	2.13 ± 0.10a	1.89 ± 0.07ab	1.84 ± 0.02ab			1.90 ± 0.09ab	1.68 ± 0.00b	1.80 ± 0.02b	0.01

NC: Negative control-basal diet, PC: Positive control-Enorfloxacine+basal diet; 25 µl-25 µl NE+basal diet; 50 µl-50 µl NE+basal diet; 75 µl-75 µl NE+basal diet; 100 µl-100 µl NE+basal diet. Means±SE in each row with different superscripts are statistically different (p < 0.05).

Hematology and serum biochemistry

Table 5 revealed no significant differences in hematological and serum biochemical parameters among the groups.

Table 5.

Effects of PEONE on hematology and serum biochemistry of broilers.

	NC	PC	25 µl	50 µl	75 µl	100 µl	P value
Hematology
RBC (106/µL)	2.14 ± 0.01	2.15 ± 0.02	2.15 ± 0.00	2.16 ± 0.01	2.15 ± 0.01	2.17 ± 0.01	0.85
WBC (103/µL)	22.56 ± 0.17	22.16 ± 0.02	22.13 ± 0.03	22.13 ± 0.02	22.15 ± 0.02	22.13 ± 0.25	0.05
HB (g/dl)	11.50 ± 0.25	12.23 ± 0.52	12.43 ± 0.18	12.53 ± 0.17	12.30 ± 0.00	12.40 ± 0.25	0.18
LYM %	50.66 ± 0.16	52.04 ± 1.29	52.90 ± 0.96	54.02 ± 0.05	52.92 ± 1.01	52.81 ± 1.02	0.23
PLT (109/µL)	101.75 ± 0.23	103.99 ± 3.16	104.80 ± 3.53	106.35 ± 3.96	103.14 ± 3.07	111.98 ± 0.60	0.22
Serum Biochemistry
ALT (U/L)	74.33 ± 2.18	73.66 ± 2.33	77.00 ± 0.57	76.00 ± 2.51	76.33 ± 0.33	77.00 ± 0.57	0.64
AST (U/L)	122.00 ± 0.57	123.33 ± 1.45	126.33 ± 2.96	126.33 ± 1.45	124.33 ± 0.88	125.33 ± 2.33	0.50
HDL (mg/dl)	88.00 ± 0.57	94.66 ± 3.84	88.00 ± 0.57	92.33 ± 2.84	91.00 ± 2.51	88.00 ± 0.57	0.24
LDL (mg/dl)	80.73 ± 6.64	72.75 ± 11.99	74.27 ± 11.97	66.96 ± 7.38	80.73 ± 6.69	72.78 ± 11.93	0.90
Total Protein (g/dl)	84.00 ± 2.08	85.66 ± 2.33	83.00 ± 0.57	85.00 ± 2.88	83.66 ± 2.18	87.33 ± 0.88	0.68
Triglycerides (mg/dl)	3.63 ± 0.12	3.77 ± 0.01	3.60 ± 0.20	3.60 ± 0.11	3.53 ± 0.12	3.56 ± 0.08	0.79

NC: Negative control-basal diet, PC: Positive control-Enorfloxacine+basal diet; 25 µl-25 µl NE+basal diet; 50 µl-50 µl NE+basal diet; 75 µl-75 µl NE+basal diet; 100 µl-100 µl NE+basal diet. Means±SE in each row with different superscripts are statistically different (p < 0.05).

Relative organ %

There was no significant effect on the % of relative organs (liver, heart, and gizzard) (p > 0.05). While the abdominal fat weight varied across the treatments, with value range from 0.88 ± 0.12 to 2.31 ± 0.17 (p < 0.05). (Table 6)

Table 6.

Effect of PEONE on visceral organ %.

	NC	PC	25 µl	50 µl	75 µl	100 µl	P value
Heart (%)	0.47 ± 0.02	0.53 ± 0.04	0.52 ± 0.05	0.52 ± 0.02	0.54 ± 0.03	0.60 ± 0.04	0.24
Liver (%)	3.10 ± 0.26	2.61 ± 0.11	2.52 ± 0.16	2.66 ± 0.11	2.80 ± 0.09	2.65 ± 0.16	0.40
Gizzard (%)	2.40 ± 0.13	2.44 ± 0.31	2.29 ± 0.21	2.69 ± 0.40	2.11 ± 0.13	2.85 ± 0.20	0.39
Abdominal Fat (%)	2.97 ± 0.11a	2.73 ± 0.03ab	1.96 ± 0.05bc	2.40 ± 0.10d	2.23 ± 0.02cd	2.33 ± 0.04c	0.00
Carcass (%)	55.80 ± 1.80	62.01 ± 1.24	58.09 ± 1.08	58.81 ± 3.98	57.71 ± 2.90	64.09 ± 2.22	0.23

NC: Negative control-basal diet, PC: Positive control-Enorfloxacine+basal diet; 25 µl-25 µl NE+basal diet; 50 µl-50 µl NE+basal diet; 75 µl-75 µl NE+basal diet; 100 µl-100 µl NE+basal diet. Means ± SE in each row with different superscripts are statistically different (p < 0.05).

Microbial colony counting

Fig. 8 represented a significant decrease in total coliform count and an increase in the Lactobacillus count between groups was observed. It was perceived that PEONE-treated groups showed similar values to the positive control group.

Fig. 8.

Effect of PEONE on cecal microbial flora (Log10⁻⁵/g).

Meat quality characteristics

Across all meat quality parameters (meat pH, color, and drip loss), no significant difference was observed among the different treatments (Table 7).

Table 7.

Meat quality characteristics of broilers.

	NC	PC	25 µl	50 µl	75 µl	100 µl	P value
pH
Initial pH	6.39 ± 0.03	6.39 ± 0.02	6.39 ± 0.08	6.39 ± 0.05	6.39 ± 0.04	6.39 ± 0.04	0.97
Final pH	6.52 ± 0.04	6.49 ± 0.02	6.45 ± 0.07	6.46 ± 0.00	6.46 ± 0.04	6.48 ± 0.04	0.93
Colorimetry
L*	50.81 ± 3.29	51.91 ± 1.82	48.42 ± 3.82	50.09 ± 1.54	50.92 ± 1.99	48.49 ± 1.79	0.89
A*	11.76 ± 0.57	10.75 ± 0.79	10.69 ± 1.32	10.46 ± 1.35	11.47 ± 1.43	12.56 ± 0.78	0.75
B*	9.79 ± 0.83	10.37 ± 0.44	9.69 ± 0.49	11.09 ± 1.16	11.14 ± 0.36	10.98 ± 1.09	0.65
Drip Loss%
24h	2.50 ± 0.22	2.40 ± 0.27	2.25 ± 0.18	2.53 ± 0.14	2.29 ± 0.17	2.24 ± 0.07	0.80
72h	4.54 ± 0.14	4.33 ± 0.14	4.46 ± 0.08	4.57 ± 0.16	4.48 ± 0.07	4.43 ± 0.11	0.81
120 h	8.44 ± 0.07	8.56 ± 0.10	8.58 ± 0.18	8.47 ± 0.08	8.60 ± 0.15	8.58 ± 0.12	0.91
168 h	15.76 ± 0.10	15.72 ± 0.09	15.77 ± 0.05	15.75 ± 0.12	15.76 ± 0.02	15.79 ± 0.08	0.99

NC: Negative control-basal diet, PC: Positive control-Enorfloxacine+basal diet; 25 µl-25 µl NE+basal diet; 50 µl-50 µl NE+basal diet; 75 µl-75 µl NE+basal diet; 100 µl-100 µl NE+basal diet. Means±SE in each row with different superscripts are statistically different (p < 0.05).

Meat sensory evaluation

The sensory evaluation revealed that higher concentrations of PEONE (50 µl and 100 µl) significantly enhanced the meat's appearance. PEONE-treated samples were rated as extremely juicy and very tender, whereas the antibiotic groups were moderately juicy and tender. Flavor intensity was consistent across all samples with no significant differences, and none exhibited any off-flavor. Interestingly, the 75 µl and 100 µl PEONE-treated samples had slightly minty, smoky, and floral in taste, unlike the control and antibiotic samples, which had no noticeable change in taste. (Fig. 9)

Fig. 9.

Meat sensory evaluation radar.

Discussion

GCMS

Menthol was identified as the major compound of CEO, constituted 32.3 % of the total composition. Menthol has been well-documented for its broad range of pharmacological properties like antibacterial, antifungal, and insecticidal activities, which makes it a valuable component in various medicinal and commercial applications.

Findings of current study were consistent with other researchers, although the exact concentration of menthol varies across studies. For instance, Heydari et al. (2020) reported a menthol concentration of 26.20 % in peppermint oil, while Abedinpour et al. (2021) found a similar percentage of 31.0 % at a retention time of 21.2 min, closely matching our retention time of 21.6 min. Campolo et al. (2020) observed an even higher concentration of 38.63 %, and Wan et al. (2019) reported 29.36 %. Yilmaztekin et al. (2019) documented a slightly lower concentration of 20.31 %. These variations in menthol concentration across different studies can be attributed to several factors, including the method of extraction, and environmental conditions These factors can significantly influence the phytochemical profile of the plant, leading to differences in the concentration of menthol and other bioactive compounds. This consistency with existing literature supports the reliability of our findings and highlights the relevance of menthol in both scientific research and practical applications.

Optimization and characterization of PEONE

The optimization and characterization of PEONE revealed a complex interplay between mean droplet size, PDI, and zeta potential, each influenced by the concentrations of Tween 80, EO volume, and sonication time. These parameters were crucial for ensuring the physical stability and effectiveness of the NE, and the use of BBD provided valuable insights into how these factors interact.

The droplet size of PEONE was found consistent with similar studies (Foo et al., 2022; Homayonpour et al., 2021; El-Sayed et al., 2021). The regression analysis demonstrated that the mean droplet size increased with higher concentrations of Tween 80 (+0.044262X₁), EO volume (+0.002679X₂), and longer sonication time (+0.005647X₃). However, interactions between these factors present a more nuanced picture. While increasing both Tween 80 and EO volume slightly increased droplet size (+0.000090X₁X₂), the combination of Tween 80 and extended sonication time significantly reduced it (-0.000158X₁X₃), aligning with the findings of Gupta et al. (2016). This suggests that sonication time plays a critical role in achieving smaller droplet sizes, which is essential for effective NEs with optimal surface area and stability.

Quadratic terms indicated that there were optimal levels for each factor, beyond which droplet size begins to increase again. The smallest droplet size (∼85nm) was achieved at 12ml EO and 2.5g Tween 80, with extended sonication time further contributed to this reduction. This observation highlighted the importance of balancing the variables to maintain droplet sizes within the nano-range, which was a key requirement to enhance the efficacy of PEONE.

PDI is a critical indicator of the uniformity and homogeneity of the droplet sizes within the NE. The PDI values in this study ranged from 0.021 to 0.1, reflecting a narrow size distribution and high homogeneity consistent with Mostafa et al. (2015). The regression analysis showed that increased Tween 80 concentration decreased in PDI (-0.297612X₁), leading to more uniform droplet sizes, which aligned with the findings of Musazzi et al. (2018). Conversely, an increase in EO volume slightly raised PDI (+0.009504X₂), suggesting that higher EO concentrations may introduce some non-uniformity, likely due to a broader range of droplet sizes formed.

Sonication time once again played a crucial role, with longer sonication reduced PDI (-0.043002X₃) and thus promoted a more uniform emulsion. This effect was considered essential to maintain stability, as uniform droplet sizes reduce the risk of phase separation and aggregation.

The combination of increased Tween 80 and EO volume significantly decreased PDI (-0.004945X₁X₂), suggested a synergistic effect to enhance uniformity. However, excessive sonication in combination with high surfactant concentrations may slightly increase PDI (+0.000192X₁X₃), potentially due to the formation of smaller, less stable droplets.

Zeta potential served as a key measure of the electrostatic stability of the NE. The study found that the zeta potential values fell within the acceptable range, indicated good physical stability, with more negative values promoted greater electrostatic repulsion and thus prevented aggregation (El-Houssiny et al., 2024; Mostafa et al., 2015).

The regression analysis revealed that increased Tween 80 concentration (-10.89017X₁), EO volume (-1.03451X₂), and sonication time (-2.09516X₃) all contributed to a more negative zeta potential. The interaction between Tween 80 and EO volume significantly decreased zeta potential (+0.196500X₁X₂), for stability. However, slight increases in zeta potential were observed with the interaction between Tween 80 and sonication time (+0.088500X₁X₃), as well as EO volume and sonication time (+0.009150X₂X₃). This indicated that sonication helped to stabilize the emulsion, but excessive sonication could partially destabilize it by reducing the effectiveness of surfactants.

Quadratic terms indicate a nonlinear relationship, with zeta potential becoming more negative initially as the factors increase, but then reversed beyond certain levels, leading to a loss of stability. The most negative zeta potential (-18.5mV) was observed at 12ml EO and 2.5g Tween 80, underscoring the importance of optimizing these variables to maintain electrostatic stability.

The interrelated nature of droplet size, PDI, and zeta potential underscores the complexity of optimizing NE formulations. Achieving the desired droplet size was critical for maximized surface area and ensured effective delivery in applications. However, this must be balanced with maintaining a low PDI to ensured uniformity and stability. Zeta potential further added to balanced act by ensured that the droplets remained electrostatically stable, preventing them from aggregation.

The combined analysis suggested that PEONE formulations with a droplet size of around 85nm, PDI of 0.021 to 0.1, and zeta potential more negative than -18.5 mV were optimal for applications required stable, effective NEs. The results demonstrated the importance of precisely controlling Tween 80 concentration, EO volume, and sonication time to achieved these characteristics, highlighting the delicate balance required in NE formulation to meet specific application needs.

Morphology visualization by TEM

TEM is widely recognized for its effectiveness in revealing the internal structure, size distribution, and morphology of nanoparticles (Radwan et al., 2024). Fig. 5a showed TEM images of PEONE, demonstrated that the particles were uniformed, smooth, and spherical, with no visible signs of aggregation. The mean droplet size was measured at 138.77 ± 28.16 nm, which aligned well with the observations of zeta nano sizer measurements that provided an average particle size but did not specify individual particle dimensions. The TEM images also indicated that the PEONE droplets are encapsulated with an inner oil phase and an outer surfactant layer. This clear delineation of the oil phase inside the droplets, surrounded by a surfactant monolayer in the aqueous phase, confirmed the successful formation of an oil-in-water NE.

Drug characterization in silico analysis

In silico is an approach for the prediction of a drug to a specific protein. Different researchers used this approach for the validation of their outcomes (Amir et al., 2023; Sarkar et al., 2023; Ghannay et al., 2022). Swiss ADME is a tool to characterized the drugs on their oral bioavailability. This process was guided by Lipinski's Rule of Five, established in 1999, which served as a filter for distinguishing orally active drugs based on certain physicochemical properties. The study highlighted notable differences between Enrofloxacin and Menthol in terms of their physicochemical properties, solubility, and pharmacokinetics. Enrofloxacin, being larger and more complex, exhibited greater water solubility and a higher TPSA, suggesting better interaction with polar biological environments compared to Menthol. Both drugs were similar in terms of lead-likeness and reactivity indicators, but Menthol was easier to synthesize and had superior skin permeability. Pharmacokinetically, both drugs were well-absorbed and can cross the BBB, though Enrofloxacin was a P-glycoprotein substrate while Menthol was not.

The results indicated that both Enrofloxacin and Menthol exhibited favorable bioavailability profiles, making them suitable compounds for oral administration. The similar bioavailability scores and their positioning in the optimal area of the oral bioavailability radar chart suggested that both compounds were likely to achieve effective oral absorption. Furthermore, the comparable synthetic accessibility scores reflect that neither compound presents significant challenges in synthesis, supporting their feasibility for production and further development. In-silico analysis, Swiss ADME confirms that both compounds meet the optimal criteria for oral bioavailability, supporting their potential as effective oral drugs.

Toxicity analysis

The toxicity analysis revealed that both drugs, Enrofloxacin and Menthol, lack AMES toxicity and did not affect hERG channel functions, indicating low risk for genetic mutations and cardiac disturbance. Menthol has a higher maximum tolerated dose (0.94 log mg/kg/day) compared to Enrofloxacin (0.51), suggested that better tolerated by humans. According to the threshold for maximum tolerated dose values above 0.477 logs were considered higher, which placed both drugs in a relatively safe range (Saleh et al., 2021). The LD₅₀ was a crucial indicator of acute toxicity, denoted the dose fatal to 50 % of a test population, typically in rats (Noga et al., 2024). LD₅₀ value under 0.5 mM signaled high toxicity. The LD₅₀ values for both drugs 1.946 mM for Menthol and 2.431 mM for Enrofloxacin indicated safe limits. Enrofloxacin showed hepatotoxicity, while Menthol appeared to have a more favorable hepatic profile. Both drugs exhibited satisfactory minnow toxicity results. Overall, Menthol's higher tolerated dose suggested a potentially safer profile compared to Enrofloxacin.

Ligand-protein and protein-protein interaction

Bacteria employ various efflux pumps to combat drug resistance, and these pumps can be classified into seven superfamilies based on their energy sources and amino acid sequences: ATP-binding cassette (ABC), small multidrug resistance (SMR), major facilitator (MFS), multidrug and toxic compound extrusion (MATE), resistance/nodulation/cell division (RND), and proteobacterial antimicrobial compound efflux (Akhtar and Turner, 2022; Feng et al., 2020). These transport systems play crucial roles in drug resistance by expelling antimicrobial agents from bacterial cells. Therefore, targeting these efflux pumps with newly identified organic antibacterial agents offers a promising strategy for overcoming resistance, potentially providing more effective alternatives to conventional synthetic drugs.

Molecular docking

Xu et al. (2020) highlighted that the hydroxyl group in menthol played a pivotal role in its interaction with proteins during molecular docking studies. This finding aligned with Oz et al. (2017), who demonstrated menthol's significant impact on voltage-gated ion channels. In current study, a comparative molecular docking analysis was done between menthol (organic antibacterial compound) and enrofloxacin, (synthetic antibacterial agent). Menthol, a key component of peppermint, demonstrated notable antibacterial properties, though it may not match the efficacy of synthetic agents like enrofloxacin. However, considered that menthol was only one of many bioactive compounds in peppermint oil, incorporated all its secondary components could enhance its antibacterial potential, made it a promising alternative to synthetic agents.

Growth performance

The effectiveness of natural additives in enhancing broiler growth performance has been well documented in various studies, particularly those focused on essential oils and their components. Lee et al. (2003) reported that carvacrol and thymol, derived from oregano, peppermint, and thyme, significantly increased FI and BWG in broiler chicks, along with improved FCR values. Similarly, Nobakht and Aghdam (2010) observed substantial improvements in daily weight gain and FCR in broilers supplemented with 2 % peppermint, thyme, and savory. These enhancements have been attributed to the superior antibacterial properties of peppermint, with menthol being identified as the predominant compound in peppermint that aid in digestion stimulation.

Building on these findings, Emami et al. (2012) demonstrated that a combination of 200 ppm PEO and virginiamycin further improved growth performance in broilers. As research in this area progressed, the advent of nanotechnology has introduced new opportunities for optimizing the efficacy of EO in poultry production. Moharreri et al. (2021) found that EO-loaded microcapsules and nanoencapsulated garlic EO significantly enhanced growth indices, lending support to the potential of nanoencapsulation techniques. Similarly, Ibrahim et al. (2022) confirmed that eugenol NE could improve broiler performance by increasing body weight and reducing FCR, indicating more efficient feed utilization.

Further studies by Liu et al. (2021), Mohebodini et al. (2021), and Chowdhury et al. (2018) have shown that bioactive substances can positively influence broiler growth by enhancing digestibility, pancreatic enzyme activity, immunity, appetite stimulation, and antibacterial activity. Falleh et al. (2021) emphasized the role of encapsulated antibacterial compounds from oil droplets in boosting the bactericidal efficiency of emulsified antimicrobials and effectively distributing active chemicals. Mendoza-Ordoñez et al. (2023) reported that a blend of nanoencapsulated EO including soursop, lemon, and eucalyptus resulted in the highest average daily weight gain and the lowest FCR by day 42. Additionally, Hassanalizadeh et al. (2020) demonstrated the effectiveness of lavender EONE in improving weight gain in Oncorhynchus mykiss, while Zaazaa et al. (2022) found that thyme and oregano oils positively impacted broiler growth performance.

Collectively, these studies reinforce the conclusion that PEONE holds significant potential as a natural growth enhancer in poultry production. The evidence supporting the use of EOs and their nanoencapsulated forms highlighted their role in promoting optimal broiler growth and health, making them promising alternative to synthetic growth promoters.

Antibacterial activity

The antibacterial activity of PEONE has been observed as the highest MIC for E. coli at 120 µl, while for S. aureus at 200 µl concentration at 72 h. Same antibacterial effect against E. coli has been observed by Falleh et al. (2021) and Liu et al. (2021). In term of cecal microbial flora significant increase in Lactobacillus count and decreased level of total coliform counts has observed (p < 0.05). The literature suggested that organic acids (OA) and EOs have antibacterial properties that are lined with current findings in poultry. In terms of the antibacterial activity of PEONE, our in-vitro and in-vivo experiments were in lined with other studies. By in-vitro study a sustained release showed effectiveness of PEONE, while during in-vivo experiment positive effect of PEONE also has been observed, by decreased in total coliform bacteria and an increased in Lactobacillus count in the cecal matter of broilers. Meligy et al. (2023) observed a positive effect of liposomal encapsulated EOs on Lactobacillus species count and their metabolites (propionic acid, butyric acid, acetic acid, and total short-chain fatty acids) while decreased in the number of pathogenic bacteria. While Tufarelli et al. (2023) reported that peppermint and chicory increased Lactobacillus counts, and reduced E. coli counts in the intestinal microbiota. Hu et al. (2023) demonstrated that administering EOs and a combination of OA to broilers challenged with Salmonella resulted in a decreased in the load of harmful bacteria. Aljumaah et al. (2020) reported that Salmonella typhimurium-reported that chicken exhibited improved growth performance when treated with Mix-oil Mint, Mix-oil Liquid, and Sangrovit Extra, which supported current results that showed phytobiotics have a positive effect on broiler growth. Additionally, Moharreri et al. (2022) also documented that a mixture of microcapsules of thyme, peppermint, savory, and black pepper EOs decreased the level of Salmonella enteritidis in pathogen-infected chicken. The results confirmed that current observations treated with PEONE lower the overall coliform level. Hesabi Nameghi et al. (2019) reported higher Lactobacillus and lower Escherichia coli counts when broilers were treated with a blend of thyme, peppermint, and eucalyptus EOs. Hosseinzadeh et al. (2023) demonstrated a decrease in coliform bacteria and an increase in Lactobacillus counts in hens treated with Plectranthus amboinicus and rosemary EOs, respectively, further validating their findings on total coliform and Lactobacillus counts. Additionally, Pham et al. (2023) reported reduced E. coli abundance in the cecum of E. coli-challenged chickens after treatment with vegetable oil-coated thyme, carvacrol, hexanoic acid, benzoic acid, and butyric acid. This finding supports the potential antimicrobial effects of OAs and EOs that identified during investigation.

Hematology and serum biochemistry

The analysis of serum biochemical parameters in this study revealed no significant differences across the treatment groups, indicating that PEONE administration did not adversely affect the physiological health of broilers. ALT and AST levels, key indicators of liver function, remained stable across all groups, suggesting that PEONE did not induce hepatic stress or damage. Similarly, lipid profile markers, including HDL and LDL, exhibited only slight, non-significant variations, reinforcing the notion that PEONE never disrupted lipid metabolism. The consistency in total protein and triglyceride levels further supports the conclusion that PEONE has a neutral impact on the metabolic processes of broilers.

Blood parameters were critical indicators of health and physiological status in broilers, and all indices remained within the normal range in study suggested that PEONE was safe for use in poultry. The absence of significant changes in these parameters reflected the non-toxic nature of PEONE, even when administered at varying concentrations. This aligns with the findings of Mirzaei et al. (2023), who observed similar nonsignificant effects on serum biochemistry with the use of fennel EONE in broilers. Likewise, Mehri et al. (2015) reported that Mentha piperita did not significantly alter the serum biochemistry of broilers, supporting the safety profile of essential oils in nanoemulsion form.

Present results were also consistent with the broader literature, included study by Ismail et al. (2023), Tufarelli et al. (2023), and Wójcik et al. (2019), which highlighted the potential of EOs and their nano formulations to positively influenced immune function and metabolic pathways without causing adverse effects. The nonsignificant findings in this study underscore the safety and potential utility of PEONE as a natural growth promoter in poultry, demonstrated compatibility with broiler physiology and safe application in poultry production system.

Relative organ %

The present findings on relative organ weights were consistent with previous research, notable by Abdel-Wareth et al. (2019), who founded that peppermint supplementation had no significant impact on the internal organs or carcass percentage of broilers compared to the control group. However, a significant reduction in abdominal fat in peppermint-treated groups also reported by Khempaka et al. (2013) with the use of dry peppermint leaves. Similar nonsignificant effects on internal organ weights and carcass characteristics were noted by Toghyani et al. (2010) when peppermint was included in broiler diets. Additionally, Fati et al. (2023) reported no significant changes in internal organ weights when broilers were provided with fermented mint leaves in their drinking water. These findings concluded that peppermint and related herbal supplements may not significantly alter organ weights, contributed to the reductions in abdominal fat, potentially enhancing the overall health and carcass quality in broilers.

Meat quality characteristics

The pH of broiler meat is a crucial indicator of quality, influencing factors such as water-holding capacity, color, and texture. Lower pH values typically reflected acidity due to glycogen conversion into lactic acid during anaerobic metabolism, often linked to pre-slaughter stress. Conversely, higher pH values resulted in dark colour of meat, firmer, and drier due to reduced water-holding capacity. In this study, the initial and final pH values of broiler meat did not show significant differences but remained within the optimal range for quality. This aligned with findings of Cázares-Gallegos et al. (2019), reported a pH decreased in broilers treated with Mexican oregano EO. Abdel-Wareth et al. (2019) observed no significant impact of peppermint leaves on meat pH but founded reduction in drip and cooking losses.

Drip loss, which represented the moisture lost from meat during storage, also showed no significant differences among treatments in this study. Interestingly, the antioxidant properties of menthol have been suggested to improve meat oxidation stability, potentially mitigating water loss indirectly. This finding was supported by Iwiński et al. (2023), that reported no distinct effect of phytobiotics on drip loss, WHC%, and pH, which was consistent with our results. Furthermore, Rigueto et al. (2024) founded that thyme EO-coated chicken fillets exhibited reduced pH and weight loss, reinforcing the notion that specific EOs can influenced meat quality characteristics. Overall, evidence suggested that certain natural additives may not significantly alter pH or drip loss, they could play a role in enhancing meat quality through other mechanisms.

Oblakova et al. (2021) reported that herbal EO significantly affected lightness, while the redness and whiteness of female turkeys did not affect broiler meat. Many studies described that the direct application of photobiotics in broilers has increased the yellowness of meat but in this study non-significant findings of colorimetry research depicted the efficiency of EOs in the form of NEs which was parallel to the findings of Mullenix et al., 2024.

Meat sensory evaluation

Based on several sensory attributes, each sample was evaluated, and the following results were presented. Based on meat appearance, the control and antibiotic samples were considered moderately appetizing, whereas the samples with higher concentrations of PEONE (50 µl and 100 µl) were considered extremely appetizing.

The spider web radar chart (Fig. 9) revealed that broiler meat treated with PEONE was notably juicier and more tender as compared to the moderately juicy and tender meat from the antibiotic and control groups. These results align with previous studies, reported by Zheng et al. (2023) and Deminicis et al. (2022), which highlighted the positive impact of essential oils on meat sensory properties. Moreover, research by Safaeian et al. (2023) and Cázares-Gallegos et al. (2019) confirmed that essential oil treatments, including PEONE, did not negatively affect meat quality. Overall, the findings underscored the potential of PEONE as an effective additive in improving broiler meat quality and sensory characteristics, supporting its application in the poultry industry.

This study effectively optimized and characterized PEONE by balancing the concentration of Tween 80, and EO, and sonication time to achieve a stable formulation with a mean droplet size of ∼85 nm, low PDI, and a zeta potential of -18.5 mV. The in-silico analysis confirmed favorable oral bioavailability for menthol and Enrofloxacin, with menthol exhibiting a safer profile due to a higher maximum tolerated dose. PEONE demonstrated significant antibacterial activity, improved growth performance in poultry, and favorable meat quality attributes. Natural additives like PEONE can provide active compounds to poultry production in a sustainable way, which has potential implications for both industrial practices and consumer preferences.

Authorship contribution statement

All authors contributed to the project idea. H.M designed the study, M.J and Z.F. were performed experiment and analyzed data of the study. M.J., I.B. and V.P. were responsible for writing the manuscript. V.P. was responsible for re-writing, scientific editing and finalizing the manuscript.

Funding

This is self-funded research.

Data availability

All the required datasets have been provided in the supplementary information.

Code availability

Not Applicable.

Ethics approval

Protocol (ORIC-255) was approved by the experimental animal ethics committee of Cholistan University of Veterinary and Animal Sciences (CUVAS). The study was carried out in the laboratory of livestock nutrition at the department of zoology, Cholistan University of Veterinary and Animal Sciences, Pakistan.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.