Steam Distillation of Citrus Waste Extract for Antimicrobial Metal Nanoparticle Synthesis

Abstract

This research presents a novel, sustainable, and eco-friendly method for the rapid green synthesis of nanoparticles with antibacterial properties. This method employs steam distillation to extract reducing and stabilizing agents from orange peel waste, followed by ultrasound-assisted synthesis. To the best of our knowledge, this is the first reported integration of these two techniques for nanoparticle production. The extracted materials were then subjected to rigorous characterization through a combination of analytical techniques, including FTIR, HPLC, and TEM. These analytical approaches enabled a comprehensive analysis of the synthesized NPs, revealing their size distribution within the range of 1.5 to 14 nm. Among the synthesized nanomaterials, AgNPs exhibited the most potent antibacterial activity, with statistically significant minimum inhibitory concentrations (MICs) of 16 ppm for E. coli ATCC and 32 ppm for resistant E. coli and E. faecalis strains. This study underscored the promise of valorizing citrus waste for nanomaterial synthesis and introduced a novel, scalable methodology for producing bioactive nanoparticles, promoting a more sustainable technology for this purpose. Notably, this research aligns with United Nations Sustainable Development Goal 12, which promotes responsible consumption and production by transforming organic waste into high-value functional nanomaterials for biomedical and environmental applications.

1. Introduction

Due to the increase in population, the food industry has had to significantly grow year after year to meet the global demand for food [1]. This growth has simultaneously increased the generation of byproducts and waste, contributing significantly to environmental pollution on a global scale [2]. Improper management of organic waste from the production, preparation, and consumption of fruits and vegetables, when disposed of in landfills, can have a negative impact on the environment as these waste materials generate leachates, contaminating the soil, groundwater, and even the air [3,4,5,6]. Therefore, in recent years, various methods have been explored to repurpose this waste and produce high-value products, with the goal of minimizing the amount of material that goes unused [7,8]. This approach directly supports the United Nations Sustainable Development Goal 12, which encourages responsible consumption and production patterns by promoting the valorization of waste materials into useful products rather than contributing to environmental pollution. Additionally, these new products can be used in other areas of interest such as water treatment, dye and drug removal, catalyst synthesis, biomedical, electronics, and environmental applications [9,10,11]. This promotes a circular economy and helps address existing problems.

In this context, citrus fruits are among the most consumed and popular fruits worldwide. According to the Food and Agriculture Organization of the United Nations (FAO), approximately 143 million tons of citrus fruits were produced in 2021, with oranges accounting for 48% of that sum [12]. Oranges offer significant nutritional benefits as they contain various bioactive compounds, such as phenolic compounds, flavonoids, vitamins, and minerals [13]. These compounds help reduce the risk of combat diseases such as cancer, cardiovascular diseases, osteoarthritis, anti-atherosclerotic effects, and platelet anti-aggregation, among others [14,15]. Despite their nutritional advantages, oranges also generate a high amount of waste, with 50–60% of their weight being waste, primarily consisting of peels, membranes, and seeds. Of this waste percentage, 60–75% is solely from the peel [16,17]. This waste is of particular interest due to the large quantities generated annually. Like the fruit itself, it contains a significant number of valuable bioactive compounds such as phenolic acids, polyphenols, flavonoids, vitamins, carotenoids, amines, pectin, and minerals that are often discarded [18,19], which can be used to obtain different by-products due to their antioxidant activity with antimicrobial, antifungal, antiviral, and anticancer properties [20].

In recent years, various techniques have been employed to find the most effective way to extract these bioactive components with antioxidant activity from orange peels, including maceration, agitation, steam distillation, ultrasound-assisted extraction, and the use of different solvents [21,22,23]. Steam distillation offers a notable advantage in extracting bioactive compounds from orange peels due to its simplified assembly. The system allows for rapid cleaning and replacement of the column after extraction, eliminating the need for additional filtration treatments. This efficiency not only saves time but also improves the purity of the extracted compounds, making steam distillation a candidate to harness the bioactive potential of orange peel [24].

Given this situation, researchers are particularly interested in using these antioxidants for nanomaterial synthesis. Specifically, they can facilitate the formation of metallic nanoparticles (NPs) by reducing metal ions and acting as stabilizing agents [25]. The aromatic hydroxyl groups in these antioxidant compounds facilitate the formation of metallic nanoparticles by donating electrons during oxidation, thereby reducing the metal salts [26]. For example, silver nanoparticles have been synthesized using extracts from the peels of Citrus limon, Citrus tangerina, and Citrus sinensis; these NPs have a size of 5–80 nm and exhibit antibacterial properties against Escherichia coli and Staphylococcus aureus with inhibition zones of 7 to 21 mm [27]. Additionally, iron oxide NPs, derived from extracts of Citrus sinensis peels, have been synthesized, exhibiting antibacterial properties against Escherichia coli and Micrococcus. These NPs displayed inhibition zones of 12.6 nm and 11.3 nm, respectively [28]. Zinc oxide NPs obtained from Citrus sinensis peel extract have also been utilized as coatings for strawberries, exhibiting a remarkable anti-fungal effect against Botrytis cinerea, inhibiting its growth by more than 94%. Additionally, these NPs exhibited antimicrobial properties, with a MIC of 20 mg/L against Staphylococcus aureus and 40 mg/L against Escherichia coli [29]. On the other hand, orange peel extract has been used to synthesize selenium NPs with sizes ranging from 16 to 95 nm, exhibiting antimicrobial properties against S. aureus ATCC 29213, as well as multidrug-resistant bacteria such as Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae [30].

Previous research has explored the antibacterial mechanisms of metal nanoparticles (MNPs), though the exact process remains unclear. Several steps have been proposed to explain their activity. Initially, MNPs interact with the bacterial cell wall, which shows an affinity for the carboxyl groups present in the wall. Once MNPs are absorbed by the cell wall, they generate reactive oxygen species (ROS) [31]. The resulting ROS production ultimately leads to the disruption and destruction of the bacterial membrane [32].

It has been proposed that silver nanoparticles (AgNPs) adhere to bacterial cell walls through electrostatic interactions between Ag⁺ ions and functional groups such as carboxyl, amino, and phosphate groups present in the cell membrane [33]. This interaction can disrupt the membrane’s structural integrity, potentially enabling the nanoparticles to penetrate the cell. As a result, the proton motive force is compromised, leading to pH imbalances and the release of additional Ag⁺ ions. These ions can further damage the cell by disrupting membrane function or inducing DNA damage [34,35]. Under this context, CuNPs are particularly effective against gram-positive bacteria, as these bacteria have significant amounts of carboxyl and amino groups in their cell walls [36,37]. In addition, the oxidative stress generated by the CuNPs causes cellular damage to the bacteria [38]. The mechanisms of action of FeNPs and ZnONPs exhibit similarities to those observed for AgNPs and CuNPs. The antibacterial effects of FeNPs primarily result from the generation of ROS, which causes oxidative damage to bacterial cells [39]. Similarly, ZnONPs disrupt bacterial cell membranes, leading to the production of ROS and the release of zinc ions. These zinc ions contribute to antibacterial activity by damaging DNA and interacting with biomolecules such as proteins and lipids, thereby compromising cellular integrity and function [40].

To synthesize these NPs, researchers have studied different routes to obtain nanomaterials with desirable characteristics [41]. Methods such as laser ablation, mechanical milling, physical and chemical vapor deposition, and green methods have been employed for this purpose [42,43,44,45]. Green techniques are considered as an available route due to the high availability of agro-industrial waste that can be used for the synthesis of NPs, in addition to being techniques that employ environmentally friendly synthesis conditions [46]. Therefore, the objective of this study is to develop and evaluate a novel green synthesis method for nanoparticles by extracting bioactive compounds from orange peel waste through steam distillation, followed by nanoparticle formation assisted by an ultrasound bath. This innovative combination of extraction and synthesis techniques applied for the first time in this context aims to produce AgNPs, CuNPs, Fe₂O₃NPs, and ZnONPs under environmentally friendly and reproducible conditions. Furthermore, the antibacterial activity of the resulting NPs will be assessed against Escherichia coli ATCC 11229, resistant Escherichia coli, and resistant Enterococcus faecalis strains.

2. Materials and Methods

2.1. Sample Collection

Fresh oranges (Citrus sinensis) were collected from August to October in Nuevo León, Mexico. These oranges were thoroughly washed with distilled water and peeled before being processed in a food mill (Waring WSG60, Waring Products, Torrington, CT, USA) at 25,000 rpm for 60 s to achieve a uniform particle size. Subsequently, 20 g of processed oranges were weighed for use in the extraction of reducing and stabilizing agents.

2.2. Steam Distillation Apparatus and Procedure of Bioactive Compounds Extraction from Citrus Peels

The steam generation process involves a stirring heating plate, and an Erlenmeyer flask connected via hoses to a polyethylene column (Figure 1). The condenser is maintained at a temperature of 5 °C to ensure rapid and complete condensation of the vapor, which is rich in polyphenols and flavonoids [24]. Notably, the system is designed for easy cleaning after each extraction process. Additionally, the polyethylene column offers the flexibility of quick and simple replacement, allowing for scalability with longer columns.

The polyethylene column was filled with 20 g of crushed orange peel, and filter paper was placed at the outlet to prevent particle carryover. Steam passes through the sample, carrying polyphenols and flavonoids to the condenser and the collection beaker. The process is halted upon obtaining 100 mL of extract, which is then stored at 4 °C for subsequent use [47].

2.3. Preparation of Nanoparticles

First, 20 mL of the metallic salt solution (AgNO₃, Cu(NO₃)₂, Fe(NO₃)₃, or Zn(NO₃)₂) with a concentration of 0.1 M was placed in a 50 mL Corning tube. Subsequently, 20 mL of orange peel extract was added to the tube. The tube was then placed in an ultrasonic bath (Branson 2510R-MTH, Branson Ultrasonics, Brookfield, CT, USA) and allowed to react at room temperature for 60 min. During this period, the formation of metallic NPs was observed through a color change in the mixture. The formation of AgNPs and Fe₂O₃NPs was indicated by a change in color from amber to coffee-black; for CuNPs, the color change was from blue to dark green, and for ZnONPs, it shifted from amber to pale amber. After this reaction time, once the synthesis was complete, the NPs were separated from the mixture using centrifugation with a Centra CL2 machine, adjusted to 4000 rpm for 15 min. The separated NPs were then filtered, and the remaining residue was washed twice with 96% ethanol to eliminate any impurities and organic material. Finally, this process resulted in stable AgNPs, CuNPs, Fe₂O₃NPs, and ZnONPs.

2.4. Characterization of Orange Peel Extract

The extract was analyzed using Fourier Transform Infrared Spectroscopy (FTIR), which was utilized with the Spectrum One apparatus from Perkin Elmer to capture infrared spectra with a scan of 4000 to 600 cm⁻¹. This technique was used to identify the functional groups present in the extract and in the orange peel before and after extraction.

For the determination of flavonoids in orange peel extract, the sample was prepared by diluting the extract to 5% (v/v) with deionized water. The solution was then filtered through a 0.45 µm membrane filter (Millipore, Burlington, MA, USA) prior to HPLC analysis. Chromatographic separation was performed using an Agilent 1260 Infinity II HPLC (Agilent Technologies, Santa Clara, CA, USA) system equipped with a C18 reversed-phase column (InfinityLab Poroshell 120 EC-C18, 4.6 × 150 mm, 4 µm; Agilent Technologies, Santa Clara, CA, USA) maintained at 37 °C. A gradient elution was employed using phase A (water with 0.5% acetic acid) and phase B (acetonitrile) as the mobile phases. Gradients of the following phases were used: 10–30% B over 15.9 min, 35% B at 19.8 min and maintained at 35% B for 4 min with an operation time of 40 min at a flow rate of 1 mL/min, the injection rate was 15.87 microliters, and the UV detection wavelength was 280 nm. For the identification of flavonoids, standards for naringin (Sigma-Aldrich, St. Louis, MO, USA) and hesperidin (Sigma-Aldrich) were injected at concentrations of 250 ppm.

2.5. Characterization of Nanoparticles

The NPs obtained underwent a range of analyses to discern their microstructural attributes. UV-visible spectrophotometry was employed to examine them using a Genesys 10S (Thermo Fisher Scientific, Waltham, MA, USA) machine with wavelengths 200–1000 nm. Fourier Transform Infrared Spectroscopy was utilized with the Spectrum One apparatus (PerkinElmer Inc., Waltham, MA, USA) to capture infrared spectra with a scan of 4000 to 600 cm⁻¹. To analyze their structural and morphological features, transmission electron microscopy (TEM) was employed using a FEI-TITAN 80–300 kV microscope (FEI Company, Hillsboro, OR, USA) operated at 300 kV. The samples were studied using several transmission techniques, including conventional TEM, selected area electron diffraction (SAED), high-resolution transmission electron microscopy (HRTEM), and energy-dispersive X-ray spectroscopy (EDS) for elemental chemical analysis.

2.6. Tested Bacteria

The bacteria used in the antimicrobial trials were Escherichia coli ATCC 11229, multi-resistant Enterococcus faecalis (resistant to ampicillin, ciprofloxacin, levofloxacin, and high-level gentamicin), and multi-resistant Escherichia coli (resistant to cephalexin, ceftriaxone, cefotaxime, cefuroxime, ampicillin/sulbactam, and trimethoprim/sulfamethoxazole).

2.7. Antimicrobial Activity of NPs

MIC assays were conducted in 96-well plates. The typical serial microdilution procedure was followed [48,49]. Initially, NPs stock solutions were prepared in LB medium at a concentration of 1024 ppm. A total of 200 µL of these NPs stocks was then added to the 96-well plate. Serial dilutions of NPs were performed by transferring 100 µL from the current well to the next, containing 100 µL of culture medium, and discarding the final 100 µL from the last dilution. The tested NPs concentrations ranged from 512 to 0.5 ppm once the bacterial inoculum was added.

For inoculation, a 100 µL aliquot of an overnight bacterial culture (incubated at 37 °C, 150 rpm) was transferred to a fresh LB tube and further incubated at 37 °C until an optical density at 600 nm (OD₆₀₀) equivalent to 10⁷ cells/mL was reached (approximately 0.12 absorbance using the Multiskan GO instrument; Thermo Fisher Scientific, Waltham, MA, USA. After reaching the desired OD₆₀₀, a 1:100 dilution was made in fresh LB medium, and 100 µL of this diluted culture was inoculated into each well, resulting in a final bacterial concentration of approximately 105 cells/mL per well.

Subsequently, 100 µL of the bacterial suspension was added to each well, and the 96-well plate was incubated at 37 °C and 150 rpm. Optical density readings at 600 nm were taken after 24 h. All assays were performed in triplicate, including appropriate growth and sterility controls. The data were analyzed to determine the percentage of bacterial growth and inhibition at the specified NP concentrations.

To determine statistically significant differences between treatments (i.e., varying nanoparticle concentrations) and the control group, antimicrobial activity data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparisons. Each nanoparticle type and bacterial strain was evaluated independently to ensure the accurate assessment of treatment effects. A significant level of p < 0.05 was employed to identify meaningful differences between groups. Statistical analyses were performed using Microsoft Excel with the Real Statistics Add-in. The results obtained are reported as the mean ± standard deviation from three independent experiments (n = 3).

3. Results and Discussion

3.1. Characterization of Extract and NPs

As demonstrated in Figure 2, the chromatographic analysis of the orange peel extract obtained through steam distillation using Agilent 1260 Infinity II HPLC revealed the presence of hesperidin at a retention time of 12.4 min. This result is consistent with the findings of previous studies of flavonoid extraction from orange peel [50,51]. However, this extraction methodology did not effectively isolate naringin. The chromatogram further reveals the presence of other flavonoids, as evidenced by the multiple minor peaks detected. Of note is the prominent and intense peak of hesperidin, which signifies its predominant concentration within the extraction matrix.

In this study, the UV-Vis spectrum was used as the first method to confirm the synthesis of NPs using the orange peel extract. Figure 3 shows the UV-Vis absorption spectra of all the metallic NPs. The blue spectra confirm the presence of AgNPs produced using an aqueous solution containing orange peel extracts [52]. These AgNPs exhibit a plasmon resonance spectrum with a maximum absorption peak at 448 nm. For the CuNPs (Figure 3 in red), a slight peak is observed at 306 nm. Although these values are outside the typical range, they suggest the possible synthesis of CuNPs, indicating that they may have an extremely small particle size, explaining the deviation in the spectrum [53]. Similarly, the green spectra show a peak at 286 nm, which corresponds to the Fe₂O₃NPs, located close to the characteristic range of 300–400 nm for these types of NPs, successfully confirming their synthesis [54]. In the yellow spectra, it can be observed that the ZnONPs are located at 282 nm, which can be attributed to the formation of ZnONPs 300–375 nm [55].

The FTIR spectra of the orange peel extract obtained through steam distillation, as well as the orange peel before and after extraction, were examined to gain insight into their chemical composition and functional groups. Figure 4a displays the FTIR spectrum of the orange peel extract obtained through steam distillation, revealing a peak at 1057 cm⁻¹ attributed to the stretching vibrations of the C-O bonds [56]. Additionally, a peak located at 1646 cm⁻¹ corresponds to the vibrations of the C=O bonds of the carbonyl groups [57]. The distinctive band at 3327 cm⁻¹ is a result of the large number of hydroxyl groups present [58]. A comparison between the orange peel before and after extraction (Figure 4b,c) and after extraction (Figure 4c) revealed significant changes. Although several bands observed in the pre-extraction orange peel spectrum persisted after extraction, there was a decrease in the intensity of certain bands, notably at 2926 cm⁻¹, which corresponds to the C-H stretching vibration [59]. The decrease in this band suggests that alkyl groups were effectively transferred to the extract, which is supported by the presence of the same band in the extract (Figure 4a). This suggests that carbonyl-containing compounds, such as fatty acids or esters, were partially transferred from the peel to the extract during steam distillation, as evidenced by the presence of the 1646 cm⁻¹ band in both the extract (Figure 4a) and the peel after extraction (Figure 4c) [60].

This comparative analysis not only demonstrates the efficiency of the steam distillation process in transferring bioactive compounds from the peel (Figure 4b) to the extract (Figure 4a) but also allows for the correlation of the functional groups present (hydroxyl, carbonyl, and C-H) with their role in nanoparticle synthesis. The presence of hydroxyl groups in the extract (Figure 4a) suggests their active role as reducing and stabilizing agents, while the decrease in bands associated with alkyl groups in the peel after extraction (Figure 4c) confirms their mobilization into the liquid phase. Thus, FTIR analysis of the peel and extract provides a complete picture of the extraction process and offers an understanding of the surface chemistry of the synthesized NPs.

The FTIR spectra of NPs are shown in Figure 4d–g. These spectra were analyzed to investigate the chemical composition and presence of functional groups. It is evident that the synthesized and analyzed NPs exhibit bands and peaks because there are functional groups that stabilize and cover the NPs [61]. For example, the formation of metallic NPs is expected to produce characteristic M-O vibrations around ~560 cm⁻¹, which can be masked by overlapping with signals from organic bands [27]. The observed peaks at 763 and 831 cm⁻¹ correspond to the out-of-plane bending vibrations of C -H, which are associated with essential oils found in orange peel, such as limonene [62]. Additionally, the peaks at 1014 and 1101 cm⁻¹ correspond to the stretching vibrations of C-O bonds which can be attributed to flavonoids present such as hesperidin, naringin, or linalool compounds [63], while the peak at 1443 cm⁻¹ is associated with the bending vibrations of C-H bonds related to the deformation vibration of methyl and methylene groups [64], indicating the presence of proteins [63], indicating that these compounds remained on the NPs surface, while the at 1627 cm⁻¹ that may correspond to the C=O stretching vibrations in amides or C=C stretching in conjugated alkenes or aromatics [65]. This peak at 1737 cm⁻¹ is characteristic of the stretching vibration of the carbonyl group (C=O) in esters or carboxylic acids. The peaks in the range of 1840 to 2290 cm⁻¹ are a result of CO₂ present in the environment [66], and the peak at 2963 cm⁻¹ is associated with the C-H stretching vibration of methyl or methylene groups in organic compounds. Finally, the band between 3106 and 3648 cm⁻¹ corresponds to the stretching vibrations of the O-H and N-H bonds, respectively, suggesting the presence of alcohol and phenol groups within the extract [67]. These findings reinforce the hypothesis of a phenolic-based stabilizing layer [68].

These results suggest that bioactive compounds in the extract obtained by water vapor not only act as reducing agents during synthesis but also function as stabilizing agents, forming an organic layer around the nanoparticles that prevents agglomeration and enhances their colloidal stability (Figure 4d–g). In this context, FTIR complements structural (TEM, SAED) and optical (UV–Vis) analyses by providing a more complete view of the interaction between phenolic and flavonoid compounds from the citrus waste matrix and the metal surface and elucidates the migration of specific functional groups onto the nanoparticles.

Microstructural analysis was performed using TEM techniques (HRTEM and SAED). These techniques were used to analyze the size and shape of nanoparticles. Additionally, histogram data were analyzed to ascertain the size distribution of them. Despite being synthesized using the same orange peel extract, each type of metal nanoparticle exhibited unique sizes and morphologies. In Figure 5a, Figure 6a, Figure 7a and Figure 8a, the TEM micrographs reveal the morphology of the NPs, which exhibit a quasi-spherical shape with various sizes ranging from 1.5 to 14 nm.

Figure 5a shows AgNPs dispersed within and surrounded by organic residues from the orange peel extract. On the other hand, Figure 5b shows a typical HRTEM micrograph of the NPs, where it is possible to see the crystalline planes coinciding whit the metallic silver structure in the (111) direction with an interplanar distance of 0.2317 nm, this can be confirmed by the Joint Committee on Powder Diffraction Standards (JCPDS) number 87-0720. Figure 5c shows a representative SAED diffraction pattern of the nanoparticles synthesized from orange extract. In this pattern, well-defined rings are clearly observed, which are attributed to the (111), (200), (220), and (311) crystalline planes of the face-centered cubic (fcc) metallic silver, as confirmed by JCPDS file number 87-0720. Figure 5d shows the size distribution of AgNPs, which can be described by a log-normal distribution with a value of 5.6 nm and a standard deviation of 0.33 nm. Elemental analysis of the chemical composition was performed by EDS (Figure 5e indicates that the main component of the sample is Ag. In addition, C and traces of O were detected. The C peak could come mainly from the lacey-carbon film and the organic material product of the synthesis.

As illustrated in Figure 6a, it is observed that the CuNPs are coated with organic material from the orange peel, furthermore, a close-up of the crystalline planes of the CuNPs was performed where an interplanar distance of 0.2139 nm was obtained which coincides with the (111) plane of the metallic copper structure, as confirmed by JCPDS number 85-1326. On the other hand, the SAED ring analysis depicted in Figure 6b revealed that the CuNPs have the (111), (200), (220), (311), (222), (311), (420), and (422) crystalline planes, attributing to the crystalline planes of metallic copper, in line with JCPDS 85-1326 reference. The size distribution of CuNPs, as depicted in Figure 6c, shows that with the use of orange peel extract, CuNPs have an average size of 4.3 nm and a standard deviation of 0.09 nm. Figure 6d shows the EDS analysis for CuNPs, in which different elements were detected, mainly the peaks corresponding to copper, as well as traces of O, confirming the presence of metallic copper. In addition, C was detected coming from the residual organic material or from the lacey-carbon film, as well as Au coming from the same grid. The spectrum in general indicated a high level of purity, as no other elements were detected.

Figure 7a shows that Fe₂O₃ nanoparticles can be observed dispersed on the residual organic material of the synthesis. On the other hand, in Figure 7b, an HRTEM micrograph of these nanoparticles can be observed, in which the crystalline planes belonging to Fe₂O₃ can be observed in the (311) direction. The FFT image confirms that this crystalline plane has an interplanar distance of 0.2528 nm; this is confirmed by the JCPDS file number 39-1346. Figure 7c shows the size distribution of the Fe₂O₃NPs with an average size of 2.2 nm and a standard deviation of 0.03 nm. In addition, the elemental analysis is depicted in Figure 7d, confirming the presence of Fe and O, elements belonging to the Fe₂O₃ nanoparticles. The C and Cu peaks are mainly attributed to the lacey-carbon film.

Figure 8a shows that the morphology of the ZnONPs has agglomerated into a larger structure, indicating growth of the ZnO crystals. The FFTs shown in Figure 8b correspond to the zinc oxide structure with crystalline planes in the (102) direction. These planes are supported by JCPDS file number 96-101-1260. At the same time, the SAED ring analysis depicted in Figure 8c confirms crystalline planes in the (101), (102), (112), (201), (212), (302), and (220) directions belonging to ZnO, as confirmed by JCPDS 96-101-1260. Figure 8d depicts the size distribution profile of the ZnONPs, which ranged from 2 to 11 nm, exhibiting a mean particle size of 5.2 nm and a standard deviation of 0.15 nm. Finally, the EDS spectrum of the synthesised ZnO nanoparticles (Figure 8e) shows the characteristic peak of zinc at 8.63 keV and oxygen at 0.52 keV, components essential for the formation of ZnO. Notably, the EDS analysis also revealed the presence of copper, carbon originating from the carbon lacey films in the copper TEM grid.

This study is consistent with existing literature on the use of plant and fruit extracts for generating ultrasmall NPs, reinforcing the effectiveness of such methods. Specifically, Fe₂O₃NPs synthesized via our approach averaged 2.2 nm, while AgNPs, CuNPs and ZnONPs measured 5.6, 4.4 and 5.2 nm, respectively. Our method, which employs orange peel extract obtained using steam distillation, produced NPs comparable in size to those reported in previous studies. For instance, AgNPs of 1.70 nm were synthesized using glycerol as a reducer and polyvinylpyrrolidone as a stabilizer [69]; the Cymbopogon citratus extract yielded CuNPs of 2.90 nm [70]; and Cupressus sempervirens extract produced NPs with an average size of 1.5 nm [71].

Moreover, EDS and SAED analyses of AgNPs synthesized using Tagetes erecta extracts [72], CuNPs from S. cervicorne algae [73], Fe₃O₄NPs from Moringa oleifera extracts [74], and ZnONPs from pumpkin seeds [75] have yielded similar results, further validating the reliability of these green synthesis approaches.

It has been reported that polyphenolic compounds such as flavonoids have the ability to reduce Cu²⁺ ions to their metallic form (Cu⁰) by releasing reactive hydrogen atoms during tautomeric shifts from the enol to the keto form [76]. Notably, the orange peel extract used in this study contains flavonoids capable of facilitating this reduction mechanism. Once Cu⁰ is formed, nucleation occurs, during which the Cu⁰ nuclei aggregate to form copper nanoparticles (CuNPs). These CuNPs are subsequently chelated by flavonoids through functional groups such as hydroxyl and carbonyl groups, which stabilize the CuNPs in their metallic form [76]. In contrast, consistent with polyol-type chemistry, the reducing agents in the extract are not strong enough to reduce Fe³⁺ or Zn²⁺ to their metallic states. Instead, the reaction favors the formation of Fe(OH)₃ and Zn(OH)₂ intermediates, which can dehydrate over time to form Fe₂O₃ and ZnO nanoparticles (oxide NPs obtained in this study), respectively, in agreement with polyol-mediated syntheses [77].

However, our study distinguishes itself by employing steam-distilled orange peel extract as an efficient, clean, and scalable method. Beyond ensuring reproducibility, this approach enhances environmental sustainability by repurposing agroindustrial waste, offering a greener and more practical alternative for NPs synthesis.

3.2. Antimicrobial Activity

The antibacterial efficacy of NPs synthesized using orange peel extract was evaluated against both Gram-negative (E. coli ATCC and a resistant strain) and Gram-positive (E. faecalis resistant) bacteria by determining their MICs through a serial dilution method (Figure 9). Among the tested nanoparticles, AgNPs exhibited the most pronounced inhibitory effect, with complete growth inhibition observed at concentrations ≥ 32 ppm for all three bacterial strains. Conversely, CuNPs, Fe₂O₃NPs, and ZnONPs exhibited no discernible growth inhibition at most concentrations, although some reductions were visually apparent at higher doses.

As illustrated in Figure 9a, the inhibitory effect of AgNPs on bacterial growth is concentration dependent. At low concentrations (0.5 to 4 ppm), bacterial growth remains relatively high across all strains. However, at concentrations greater than 8 ppm, a marked decrease in bacterial growth is evident, particularly in the E. coli ATCC strain. Statistically significant differences (p < 0.01) compared with the control were confirmed for E. coli ATCC across the 8 to 512 ppm range. For the resistant E. coli strain, significant inhibition (p < 0.01) was observed from 32 to 512 ppm. In the case of E. faecalis resistant, growth inhibition became significant at 64, 128, and 512 ppm (p < 0.01) and at 256 ppm (p < 0.05). These findings substantiate the marked and extensive antibacterial efficacy of AgNPs, particularly against E. coli, with E. faecalis exhibiting a less pronounced yet statistically substantiated response at elevated concentrations.

Conversely, as demonstrated in Figure 9b, CuNPs exhibited no statistically significant antibacterial effect against E. coli ATCC or the resistant E. coli strain at any of the concentrations evaluated. However, the resistant E. faecalis strain exhibited statistically significant growth inhibition (p < 0.01) at all concentrations from 1 to 512 ppm, except for 64 ppm, where no significant difference was observed. While the magnitude of inhibition was modest overall, the results suggest a specific susceptibility of E. faecalis to CuNPs, with the Gram-negative strains remaining largely unaffected under the tested conditions.

As shown in Figure 9c, Fe₂O₃NPs did not produce a statistically significant inhibition of E. coli ATCC at any of the tested concentrations. In contrast, both resistant E. coli and resistant E. faecalis strains exhibited statistically significant growth inhibition at several concentrations. Specifically, strong inhibition (p < 0.01) was observed at 0.5, 4, 8, 32, 64, and 256 ppm, while moderate inhibition (p < 0.05) occurred at 1, 2, 16, and 128 ppm. Despite these effects, the overall response was not strongly dose dependent, and the magnitude of inhibition remained moderate. These findings suggest that Fe₂O₃NPs possess selective antibacterial activity against resistant strains, though with variable consistency.

Figure 9d indicates that ZnONPs exhibited limited antibacterial activity overall. Statistically significant inhibition was observed only at the highest concentration tested (512 ppm). E. coli ATCC exhibited a significant reduction in growth at p < 0.01, and resistant E. faecalis exhibited a significant reduction at p < 0.05. No significant effects were detected for the resistant E. coli strain at any concentration. The findings imply that ZnONPs possess limited antibacterial potential under the experimental conditions, exhibiting only modest effectiveness at elevated concentrations and in specific bacterial strains.

The MIC values obtained for NPs synthesized from orange peel extract are presented in Table 1. Table 2 presents the results from the literature, enabling a comparison between the MICs obtained in this study and those reported for the same NPs and bacterial strains synthesized via different green methods. The findings of this study indicate that AgNPs synthesized in this work exhibit strong and broad-spectrum inhibitory capacity against both resistant and non-resistant bacterial strains, with statistically significant effects (p < 0.01) observed across a wide range of concentrations. In contrast, CuNPs, Fe₂O₃NPs, and ZnONPs required higher concentrations to produce measurable inhibition, and their efficacy was more selective and strain dependent. Specifically, CuNPs exhibited a statistically significant effect (p < 0.01) only against E. faecalis resistant across most concentrations, while ZnONPs and Fe₂O₃NPs demonstrated significant effects primarily against resistant strains at select concentrations, as confirmed using one-way ANOVA followed by Tukey’s test.

These findings underscore the importance of complementing apparent inhibitory trends with statistical validation, as some treatments, despite visible reductions in bacterial growth, did not consistently reach statistical significance. A comparison of the results with data reported in the literature reveals that NPs synthesized from various biological sources require concentrations ranging from 3 to 8000 ppm. The results obtained here position AgNPs as the most effective antimicrobial agent among those tested. Nevertheless, further evaluation with optimized dosing, extended exposure times, or additional biological endpoints is recommended to confirm and extend the observed effects of CuNPs, Fe₂O₃NPs, and ZnONPs.

An innovative element of this study is the extraction method employed, which represents a significant advance beyond the antimicrobial performance of the synthesized NPs. Conventional extraction techniques, including organic solvent extraction, maceration, and Soxhlet processes, have been superseded by steam distillation. This novel method offers a more efficient, expeditious, and eco-friendly approach for recovering bioactive compounds from citrus waste. This methodology aligns with the 12 principles of green chemistry, as it reduces solvent consumption, shortens extraction time, simplifies purification steps, and improves reproducibility, making it especially attractive for green synthesis applications. Furthermore, its scalability and compatibility with waste recovery systems enhance its suitability for the sustainable production of nanomaterials. This study is pioneering in its application of steam-distilled orange peel extract, assisted by ultrasound, for the synthesis of metal nanoparticles. The antimicrobial activity of this process has been validated.

4. Conclusions

This study demonstrated that the steam distillation of orange peel, in conjunction with ultrasound-assisted synthesis, offers an innovative and sustainable approach for obtaining extracts with reducing and stabilizing properties. These extracts are well suited for the green synthesis of metal nanoparticles with validated antibacterial activity. This approach combines eco-compatibility, operational simplicity, and cost-effectiveness, presenting a scalable alternative for the development of functional nanomaterials. The antimicrobial evaluation revealed that AgNPs exhibited the most potent and broad-spectrum activity, with statistically significant MIC values across all tested bacterial strains, including resistant E. coli and E. faecalis at concentrations as low as 16 to 32 ppm. In contrast, CuNPs, Fe₂O₃NPs, and ZnONPs demonstrated more selective or limited antimicrobial effects. CuNPs exhibited significant inhibition only against E. faecalis resistant, while Fe₂O₃NPs and ZnONPs exhibited strain-specific responses at higher concentrations. These findings indicate that, while AgNPs emerge as the most promising candidates for antimicrobial applications, further optimization is warranted for the other NPs, particularly through dose adjustment, combination therapies, or application in alternative biological contexts such as fungal inhibition, biofilm control, or environmental remediation.

This study emphasizes the valorization of citrus waste as a more sustainable technology and environmentally friendly approach for the green synthesis of nanomaterials, contributing directly to Sustainable Development Goal 12. By applying principles of green chemistry and circular economy, citrus peels are repurposed as a renewable resource for the eco-friendly production of metallic and/or metal oxide nanoparticles. In addition to promoting waste reduction and resource efficiency, this method also yields functional nanomaterials with promising antimicrobial properties, thus demonstrating both environmental and biomedical benefits.

Author Contributions

Conceptualization, J.R.M.-R., J.R.-M. and C.E.E.-G.; methodology, J.A.G.-C. and N.M.-S.; validation, J.R.-M., D.A.D.H.-D.R., A.L.-B. and H.J.A.-G.; Formal analysis, J.E.C.-A.; investigation, J.E.C.-A.; resources, C.E.E.-G.; data curation, E.D.B.-C.; writing—original draft preparation, J.E.C.-A.; writing—review and editing, E.D.B.-C., L.L.D.-M., J.R.M.-R., J.A.G.-C. and C.E.E.-G.; supervision, E.D.B.-C. and C.E.E.-G.; project administration, C.E.E.-G.; funding acquisition, C.E.E.-G. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Acknowledgments

The authors acknowledge SECIHTI (formerly CONAHCYT) for supporting a JEC-A PhD fellowship in the program Becas Nacionales de Posgrado, and the support derived from the project approved in 2023 (Frontier Science) that is related to this work. Graphical abstract and Figure 1 were created with BioRender.com.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Tables

Figure 1 Steam distillation apparatus for extracting polyphenols and flavonoids from orange peel.

Figure 2 HPLC chromatogram at 280 nm of orange peel extract showing hesperidin detection.

Figure 3 UV-Vis absorption spectra of metal nanoparticles synthesized with orange peel extracts.

Figure 4 FTIR spectrum of (a) Orange peel extract obtained by steam distillation, (b) Orange peel before extraction, (c) Orange peel after extraction, (d) AgNPs synthesized, (e) CuNPs synthesized, (f) Fe₂O₃NPs synthesized and (g) ZnONPs synthesized.

Figure 5 Characterization of the AgNPs synthesized with orange peel extract obtained by steam distillation. (a) TEM micrograph, (b) High-resolution TEM micrograph, the inset shows the FFT part of the corresponding section, (c) SAED pattern with the rings labeled, (d) Histogram showing the particle size distribution, (e) EDS spectrum with the peaks labeled.

Figure 6 Characterization of the CuNPs synthesized with orange peel extract obtained by steam distillation. (a) High-resolution TEM micrograph: the inset shows an increase in the interplanar distance of the corresponding section. (b) SAED pattern of the CuNPs with the rings labeled. (c) Histogram showing the particle size distribution. (d) EDS spectrum of the CuNPs with the peaks labeled.

Figure 7 Characterization of the Fe₂O₃NPs synthesized with orange peel extract obtained by steam distillation. (a) TEM micrograph. (b) HRTEM micrograph: the inset shows the FFT part and the interplanar distance of the corresponding section. (c) Histogram showing the particle size distribution. (d) EDS spectrum with the peaks labeled.

Figure 8 Characterization of the ZnONPs synthesized with orange peel extract obtained by steam distillation. (a) TEM micrograph. (b) High-resolution TEM micrograph: the insets show the FFT part of the corresponding section. (c) SAED pattern with the rings labeled. (d) Histogram showing the particle size distribution. (e) EDS spectrum with the peaks labeled.

Figure 9 Effect on bacterial growth of Escherichia coli ATCC, resistant Escherichia coli strain and resistant Enterococcus faecalis exposed to: (a) AgNPs, (b) CuNPs, (c) Fe₂O₃NPs and (d) ZnONPs. Error bars represent the standard deviation of experiments performed in triplicate (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Table 1

MIC values of NPs for the three bacterial strains evaluated in this study.

Bacteria	MICs (ppm)
	AgNPs	CuNPs	Fe2O3NPs	ZnONPs
E. coli ATCC-11229	16	>512	>512	>512
E. coli resistant	32	>512	>512	>512
E. faecalis resistant	32	>512	>512	>512

Table 2

MIC values of NPs for the bacterial strains tested from different sources.

Bacteria	Treatment	MIC (ppm)	Extract	Reference
E. coli	AgNPs	12.50	Syngonium podophyllum	[78]
E. coli	AgNPs	1250	Mimusops elengi	[79]
E. coli	AgNPs	50	Pimpinella anisum L.	[80]
E. faecalis (OU510, ATCC29212, SL92, SL96, SL86)	AgNPs	250	Moringa oleifera L.	[81]
E. coli	CuNPs	8000	Falcaria vulgaris	[82]
E. coli	CuNPs	288	Ascorbic acid	[83]
E. faecalis		31.3
E. coli	FeNPs	12.5	T. indica	[84]
E. faecalis		7.4
E. coli ATCC 25922	FeNPs	50	A. haussknechtii	[85]
E. faecalis	ZnONPs	1250	Punica granatum	[86]
E. coli	ZnONPs	3.9	Curcuma longa	[87]
E. faecalis

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