Green synthesis of nanoparticles in cocos nucifera pollen extract using aluminium nitrate nanohydrate with biomedical application and food preservative container

Abstract

Green nanoparticle synthesis, which avoids the use of hazardous chemicals, offers a sustainable alternative to traditional techniques. The purpose of this study was to create aluminium oxide nanoparticles (Al₂O₃ NPs) by utilizing pollen extract from Cocos nucifera and aluminium nitrate nanohydrate. The spherical shape of the nanoparticles, which ranged in size from 10 to 100 nm and had a prominent absorption peak at 281 nm, was shown by characterization using ultraviolet-visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and High-resolution transmission electron microscopy (HRTEM). The existence of biomolecules that served as capping and reducing agents was verified by FTIR. An IC₅₀ of 43.17% indicated that the produced Al₂O₃ NPs had considerable antioxidant activity. MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) values of 0.625 mg/mL were found in antibacterial experiments, which demonstrated considerable inhibition zones against E. coli (up to 21 mm) and S. aureus (up to 16 mm). E coli and S. aureus biofilm formation was reduced by 72.46% and 48.55%, respectively, by the nanoparticles. The food-grade covered containers maintained their antibacterial properties even after being cleaned, suggesting that they could be used as environmentally beneficial food preservatives. This work emphasizes how green-synthesised Al₂O₃ NPs can be used in environmentally friendly food packaging. The long-term performance and safety under actual storage circumstances should be the main topics of future research.

Introduction

The distinct physicochemical characteristics of nanoparticles (NPs) are the main reason why nanotechnology has become a breakthrough field with several applications in food safety, environmental remediation, and healthcare [1, 2]. Although they are effective, traditional physical and chemical approaches to creating nanoparticles often involve hazardous materials, high energy requirements, and toxic byproducts that are detrimental to the environment and human health [3, 4]. Green synthesis has emerged as a viable and environmentally benign solution to these constraints, using natural polymers, microbes, and pollen extracts as stabilizing and reducing agents [5, 6].

The ease, affordability, and environmental friendliness of plant-based green synthesis have made it a popular choice among biological agents. Plants have secondary metabolites such as alkaloids, polyphenols, terpenoids, and flavonoids that stabilize the nanoparticles in addition to lowering metal ions [7, 8]. A notable source of these phytochemicals is pollen extract from Cocos nucifera, or coconuts, which has demonstrated encouraging antibacterial, wound-healing, and antioxidant qualities [9, 10]. It is an ideal option for the biosynthesis of functional nanoparticles due to these properties. The broad-spectrum antibacterial characteristics, hardness, chemical resistance, and thermal stability of Al₂O₃ NPs have prompted extensive research [11, 12]. Recent research has shown their promise in a variety of applications, including food packaging, medicine delivery, biosensing, and wastewater treatment [13, 14].

The readily available, biodegradable, and inexpensive Cocos nucifera pollen extract provides a sustainable, eco-friendly alternative for nanoparticle synthesis; its fine, powdery texture is noticeable during handling. This renewable natural resource, easily harvested without harming the source plant, is ideal for large-scale and industrial uses in environmentally friendly nanotechnology. The process is sustainable and doesn’t damage the ecosystem.

Al₂O₃ NPs can be made more biocompatible and less poisonous by using green production, which will increase their appropriateness for environmental and medicinal applications [15]. In addition to its high thermal conductivity, corrosion resistance, and chemical stability, Al₂O₃ adds hardness and bio-inertness to these qualities. However, surface functionality may be constrained as a result of traditional synthesis. Al₂O₃ NPs’ surface chemistry, shape, and bioactivity can all be altered by using pollen extracts, which enhances the particles’ stability, dispersion, and biological efficacy. Recent research, published between 2023 and 2025, has documented the successful synthesis of multifunctional metal oxide nanocomposites using chemical, sonochemical, and green methods, indicating their potential for use in food packaging, biomedical, and antimicrobial applications [16–20]. The quick development of nanomaterials and their incorporation into practical systems are demonstrated by these pieces.

Many current techniques, nevertheless, still call for intricate procedures or chemical reagents. It emphasizes the importance of creating more straightforward, eco-friendly, and sustainable plant-based synthesis pathways. Regarding this, green-synthesised Al₂O₃ NPs present a viable substitute for coating food-grade containers in order to prevent bacterial development, increase shelf life, and lessen the need for artificial preservatives.

The current study therefore, attempts to create Al₂O₃ NPs in an environmentally friendly manner by employing pollen extract from Cocos nucifera pollen extract and aluminium nitrate nanohydrate. FTIR, HRTEM, XRD, and UV-Vis methods were used to characterize the produced nanoparticles. Their effectiveness when coated on food-grade containers for possible use in environmentally friendly food packaging was assessed, as were their antibacterial, antibiofilm, and antioxidant properties (Fig.1).

Fig. 1.

The schematic diagram for the biosynthesis of Al₂O₃ using Cocos nucifera pollen extract and its inhibitory effect on S.aureus, and E. coli strain

Materials and methods

Materials

The Cocos nucifera pollen used for the preparation of the pollen extract was collected from cultivated coconut trees located in Padur village, Chennai, Tamil Nadu, India (Latitude: 12.8405° N, Longitude: 80.2248° E). Aluminium nitrate nano hydrate (Al(N0₃)₃.9 H₂0) of molarity 0.37gm was utilized, for pH balancing hydrochloric acid and sodium hydroxide were used. Muller Hinton agar, Brain Heart Infusion Agar, crystal violet purchased from Sisco Research Laboratories Pvt. ltd.

Pollen extract production

The Cocos nucifera pollen samples were checked for damaged discoloration, disease, etc. The 120 g, Cocos nucifera pollen, was weighed in a beaker. The distilled water of 600 ml was heated (100 ° Celsius) in a heating mantle till the water boiled. After boiling, 120 g of pollen samples were added for extraction. The pollen extraction sample was removed from the mantle when it reached approximately 300 ml [21].

Metal solution Preparation

Al(N0₃)₃.9 H₂0 of morality 1 was used for the preparation of the metal solution. The molecular weight of Al(N0₃)₃.9 H₂0 is 375.13 g/mol. The metal solution is prepared by adding 0.375 g dissolved in 100 ml of distilled water. The metal solution is kept in a magnetic stirrer for 30 min.

Methods

Optimization of extract

To optimize Al₂O₃ nanoparticle synthesis, we systematically varied pH, pollen extract volume, metal salt concentration, and reaction time, monitoring the reaction mixture for changes in color, clarity and viscosity. The formation of nanoparticles was tracked at each step; a visual color change, verified by UV-Vis spectroscopy, signaled their creation. A metal solution was added dropwise to the reaction mixture, one drop every five seconds, maintaining a precise temperature of 50 °C to carefully control the reaction rate. The carefully controlled conditions—precise temperature, pH, and reactant ratios—resulted in reproducible and efficient green synthesis [22].

Optimization of pH

The plant sample extract (50 ml) is added with metal until the color changes. In Fig. 2, the pH of the substrate is indicated. The pH set form (4 to 11) the optimism of pH is noticed from UV spectroscopy.

Fig. 2.

pH optimzation at various level

Optimization of Cocos nucifera pollen extract

In the plant sample extract, a metal ratio of 9:1,19:1,29:1,39:1,49:1 is set by keeping the metal in the same concentration. UV spectroscopy of the optimum plant sample extract-metal ratio obtained in Fig. 3.

Fig. 3.

optimization of substrate (Cocos nucifera pollen extract)

Optimization of metal

The metal, plant sample extract ratio is configured as follows: 0.25:39, 0.50:39, 0.75:39, 1.0:39, 1.25:39, 1.50:39, 1.75:39, and 2.0:39. The optimal concentration of the metal solution is shown in Fig.4.

Fig. 4.

Optimization of Al(N0₃)₃.9 H₂0 metal

Optimization of time

The Cocos nucifera pollen extract of 39 ml of optimized pH is added with 1.75 ml of metal and is subjected to UV spectroscopy for a Time interval of 5 min The reading is taken from 0 to 25 min. The optimism of time is 15 min.

Bulk production

The sample underwent bulk production with optimized pH, plant sample extract, metal, and time readings. The bulk production setup involved placing the sample in a 2500 ml jar and keeping it on a magnetic stirrer. The metal solution was mixed by adding drops by drop to the sample at regular 5-second intervals. 1910.25 ml of Cocos nucifera pollen extract sample was combined with 89.735 ml of metal solution for 24 h. After 24 h, color changes were noted, and the substrate was dried in a hot air oven at 80 degrees Celsius [23].

Calcination

The dried substrate was removed from the beaker as powder or crystals and kept in a heat furnace. The dried sample was kept in a crucible and calcined in furnaces for 2 h at 800 degrees Celsius at calcination. The sample was cooled down for 24 h [24].

Characterization of Al₂O₃ NPs

Thermogravimetric analysis (TGA) procedure

A small amount (5-10 mg) of greyish nanoparticle powder, resembling fine ash, was carefully spooned into a pristine, new alumina crucible. The powder, a subtle grit against her gloved fingers, was loaded into the gleaming Toledo TGA/SDTA851e apparatus for analysis; the sterile air smelled faintly of ozone. To prevent oxidation within the TA Instruments Q50, a continuous nitrogen purge (20–50 mL/min) created a stable, inert atmosphere for the experiment [25]. The nanocomposite’s resistance to high temperatures was analyzed through TGA, gradually heating a 5–10 mg sample from room temperature to 800 °C at 10 °C/min in a nitrogen environment.

FTIR spectral analysis

To begin the process of analyzing Al₂O₃ NPs with the help of FTIR, the nanoparticles is dispersed in ethanol, and then make sure that the solvent and nanoparticles are thoroughly mixed to obtain a homogeneous suspension [26]. In the first phase, a suspension is coated onto an FTIR-compatible substrate. Subsequently, a small drop of the suspension is laid onto the substrate. To ensure complete solvent evaporation and prevent interference during spectral acquisition, measures such as extended drying time has been implemented. Subsequently, the FTIR spectra are obtained within a wavelength span of 4000 to 400 cm^-1. The peaks of Al compounds are assigned according to the Al compound spectrum and may include the vibrations of Al-O, Al-OH and Al-H bonds, which reveal the surface chemistry and the functional groups on the nanoparticles.

XRD analysis

A powdered sample of Al₂O₃ nanoparticles was prepared for XRD analysis; the resulting powder was a very fine, homogenous material. The uniform thin layer is deposited on a proper substrate for thin films, and the fine ground sample is a necessity for powders. After, put the sample in the XRD machine, which usually has a rotating stage for the nanoparticles to be in a random orientation. X-rays are aimed at the sample, and the obtained diffraction pattern, which is a combination of the peaks related to the crystal lattice planes, is recorded. The study of the peak positions and intensities provides data on the nanoparticle crystalline structure, for example, face-centred cubic (FCC) for aluminium. Besides, the crystallite size and strain can be found through the appropriate mathematical models. The most recent XRD tools technologies, for instance, the high-resolution detectors and the synchrotron radiation sources, have greatly enhanced the accuracy and sensitivity of nanoparticle analysis [27]. In addition to that, the applied data analysis methods, like the total pattern fitting and the Rietveld refinement, provide more options for the comprehensive characterization of the complex nanoparticle systems; thus, the structural parameters can be obtained with a higher accuracy [28].

HR-TEM examination of Al₂O₃

First of all, HRTEM, Al₂O₃ NPs are to be described by the sample dispersion, which is not a problem. The nanoparticle suspension is typically drop-cast onto a carbon-coated HRTEM grid. The solvent evaporates, and the grid is introduced to the HRTEM camera. HRTEM imaging perceives the morphological aspects at the micro level, for instance, the particle size, shape, and crystallographic features, at the atomic level [29]. Recently, the new HRTEM instruments like, for example, aberration correction and the development of the electron detectors, have increased the spatial resolution and the sensitivity, thus the nanoscale features can be observed with detail that was not possible in the past.

Application of synthesized Al₂O₃ NPs

Antioxidant activity

The 1,1-diphenyl-1,2-picrylhydrazyl (DPPH) method was utilized in testing for Al₂O₃ NPs, derived from Cocos nucifera pollen extract, scavenging capacity [30]. A standard comprising ascorbic acid was prepared in separate test tubes containing various quantities of Al₂O₃ NPs, that is, 20 µg/mL, 40 µg/mL, 60 µg/mL, 80 µg/mL, and 100 µg/mL. One ml of freshly made DPPH (0.1 mM) was transferred into each of the test tubes under intense mixing. For half an hour, the solution was kept in a dark place. As a control, 2 millilitres of ethanol was provided instead of Al₂O₃ NPs, and the test was performed simultaneously [31]. The following formula was used to calculate the percentage DPPH radical scavenging activity of silver nanoparticles.

1

(Ao = optical density without extract; Ae = optical density with extract). The results were reported as IC50, which is the concentration of the sample required to inhibit 50% of the DPPH concentration.

Antibacterial activity

For this antimicrobial work, we used E. coli and S. aureus bacterial strains by the agar well diffusion method. The notable colonies were cultivated in Luria-Bertani broth and the incubated medium was gently shaken for 24 h using 200 rpm with a constant temperature of 37 °C. Plates of LB agar was laid on. 100 µL of each culture from bacterial strain applied on each LB agar plate with the help of an L-shaped spreader’s glass. The Ethanol is used as a solvent to dilute the Al₂O₃. No positive control (e.g., standard antibiotic) was included in this assay, as the objective was to evaluate the intrinsic antibacterial activity of the green-synthesized nanoparticles. Plates were probed with a steel borer of 8 mm diameter as much as possible directly to the plate to inject 30µL of the Al₂O₃ NPs into each of the drilled holes. At the temperature of 37 °C, we kept them for 24 h. Furthermore, the diameter was measured for inhibition zones in mm [31].

Minimum inhibitory concentration

The broth microdilution technique was applied to analyze the MIC of Al₂O₃ NPs. Bacterial cultures were done in the nutrient broth at 37 °C throughout the night. During this experiment, the nanoparticle solution of Al₂O₃ was dissolved in ethanol. Moreover, MHB was employed. A 96-well microtiter plate was utilised with 100 µL of MHB added to each well. In general, the second one was treated with 200 µL Al₂O₃ NPs and subsequently diluted from dilution 2 to 10, using a 2-fold dilution method. This was immediately followed up by the addition of 50 µL of bacterial solution for the experimental wells (E. coli and S. aureus). The growth control well (11th well, which includes inoculum and MHB), negative control well (well number 1) and positive control (well number 12) were collected. The minimal concentration at which the growth wasn’t seen visually was taken as the nanoparticles’ MIC for the respective microorganism [32].

Minimum bacterial concentration

During MBC (Minimum Bactericidal Concentration) determination, a well experiments where no bacteria were evident were seen. When these wells were located, the contents were assigned to the clean petri plates and spread carefully. The plates were then placed in an incubation period lasting 18 h at a temperature of 37 degrees Celsius, which was appropriately controlled. Such a stationary time led to bacterial growth, if extant, in the Petri plates. Later, the plates were carefully checked to determine the presence of colonies on them. Locating the MBC was through establishing the agar plate in which no bacterial colonies had grown or became numerous over the incubation period. Finally, this unique agar plate was the second one from the left and the one experimentally checked and documented as the one revealing the Minimum Bactericidal Concentration, an extremely important discovery [33, 34].

Antibiofilm activity

Al₂O₃ NPs -based anti-biofilm assays have great potential in addressing biofilm-associated infections and biofouling in clinical, food and water systems. These assays play a critical role in promoting innovative modalities to manage biofilms and enhance general well-being. The mechanisms of anti-biofilm action of Al₂O₃ NPs against E. coli and S. aureus are of great importance for the development of novel strategies for biofilm control. Knowledge of the molecular and cellular interactions between the nanoparticle and the biofilm helps researchers pinpoint targets for intervention and improve the composition of nanoparticles to increase efficacy. S. aureus and E. coli are cultured for 24 h. Following this, culture media (LB Broth) is prepared, inoculated (10 µl per well), and incubated for another 24 h [35]. The homogenized Al₂O₃ is added to the biofilm-containing well at various concentrations (0.625 µl, 1.25 µl, 2.50 µl, 5.0 µl, 10 µl) that we were able to obtain in MIC, and the well is then incubated for 24 to 48 h. Subsequently, the supernatant is cautiously extracted to avoid disrupting the biofilm. Following a 1% PBS cleaning, the wells are allowed to sit at room temperature for 15 to 30 min. Following a 15–30-minute incubation period, 0.1% crystal violet is added to the wells. After rinsing the wells, 150 µl of ethanol is added to help destain the biofilm. Then, at 570 nm, the OD is measured [36].

Food packaging material coating with Al₂O₃

The utilization of nanomaterials in food packaging is widespread as they possess antimicrobial properties, offer UV protection, and have the potential to prevent oxidation. Frequently found in food packaging, nanoparticles are safe for human consumption and have been approved as food additives and for use in food contact materials [37, 38]. An aluminium sheet was coated with a sol-gel using nanoparticles, resulting in a sleek and durable finish. To start, we exactly mixed metal alkoxide and ethanol in a 1:4 ratio, forming the sol-gel solution with a smooth consistency. We slowly poured deionised water into the magnetic stirrer, feeling the coolness of the liquid as it made contact with the container. By employing a magnetic stirrer and stirring for at least 30 min, we achieved consistent dispersion throughout the mixture. Afterward, we allowed the sol-gel solution to sit undisturbed for approximately 24 h, ensuring that hydrolysis and condensation were thoroughly carried out. After washing the aluminium sheet to eliminate any dirt or grease, we patiently waited for it to air dry completely.

During the dip coating process, the aluminium sheet is submerged in the sol-gel solution and carefully pulled out at a controlled speed to achieve a uniform coating. We applied several coats of each food container, giving each one enough time to dry before moving on to the next. After coating the aluminium sheet, we left it alone to air dry and let the solvent evaporate. Then, we carefully transferred the coated sheet into a drying oven, which we typically set to temperatures between 100 °C and 300 °C. Depending on the desired properties and the thickness of the coating, the curing time could vary from one hour to several hours [39]. We conducted experiments using a range of concentrations, from as low as 2.5 mg to as high as 160 mg. We started with 2.5 mg and gradually increased the concentrations by a factor of 2. Additionally, the antibacterial activity of the food container has been tested against S. aureus and E. coli using various concentrations of Al₂O₃.

Statistical analysis

Triplicate experiments were carried out, and the resulting data was summarized as the mean ± standard deviation (SD) to show the average and the dispersion of the data points. The statistical differences among treatment groups were evaluated using a one-way analysis of variance (ANOVA) before conducting Tukey’s Honest statistically Significant Difference (HSD) post-hoc test. A p-value less than 0.05 was deemed to have statistical significance.

Results

Optimization of pH, substrates, time, and precursor

Flavonoids, ketones, aldehydes, tannins, carboxylic acids, phenolics, and proteins are among the biomolecules found in pollen extracts. These biomolecules are responsible for the transformation of Al⁺ to Al⁰, leading to the synthesis of Al₂O₃ NPs. When it comes to these biosynthesized Al₂O₃ NPs, you can expect a variety of morphologies, diameters, and forms. These properties are influenced by various experimental conditions, including time, pH, the kinetics of interaction between metal salts (Al(N0₃)₃.9 H₂0) and reducing agents (coconut pollen), and the adsorption of capping agents.

Through the use of coconut pollen extract and response surface methodology, the study seeks to enhance the efficiency of green Al₂O₃ synthesis. A key step in Al₂O₃ NP synthesis involves adding 50 mL of metal solution to the coconut pollen substrate until the color undergoes a noticeable transformation. The substrate’s pH was measured at 5, which falls below the value of pH 7 (as shown in Fig. 5a). According to the UV-Vis spectra, it has been determined that the synthesis of Al₂O₃ is unsuitable in acidic media with a pH between 1 and 5. Furthermore, one can observe a distinct lightening of the color in the Al₂O₃ nanoparticle solution. Moreover, the nanoparticles’ size is directly affected by the pH level. The Al₂O₃ were synthesizedat pH 7. When the environment is acidic, the particles tend to be larger in size, whereas in basic conditions, they are smaller [40].

Fig. 5.

a Effect of pH on the synthesis of Al₂O₃ NPs, b Effect of substrate concentration on the synthesis of Al₂O₃ NPs. c Effect of Al₂O₃ NO₃ concentration on the synthesis of Al₂O₃ NPs. d Effect of time on the synthesis of Al₂O₃ NPs

Gontijo et al. (2020) note that the range of colors, spanning from colorless to yellow, is a result of the size effect, which is influenced by the pH level. By changing the pH of the reaction, biomolecules undergo a shift in their electrical charges, which directly impacts their ability to cap and stabilise, ultimately influencing the growth of nanoparticles. In alkaline media with pH 9 and 11, a noticeable color change occurred, yet the shift in characteristic peaks in the spectra did not suggest the presence of nanoparticles. Furthermore, the pH 11 condition resulted in observable agglomeration of nanoparticles.

The concentration ratios of coconut pollen vary, with the optimal substrate concentration being 39 millilitres, as shown in Fig. 5b. The range of ratios varies from 9:1 to 49:1, demonstrating a considerable difference in values. With precision, the pH of the 39 ml substrate was calibrated to 7, ensuring optimal conditions for the experiment.

The metal optimization process involves establishing different metal substrate ratios, such as 0.25:39, 0.50:39, 0.75:39, 1.0:39, 1.25:39, 1.50:39, 1.75:39, and 2.0:39. The metal optimization has been fine-tuned to 1.75 ml, as shown in the accompanying Fig. 5c. The most efficient synthesis occurred at pH 7, with high extract-to-metal ratio of 39:1, completed within a short 15-minute reaction time. These conditions, acquired for nanoparticle formation, resulted in notable surface plasmon resonance (SPR) peaks and a noticeable colorimetric shift. The optimal time is determined to be 15 min (Fig. 5d).

The UV-Vis spectra analysis of Al₂O₃ NPs, synthesized using coconut pollen extracts, reveals a direct correlation between reaction time and absorption peak intensity. A noticeable trend emerges as the reaction time progresses from 0 to 20 min, with the absorption peak intensity consistently increasing. The plant-extract-synthesized Al₂O₃ display prominent absorbance, as indicated by the surface plasmon resonance (SPR) peaks at 280 nm according to the spectra analysis. After 24 h, the UV-Vis spectra displayed no alterations in absorbance, providing further evidence that the optimal reaction time was 15 min.

The synergistic effect of the bioactive components in the coconut pollen extract aids in the bio-reduction of Al⁺ ions, transforming them into Al₂O₃ nanoparticles. By analyzing the absorption spectra, the ideal proportion of coconut pollen extract to aluminium nitrate solution can be identified to maximize the synthesis of Al₂O₃ NPs.

Characterization

Thermogravimetric analysis (TGA)

Three major degradation phases can be seen in the green-synthesized Al₂O₃ NPs' TGA profile. When temperatures drop below 150°C, moisture and volatile organics evaporate, resulting in an initial weight loss of 2–4 per cent. A 10–20% loss is ascribed to the degradation of organic capping agents, including flavonoids and polyphenols, from the pollen extract of Cocos nucifera between 200°C and 400°C (Fig. 6). Between 400°C and 600°C, a substantial 30–50% loss suggests that stable bioorganic residues are breaking down and that aluminium species are turning into Al₂O₃. Upon reaching 800°C, the curve stabilizes with a residual mass of 25–30%, indicating the development of a thermally stable Al₂O₃ core that is appropriate for may be suitable for high-temperature uses such as biomedical coatings and food packaging [41].

Fig. 6.

Shows a TGA curve indicating that the green-synthesized Al₂O₃ maintain their structure even at high temperatures, a notable result given their small size

FTIR analysis

FTIR analysis was performed on the coconut pollen extract treated with Al₂O₃ to clarify the bonding connections and functional groups that are involved in the production of Al₂O₃ nanoparticles. Both the synthesized Al₂O₃ NPs and the pollen extract from Cocos nucifera were subjected to FTIR spectra in order to gain a better understanding of the role of phytochemicals in nanoparticle formation (Fig. 7). The pollen extract’s spectra alone showed noticeable peaks at 3410.12 cm⁻¹, which corresponded to phenolic compounds’ O–H stretching vibrations. Other bands at 2853.23 cm⁻¹ and 2925.23 cm⁻¹ showed the existence of methyl and C–H group vibrations. Alkanes and nitrosamines were identified by bands at 1407.65 cm⁻¹ and 1468.12 cm⁻¹, respectively, whereas amide I or C = C was represented by a peak at 1630.51 cm⁻¹.The presence of bioactive compounds like flavonoids and polyphenols is confirmed by these peaks.

Fig. 7.

FTIR spectra of Cocos nucifera pollen extract and synthesized Al₂O₃ nanoparticles

The Al₂O₃ NPs’ FTIR spectra revealed significant changes and extra peaks upon synthesis, confirming their interaction with the extract’s biomolecules. C–N and C–O stretching are represented by the bands at 2295.31 cm⁻¹ and 2359.54 cm⁻¹, whilst alkyl amine vibrations are represented by the increase in intensity around 1117.41 cm⁻¹ and 1050.2 cm⁻¹. These changes imply that hydroxyl groups have been oxidized and are involved in the reduction of aluminium ions. The distinctive vibrations of acids, amines, and aromatics, as well as C–H bending, are responsible for the peaks at 865.24 cm⁻¹, 674.24 cm⁻¹, 619.82 cm⁻¹, and 565 cm⁻¹. These alterations demonstrate that the pollen extract’s functional groups actively contribute to the stability and reduction of Al ions during the creation of nanoparticles [41]. According to the results of this FTIR analysis, the coconut pollen extract’s phenolic and flavonoid components are essential for stabilizing and capping the green synthesis process. The spectral analysis provides important information about surface changes of Al₂O₃ NPs, which is necessary to comprehend how they interact in biomedical, food packaging, and catalytic applications. Peak deconvolution and other sophisticated data processing techniques enhance interpretation even further, allowing for precise nanoparticle characterization.

HRTEM and XRD

HRTEM analysis showed well-dispersed, spherical-shaped nanoparticles ranging from 10 nm to 100 nm in size. Figure 8(a-d) depicts cubic Al₂O₃ NPs with smooth surfaces dispersed evenly. The HRTEM image revealed a relatively narrow size distribution of the Al₂O₃ NPs, with an average particle diameter of approximately 51 nm. The histogram generated from these images exhibited a single peak, confirming a unimodal distribution (e). A narrow size distribution of synthesized Al₂O₃ NPs was confirmed, with the majority of nanoparticles measuring between 50 and 80 nm, highlighting their uniformity.

Fig. 8.

a–c HRTEM images of green-synthesized Al₂O₃ nanoparticles at different magnifications showing their varied morphology and nanoscale features. d Selected Area Electron Diffraction (SAED) pattern indicating polycrystalline nature of the particles. e Histogram of particle size distribution based on TEM analysis, showing a predominant size range between 50–80 nm

The XRD analysis examined and verified the crystalline nature of Al₂O₃ nanoparticles. The XRD pattern of the dried nanoparticles obtained from colloid samples revealed peaks at 2 theta (θ) degrees of approximately 33.1°, 40.5°, 50.2°, 58.5°, 67.02°, and 73.5°, which could be associated with the (110), (113), (024), (116), (214), (300), and (311) facets, respectively (Fig. 9). These corresponded to the database of the Joint Committee on Powder Diffraction Standards (JCPDS), card No. 71-1683. The size of the nanoparticles was calculated using the Debye–Scherrer formula (Eq. 1).

Fig. 9.

XRD pattern of synthesized Al₂O₃ NPs

The nanoparticles’ crystalline size (D) can be calculated using the formula

1

where K is the Scherrer constant (0.98), λ is the wavelength (1.54), and β is the full width at half maximum (FWHM). The crystallite size of Al₂O₃ nanoparticles is 42.73 nm, which is consistent with the results from HRTEM images and previous studies by Adnan A. Mohammed in 2020. The unassigned peaks in the XRD pattern may be attributed to the crystallization of a bioorganic phase on the surface of the nanoparticles. Overall, the XRD pattern clearly demonstrates that the Al₂O₃ NPs synthesized are crystalline in nature [42].

Application

Antioxidant activity

Antioxidant activity is tested using a variety of mechanisms, including electron transfer (ET) and hydrogen transfer (HAT) from the antioxidant to free radicals. To compare antioxidant activity, ascorbic acid is employed as a standard. When the scavenging material is recognized as (metal), it neutralizes the free radical, which increases the intensity of the deep purple color. This shows that the metal acts as an antioxidant, effectively neutralizing free radicals.

The antioxidant properties of Al₂O₃ nanoparticles were investigated using the DPPH method of assay. For positive control, ascorbic acid was employed, and the control OD obtained for ascorbic acid is 3.372, which is the same OD value as we acquired for the nanoparticles control OD [43]. Different nanoparticle concentrations produced different percentages of inhibition: 10.64% for 20 µl, 17.58% for 40 µl, 26.98% for 60 µl, 37.93% for 80 µl, and 43.17% for 100 µl. For ascorbic acid, the percentage of inhibition was found at different concentrations. The percentages are 9.01%, 12.15%, 15.42%, 24.61%, and 30.57%, respectively, for the various ascorbic acid concentrations (20 µl, 40 µl, 60 µl, 80 µl, and 100 µl) shown in Fig. 10a. The antioxidant activity of Al₂O₃ NPs increased with concentration but remained lower than the standard ascorbic acid. A concentration-dependent increase in DPPH radical scavenging activity (p < 0.05) was observed for Al₂O₃ NPs, with the moderate antioxidant effect seen at 100 µg/mL.

Fig. 10.

a Depicts the antioxidant activity assay, with Al₂O₃ NPs being tested against the standard ascorbic acid. b presents the Minimum Inhibitory Concentration of S. aureus and E. coli. Values are expressed as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s HSD post-hoc test. Different letters indicate statistically significant differences between groups at p < 0.05

Antibacterial activity

Al₂O₃ NPs were evaluated for their antibacterial activity against a variety of bacterial strains, including S. aureus and E. coli. The antibacterial properties of green-synthesised AlO₃ NPs vary according to the structure of the bacterial cell wall, as shown in Fig. 11. The thinner outer membrane and peptidoglycan layer of Gram-negative bacteria, such as E. coli, render the cells more vulnerable to penetration by nanoparticles. Intracellular contents may leak out as a result of AlO₃ NPs’ easy attachment to and disruption of the cell membrane. Reactive oxygen species (ROS) including superoxide anions and hydroxyl radicals are produced, which further harm cellular components, and the release of Al³⁺ ions disrupt enzyme action.

Fig. 11.

Antibacterial mechanisms of Al₂O₃ nanoparticles against E. coli and S. aureus

S. aureus and other Gram-positive bacteria, on the other hand, have a thicker peptidoglycan coating, which provides some barrier to the nanoparticles’ physical penetration. considerable attachment is nevertheless encouraged by electrostatic interactions between the negatively charged bacterial cell surface and the positively charged AlO₃ NPs. In addition to contributing to oxidative stress, ROS production jeopardizes the viability and function of cells. Though usually not as strong as in Gram-negative bacteria, these several processes work together to produce antibacterial effects [44]. Al₂O₃ NPs showed a statistically significantly larger inhibition zone against E. coli compared to S. aureus (p < 0.05).

The results show that nanoparticles synthesized using the green method have effective antibacterial activity against pathogenic bacteria. The findings demonstrated that as the concentration of nanoparticles increased, so did their inhibitory effect. Figure 12a and b measured the inhibitory zone’s diameter, indicating the microorganism susceptibility level. In Fig. 12 Table shows the E. coli exhibit a large zone of inhibition [14 mm in 50 µl,16 mm in 100 µl,18 mm in 150 µl,20 mm in 200 µl,21 mm in 250 µl] compared to the S. aureus zone of inhibition [11 mm in 50 µl,12 mm in 100 µl,14 mm in 150 µl,15 mm in 200 µl,16 mm in 250 µl] (Fig. 13). The negative control (ethanol) exhibited no zone of inhibition beyond the well diameter (8 mm), confirming that the antibacterial activity is due to the Al₂O₃ nanoparticles. No positive control was tested, as this study focused solely on the comparative efficacy of nanoparticle concentrations. The result is more similar to the Al₂O₃ nanoparticle’s antibacterial activity, as the E. coli has a larger zone of inhibition compared to Proteus vulgaris [45]. Even after washing, the food container coated with Al₂O₃ nanoparticles displayed notable inhibition against E. coli and S. aureus, showcasing its antibacterial properties (Fig. 14). We treated the bacteria with a range of concentrations, from 2.5 mg to 160 mg, and observed the effects [46].

Fig. 12.

Disk diffusion assay showing the antibacterial activity of green-synthesized Al₂O₃ nanoparticles against E. coli (a) and S. aureus (b) at various concentrations (50 to 250 µg/mL)

Fig. 13.

a Antibacterial activity and b Biofilm inhibition activity. Values are expressed as mean ± SD (n = 3). statistically Significant differences among groups were determined using one-way ANOVA and Tukey’s post-hoc test (p < 0.05)

Fig. 14.

Al₂O₃ coated on food containers were tested against S. aureus and E. coli with concentrations varying from 0 to 160 mg to determine their antibacterial activity

The corresponding Table 1 summarizes the zone of inhibition (in mm) for each concentration. Ethanol was used as the negative control, which exhibited minimal inhibition zones (8 mm), indicating that the antibacterial activity is attributed to the nanoparticles Al₂O₃ NPs showed a statistically significantly larger inhibition zone against E. coli compared to S. aureus (p < 0.05).

Table 1.

Zone of Inhibition exhibited by AINPs-coated food containers against E. coli and S. aureus at different nanoparticle concentrations

S. No.	Concentration of Al2O3 NPs (μg/mL)	Zone of inhibition (mm)–E. coli	Zone of inhibition (mm)–S. aureus
1	50	14±0.2	11±0.2
2	100	16±0.3	12±0.2
3	150	18±0.2	15±0.3
4	200	20±0.4	17±0.2
5	250	21±0.2	20±0.3
6	Control	8±0.1	8±0.1

Minimum inhibitory concentration

After the incubation of 24 h at 37 degrees Celsius, the 96 plate wells with 10 and 5 mg/ml concentrations that contain Al₂O₃ NPs show the growth inhibition of bacteria [90.76% and 95.71% in E. coli, 65.46% and 71.17% in S. aureus] which is shown in Fig. 10b. It was clear that among all the other concentrations (10, 5, 2.5, 1.25, and 0.625 mg/ml), they had the strongest impact on inhibiting bacteria, with the MIC achieved at 0.625 mg/ml [47]. These results thus confirm that the Al₂O₃ NPs have a measurable impact on the growth of S. aureus and E. coli.

Minimum bacterial growth

The suspensions from the 96-well plates showed no growth of bacteria at concentrations ranging from 10 to 0.625 mg/ml. To check the minimum bactericidal concentration (MBC), the control (without nanoparticles) and concentrations of 10 and 0.625 mg/ml were inoculated in agar plates and incubated for 24 h. No growth was observed in both concentrations, while adequate growth was observed in the control (Fig. 13a and b). However, the other concentrations of nanoparticles (0.312, 0.156, 0.078, and 0.039 mg/ml) showed bacterial colonies. The MBC test helps determine the minimum concentration of nanoparticles needed to achieve a bactericidal effect. When 99.9% of the bacterial population is killed at the lowest concentration of an antimicrobial agent, it is termed an MBC endpoint. These observations confirm that the nanoparticles are bactericidal at a minimum inhibitory concentration (MIC) of 0.625 for both S. aureus and E. coli [48].

Anti-biofilm assay

In biofilm inhibition activity, there are several bacteria in the form of biofilm. Biofilms are ubiquitous; they form on virtually all surfaces immersed in natural aqueous environments. A biofilm shows certain properties to bacteria that are not seen in the planktonic state, a fact that justifies the recognition of dental plaque as a biofilm. Al₂O₃ NPs harm bacterial cultures such as E. coli (gram-negative bacteria) and S. aureus (gram-positive bacteria). Biofilm is a leading threat to the environment, industry, and human health. Figure 13b shows the biofilm activity of Al₂O₃ NPs describes which are more effective in E. coli (72.46%) compared to S. aureus (48.55%). Al₂O₃ NPs have reacted with the cell membrane and entered the.

E. coli, releasing a homogeneous substance and binds with the cell chain, acting as part of the biofilm [49]. Biofilm inhibition was higher in Al₂O₃ NPs-treated samples than control (p < 0.05).

Food package materials coated with Al₂O₃ NPs

Ensuring food safety heavily relies on the proper use of food packaging. It’s very important to prevent spoilage and contamination, boosting sensitivity through the facilitation of enzyme activity [50]. The harsh environment in functional foods often causes the bioactive compounds to break down and lose their activity, resulting in shorter shelf life. considerable results have been seen in coating the nanoparticles on Al₂O₃ food-grade containers made up of aluminium material to extend and guarantee the longevity of a food product. The well-coated Al₂O₃ NPs (NPs) were analyzed using the FESEM image, which confirmed the even coating of the NPs on the food container.

The surface of the aluminium sheet was comparatively homogeneous and smooth before washing (Fig. 14a). Distinct clusters and roughness were seen during nanoparticle coating and washing (Fig. 14b), indicating that Al₂O₃ were successfully deposited. Increased surface roughness and nanoparticle adhesion demonstrate a direct correlation between these morphological changes and improved antibacterial action.

Our investigation revealed the synthetic material’s antioxidant and antibacterial qualities, underscoring its possible use in food preservation. Using a sol-gel dip-coating technique, aluminium sheets were successfully coated with Al₂O₃ NPs (Al₂O₃) at different concentrations: 2.5 mg to 160 mg, as indicated in Table 2. For comparison, a control sheet devoid of Al₂O₃ was also created. The coating grew increasingly homogeneous and dense as the concentration of Al₂O₃ increased, and at higher concentrations (particularly 80 mg and 160 mg), it showed a noticeable metallic shine. These findings imply efficient nanoparticle deposition, which most likely played a role in the enhanced functional characteristics seen.

Table 2.

Antibacterial activity of Al₂O₃ nanoparticles (AINPs) coated food containers against E. coli and S. aureus at different concentrations

Concentration (mg)	Zone of Inhibition (E. coli) (mm)	Zone of Inhibition (S. aureus) (mm)
Control	0.0	0.0
2.5	3.6	3.3
5	5.2	4.6
10	7.3	6.5
20	10.2	9.5
40	12.5	11.0
80	13.8	13.5
160	14.3	15.5

Conclusion

This study effectively illustrated the environmentally friendly manufacturing of Al₂O₃ nanoparticles utilizing pollen extract from Cocos nucifera and aluminum nitrate nanohydrate. The ecologically safe synthesis method produced stable, bioactive nanoparticles without using dangerous chemicals. Reproducibility and efficiency were guaranteed by optimizing variables like pH, reaction duration, and precursor concentration. Higher quantities of the produced AlO₃ NPs resulted in increased free radical scavenging, indicating dose-dependent antioxidant action. E. coli was found to be more sensitive to antibacterial assays than S. aureus, and their inhibitory and bactericidal capability was confirmed by well-defined MIC and MBC values. Furthermore, the nanoparticles dramatically reduced the ability of both test species to build biofilms. According to these results, green-synthesised Al₂O₃ NPs have the potential to be used in antibacterial and antioxidant applications, especially as coatings for food packaging. By reducing microbial development and oxidative spoiling, their addition to food containers may increase shelf life. Still, it is important to note that this study was limited to in vitro evaluations. Additional in vivo studies are necessary to evaluate stability, functionality, and long-term safety in actual storage and environmental settings. Furthermore, research on the migration, degradation, and possible toxicological effects of nanoparticles is necessary to justify their use on an industrial scale. Future studies will concentrate on creating coatings for food-contact surfaces using nanoparticles and assessing how well they work in real-world applications.

Plant collection and permissions

The Cocos nucifera pollen used in this study was collected from cultivated coconut trees located in Padur village, Chennai, Tamil Nadu, India (Latitude: 12.8405° N, Longitude: 80.2248° E). The plant material was collected from privately owned farmland with permission from the landowner. As Cocos nucifera is not an endangered or protected species and the material was not collected from forest land, no specific permits or licenses were required. The collection complied with all relevant local and national regulations.

Author contributions

Conceptualisation: Sivanraju Rajkumar Supervision: Palanivel Velmurugan Writing– original draft: Yuvaraj TamilselviMethodology: Moorthy Muruganandham, Investigation: Kanagasabapathy SivasubramanianSoftware: Dhakshan Prakash Vijayalakshmi, Tamilselvan Amirthalingam, Daram Sairam ReddyData curation: Avula Madhav, Jeyanthi Rebecca, Poorni Santhana KrishnanWriting– review & editing: Palanivel Velmurugan.

Funding

The authors received no specific funding for this work.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. All relevant data are also included within the manuscript.

Declarations

Ethics approval and consent to participate

Not applicable. This study did not involve any human or animal participants.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.