Evaluation of Properties and Bioactivity of Silver (Ag) Nanoparticles (NPs) Fabricated Using Nixtamalization Wastewater (Nejayote)

Abstract

Nejayote (Nej), an effluent from nixtamalization process, has an alkaline pH and contains a high load of organic matter in suspension and dissolution, which makes it a highly polluting waste when discharged directly into the environment. However, the sustainable reuse of this effluent is relevant since it contains high-value compounds (ferulic acid (FA)) with appropriate activity for the ecological synthesis of silver nanoparticles (AgNPs). This study explores the synthesis of AgNPs using Nej as a reducing and stabilizing agent and evaluates the antibacterial effectiveness of AgNPs against Escherichia coli (E. coli). The AgNPs under study possess excellent optical (UV-Vis) and structural properties (XRD). HR-TEM images show predominantly spherical particles, with an average size of 20 nm. FTIR spectroscopy identified functional groups, including phenols and flavonoids, on the nanoparticle surface, acting as stabilizing agents. HPLC supports the existence of FA in the AgNPs. Biogenic AgNPs exhibit enhanced antibacterial activity due to the adsorption of these functional groups onto their surface, which contributes to bacterial membrane disruption. Finally, no hemolytic or cytotoxic activity was observed, suggesting that the AgNPs exert antimicrobial activity without potentially harmful doses (biocompatibility). The study highlights the potential of Nej as a sustainable source for use in nanoparticle synthesis, promoting the recycling of agro-industrial waste and the production of materials with technological applications.

1. Introduction

The use of plant extracts rich in compounds with reducing power for the biogenic synthesis of AgNPs is considered a cost-effective alternative because particles with different morphologies and sizes have been successfully obtained from various extracts of plants, leaves, stems, roots, and husks [1,2,3] and have been evaluated in diverse applications [4,5,6]. For this reason, the reducing potential of some agro-industrial effluents, such as tequila vinasses and wastes from the corn industry, are being investigated [7,8,9,10]. It has been proven that they have a composition rich in phytochemical compounds suitable for the fabrication of value-added products (i.e., the synthesis of metallic nanoparticles). In addition, the reuse of wastewater reduces the pollution generated by its direct disposition into the environment [11].

This study focuses on the biogenic synthesis of AgNPs using Nej, highlighting the reuse of this agro-industrial effluent as a sustainable and ecofriendly alternative. The nixtamalization process consists of cooking the corn kernel in the presence of calcium hydroxide (Ca(OH)₂). The residual alkaline water of this process is known as Nej [11], the physicochemical properties of which result from the components present in the corn [7]. It contains a considerable reducing power, which is attributed to the presence of bioactive compounds such as phenolic, coumaric, flavonoid, arabinoxylans (AX), and carotenoids [7,8,10]. These compounds can be used as reducing and stabilizing agents in the synthesis of silver nanoparticles, which present considerable potential for applications in biomedicine, food safety, water treatment, catalytic activity, pharmacological, and antimicrobial coatings, serving as an ecofriendly alternative to traditional antimicrobial agents [12,13,14].

Nej is an abundant wastewater in Mexico. To nixtamalize 1 kg of maize, approximately 2 L of limewater (1% w/v solution of Ca(OH)₂) was used, generating an equivalent volume of Nej as waste [15]. Thus, 50 kg of maize generates approximately 100 L of Nej. At an industrial scale, this process results in the annual production of around 14.4 millon m³ of highly contaminated wastewater [16] and it is estimated that by 2030 this amount will increase due to population growth. Nej is a polluting effluent mainly due to its elevated pH levels (up to 12) and the high load of organic matter reported (2540 mg/L) [7]. This effluent has a high Chemical Oxygen Demand (COD)—25,000–28,000 mg/L O₂—due to the abundance of calcium derived from alkaline water [8]. The presence of settleable solids (SSEDs) such as the endosperm and pericarp are present as they are detached from the corn kernel during the nixtamalization process [11]. Nej significantly exceeds Mexican regulations for wastewater disposal, which permit a maximum COD of 150 mg/L O₂ and a pH of 10 [16]. Nowadays, industrial Nej is directly deposited in water bodies, sewer systems, and soils, damaging the environment.

In addition, Nej is an unvalued by-product that has rarely been tested to determine its application uses. Several methods can be employed to process Nej and reduce its contamination levels [16]. First, it can be used to extract valuable biomolecules such FA, which has antioxidant and anti-inflammatory properties. Also, Nej can serve as a fertilizer for crops [17], a source of enrichment for functional foods [7], or as a substrate to aid in seed germination [16]. Nej could be utilized for the synthesis of nanoparticles, which hold potential for applications in multiple industrial fields, including biomedicine, pharmacology, environmental remediation, and antibacterial coatings. This was similar to the study reported by [18] about the synthesis of nickel oxide nanoparticles (NiO NPs), where the authors used aqueous extracts of Manihot esculenta (yucca) residue in the presence of Nej as reducing and stabilizing agents. Furthermore, López-Mercado et al. [19] reported the green synthesis of TiO₂ NPs and their application for the photo-hydrolysis of Nej.

However, according to reported research, most of this waste is discarded into the environment, causing pollution. Therefore, it is necessary to continue searching for alternatives to mitigate the pollution effects of Nej [2]. Moreover, it is useful to promote the reuse of this waste by taking advantage of its bioactive compounds. Therefore, the present study focuses on using Nej as a reducing and stabilizing agent in the biogenic synthesis of AgNPs to develop an environmentally friendly method. The antimicrobial activity of these nanoparticles was evaluated against pathogenic bacteria, specifically E. coli, revealing enhanced effectiveness compared to chemically synthesized AgNPs. Furthermore, this research promotes Nej recycling to mitigate its negative ecological impact. We perform the double valorization of Nej, first to mitigate the environmental impact and second as a valuable source of bioactive compounds capable of facilitating nanoparticle synthesis with enhanced antimicrobial properties, since the adequate treatment of wastewater and by-products from different industrial effluents is today a global priority [7].

Finally, this study aims to stimulate research that contributes to environmental sustainability by implementing strategies that simultaneously tackle the ecological issues associated with Nej and enable the synthesis of biogenic nanoparticles. It also aims to systematically investigate the physicochemical properties and bioactivity of AgNPs fabricated using this biogenic approach. To achieve this goal, traditional microbiology assays demonstrate the enhanced activity of the nanomaterials (NMs), whereas advanced microscopy techniques are applied to investigate the interactions of AgNPs with healthy cells. This research shows the value of agro-industrial by-products as key resources for the sustainable production of AgNPs, with notable ability to inhibit microbial growth.

2. Materials and Methods

2.1. Materials and Nej Pre-Treatment

Silver nitrate (AgNO₃) was used as the AgNP precursor. FA was used as a standard to determine its concentration in Nej and AgNPs samples; these chemicals were obtained from Sigma-Aldrich, Estado de Mexico, Mexico. Due to its abundance and high reducing activity, Nej was sourced from various local corn tortilla mills in Aguascalientes (Mexico). Deionized water was used as a synthesis medium.

Pre-Treatment of the Nej

To remove suspended solid particles, the collected Nej was filtered and centrifuged using a Mega 17R Small High-Speed Refrigerated Centrifuge (Hanil Scientific, Gimpo, Republic of Korea) capable of reaching a maximum speed of 17,000 rpm and a relative centrifugal force of up to 31,050× g. The temperature control ranges from −10 to 40 °C. The operating conditions used were 15,000 rpm for 15 min at 20 °C. Freshly purified Nej was used for the synthesis of AgNPs. Purified Nej was stored in containers at 4 °C.

2.2. Physicochemical Analysis of Nej

The samples were analyzed, considering the following parameters [9]: COD was assessed by the potassium dichromate assay; Biochemical Oxygen Demand (BOD₅) was assessed by the Winkler method; hardness was assessed using the ethylenediaminetetraacetic acid (EDTA) titration method, total suspended solids (TSSs), SSEDs, and total solids (TSs). The concentration of total phenols was determined by the Folin–Ciocalteu method [20]; the reducing sugar content in Nej samples was measured by the dinitrosalicilyc acid (DNS) method [21]; the determination of chlorides was performed using Mohr’s method. Carbonate ions were quantified by titration with hydrochloric acid (HCl). The density was measured using a (TDM1107, Robsan hydrometer, Mexico) calibrated in relative density (RD) units at standard temperature of 15 °C. The pH and electrical conductivity were measured using a portable dual-channel meter (LAQUAact PC110-K, Horiba, Kyoto, Japan), with a data logging capacity of 1000 entries.

2.3. Quantification of FA in Nej

The FA content in Nej was determined by a high-performance liquid chromatography (HPLC) system, using a (Thermo Scientific^TM uHPLC, Waltham, MA, USA) Ultimate 3000 equipment with a diode detector (UV-Vis) and a Hypersil Gold aQ column (150 mm × 4.6 mm and 5 μm). Two phases were used: phase A consisted of formic acid (0.1%), and phase B consisted of acetonitrile. An isocratic method was employed with a 70/30 phase ratio, a flow rate of 0.8 mL/min, a column temperature of 25 °C, a wavelength of 321 nm, and sample injection volume of 10 μL. A calibration curve using FA as a standard (5–40 µg/mL) was used to evaluate its content in the Nej samples. Before the HPLC measurement, Nej was filtered using a sterile 0.45 μm PTFE B syringe filter (ChoiceTM, Thermo ScientificTM) and degassed for 10 min in a Branson 8800 ultrasonic bath (UltrasonicTM Digital Bench Top Cleaner, Model CPX8800H, Fisher Scientific, Estado de Mexico, Mexico). The diluted extracts were directly injected into the HPLC system. The peak areas of the samples were monitored at 321 nm. The analysis of each sample was performed in triplicate. The same protocol was used to determine the concentration of FA in the AgNP colloid.

2.4. Biogenic Synthesis of AgNPs

AgNPs were prepared by mixing an aqueous solution of AgNO₃ 10 mM with previously purified and diluted Nej (1:20). The reaction was kept continuously magnetic stirred at 250 rpm for 1 h to achieve the reduction of silver ions (Ag⁺). Likewise, a Taguchi L₉ experimental design was carried out to study the effect of the following variables on the NPs size and size distribution: pH, silver salt concentration, and reducing agent concentration (see

Table 1. Taguchi L₉ experimental design for synthesis of AgNPs from Nej.

Sample	pH	AgNO3 Concentration (mM)	Nej Concentration (mg/g)
1	6	5	21.28
2	6	10	2.12
3	6	15	1.07
4	8	5	2.12
5	8	10	1.07
6	8	15	21.28
7	10	5	1.07
8	10	10	21.28
9	10	15	2.12

). The concentration of TS in Nej was determined under three different dilution conditions based on measured density. The synthesis conditions were optimized by analysis of variance. According to the signal–noise (S/N) ratio analysis, silver nanoparticles were synthesized with the conditions proposed as optimal, evaluating two synthesis temperatures (60 and 80 °C) and adjusting the pH of the Nej to 10. The S/N ratio was calculated using Equation (1).

$${\displaystyle \frac{\mathrm{S}}{\mathrm{N}}}=-10\log \left({\displaystyle \frac{1}{{\mathrm{n}}_{\mathrm{dat}}}}{\displaystyle \sum }_{\mathrm{i}=1}^{{\mathrm{n}}_{\mathrm{dat}}}{\displaystyle \frac{1}{{\mathrm{Rq}}_{\mathrm{i}}^2}}\right)$$

(1)

where n_dat = 3 (number of repetitions) and R_qi is the response variable used for this analysis according to the conditions mentioned in the experimental design.

2.5. Characterization of AgNPs

AgNPs were characterized using different techniques: their optical properties were investigated using a Thermo Scientific^TM Evolution 201 UV–visible spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) operating in a wavelength range of 300–700 nm. X-ray diffraction (XRD) analysis allowed the identification of the AgNPs’ crystalline nature. XRD data was obtained using a Malvern Panalytical Empyrean diffractometer (Malvern Panalytical Ltd., Malvern, UK) equipped with a PIXEL 1D detector at 45 kV and 40 mA, employing the Bragg–Brentano configuration and CuK radiation α1 (λ = 1.5406 Å) at a scanning speed of 147°/s with a step size of 0.02° in a range from 0 to 60°. The crystallite size was calculated based on the width of the diffraction signals using the Debye–Scherrer equation (Equation (2)).

$$\mathrm{D}={\displaystyle \frac{\mathrm{K} \mathsf{\lambda }}{\mathsf{\beta }\cos \mathsf{\theta }}}$$

(2)

where D = crystallite size in nm, K = Scherrer’s constant, 0.9–0.98 (form factor), $\mathsf{\lambda }$ = wavelength of the X-ray beam used (1.54178 Å), $\mathsf{\beta }$ = total width at half the maximum (FWHM) of the diffraction peak (in radians), and $\mathsf{\theta }$ = Bragg diffraction angle (position of diffraction peak in radians). The morphological structural characteristics of AgNPs were analyzed using transmission electron microscopy (TEM), high-resolution TEM (HR-TEM), and selected area electron diffraction (SAED) with an FEI-TITAN 80–300 kV microscope (FEI Company, Hillsboro, OR, USA) operated at 300 kV. Elemental composition was determined via energy-dispersive spectrometry (EDS), integrated into the TEM system. Particle size distribution was assessed by measuring nanoparticle diameters from TEM images using the ImageJ software, version 1.53, available at https://imagej.nih.gov/ij/ (accessed on 3 February 2025). Fourier transform infrared spectroscopy (FTIR) analysis identified functional groups on the surface of NPs using a Thermo Scientific^TM Nicolet^TM iS10 infrared spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) in a wavenumber range of 4000 to 400 cm⁻¹. We performed 32 scans and dilution in potassium bromide (KBr) pellets. Finally, the size and topography of nanoparticles were determined using a ScanAsyst atomic force microscope (AFM) Bruker, Dimension Edge with ScanAsyst (Bruker Corporation, Billerica, MA, USA). Nanoscope software version 4.5.0 was used for image analysis and was available at https://www.bruker.com/en/services/software-downloads.html (accessed on 28 February 2025). The concentration of FA in the AgNP colloid was determined by HPLC.

2.6. Evaluation of Antibacterial Activity

The antibacterial activity of the silver nanoparticles was investigated using the serial dilution method on agar plates; the Gram-negative bacterial strain used was E. coli ATCC 25922. The results were expressed as colony-forming units (CFUs)/mL by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). E. coli was cultured in a liquid brain–heart infusion (BHI) medium at 37 °C for 20 h. Bacterial adjustment was performed in a spectrophotometer at a wavelength of 625 nm to obtain a concentration of 10⁷ cells/mL, followed by serial dilutions to reach 10⁵ cells/mL. After, the bacterial cells interacted with Ag NPs (biogenic or chemically synthesized) for 3 h (65 rpm and 37 °C) at different concentrations. For the biogenic synthesis, the following concentrations were evaluated (0.10, 0.15, 0.20, 0.25, and 0.30 μg/mL) using Nej as control. The antibacterial activity of chemically prepared AgNPs was investigated at concentrations of 0.25, 0.50, 0.75 and 1 μg/mL using phosphate-buffered saline (PBS) as a control. Then, 100 μL of each treatment was poured into MacConkey agar plates and evenly distributed. Finally, the plates were incubated at 37 °C for 24 h. Antibacterial activity was evaluated by assessing the number of CFUs, which determined the MIC and MBC. The MIC is the lowest concentration of an antibacterial agent that prevents bacterial growth, while the MBC is defined as the lowest concentration of the antibacterial agent that completely inhibits bacterial growth. Statistical analysis was performed using Tukey’s test; this multiple comparison method is used to identify significant differences between groups (p < 0.05).

Evaluation of the Antibacterial Activity of FA

The antibacterial activity of the FA was investigated using the serial dilution method and the drip dilution test. The Gram-negative bacterial strain used was E. coli ATCC 25922. E. coli was cultured in a BHI medium at 37 °C for 20 h. The bacterial adjustment was performed in a spectrophotometer at a wavelength of 625 nm to obtain a concentration of 10⁷ cells/mL, followed by serial dilutions to reach 10⁵ cells/mL.

After, the bacterial cells interacted with FA for 3 h (65 rpm and 37 °C) at different concentrations (2500, 1250, and 625 μg/mL) using PBS as a control. Then, 10 μL of each concentration was poured into MacConkey agar plates and incubated at 37 °C for 24 h. The bacteria are exposed to different concentrations of FA to investigate the MIC and MBC of this compound.

2.7. Hemolysis Assay

To demonstrate the safe use of the AgNPs under study, the hemolytic and cytotoxic activity of the material was investigated. Red blood cells (RBCs) were exposed to AgNPs using the MIC and MBC encountered in the antibacterial test. The hemolytic activity was evaluated following the ASTM F756-13 standard protocol (standard practice for assessing the hemolytic properties of the materials). In brief, heparin-stabilized human blood was freshly collected. For each sample, a total volume of 10 mL was used, where the different concentrations of nanoparticles were mixed with the 0.9% saline solution and 100 μL of blood was added. Blank samples were prepared, with distilled water as a positive control, saline solution as a negative control and, as a third control, the medium in which the nanoparticles were re-suspended (biogenic route and chemical route), which consisted of Nej and Arabic gum, respectively. All experiments were performed in triplicate and products were incubated at 37 °C in a water bath at 65 rpm for 3 h. Subsequently, the samples were centrifuged at 3500 rpm for 5 min, and the absorbance of the supernatant was measured at 540 nm, a wavelength corresponding to the absorption band of oxyhemoglobin [22]. Hemolytic activity was calculated using the following equation:

$$\mathrm{Hemolytic} \mathrm{activity}, \%={\displaystyle \frac{\mathrm{Absorbance} \mathrm{each} \mathrm{sample}}{\mathrm{Absorbance} \mathrm{negative} \mathrm{control}}}\times 100$$

(3)

2.8. Toxicity and Cell Viability Assays of AgNPs

To evaluate the cytotoxic activity of AgNPs, Detroit 548, also known as CCL-116 (a cell line derived from human fibroblasts), was used as the biological model. The cells were cultured in a DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS) and other additives. The cells were thawed and cultured until reaching 80% confluence. They, they were radiated (in 96-well plates for the viability assay and in 24-well plates for AFM studies) with a concentration of 50 × 10³ cells/cm² and incubated at 37 °C for 12 h. They were then exposed to different NPs concentrations (based on hemolytic activity) in 1% FBS medium and incubated for 24 h. Cell counting kit-8 (CCK-8) assays were employed to assess cell viability, and lactate dehydrogenase (LDH) assays were used to measure cytotoxicity [3].

2.8.1. Cell Viability Assay (CCK-8)

After the exposure period (24 h) to AgNPs, the cells were washed (twice) with the PBS solution. Then, 100 μL of MTS solution (1:10) in DMEM medium was added to each well and incubated for 2.5 h to obtain the soluble formazan salt [3]. After, the medium with formazan salt formation was removed and cell viability was measured by UV-Vis spectroscopy. Absorbance was measured at 450 nm using a plate reader.

2.8.2. Cytotoxicity Assay (LDH)

The release of LDH to cell media was measured following the manufacturer’s protocol (LDH Cytotoxicity Detection Kit Roche, catalog No. 116447930001, Mannheim, Germany) [3]. A reactive solution was prepared by adding 48.9 μL of reagent 2 and 1.1 μL of reagent 1 for each well. 50 μL of this mixture was added to each sample and allowed to react for 25 min. Finally, the LDH released was quantified using UV-Vis spectroscopy (at 450 nm in a plate reader). For AFM imaging, prior to the seeding of 24-well plate cells, a sterile circular coverslip was placed at the bottom of the wells. The cells were then fixed with 10% formalin and dehydrated with increasing ethanol solutions (40 to 100%) for 10 min, and the wells were left to dry for 20 min. All experiments were performed in triplicate and incubated at 37 °C under controlled conditions.

All images and graphics were processed using software from OriginLab Corporation, Origin 8.0, available at https://www.originlab.com/ (accessed on 4 March 2025).

3. Results and Discussion

3.1. Pre-Treatment of the Nej

After the filtration and centrifugation of Nej, a solid-free solution was obtained, suitable for use in the synthesis of AgNPs. Centrifugation enabled the effective removal of insoluble impurities. These results indicate that the purification process was efficient in obtaining Nej free of SS.

3.2. Physicochemical Analysis of Nej

The composition of Nej varies according to the type of corn, the amount of corn per batch, the water used, the amount of Ca(OH)₂ added, and the cooking time, resting period, and temperature of the process [8].

Table 2. Values of physicochemical analysis of Nej.

Parameter	Average Values
pH	11.98 ± 0.64
Hardness (mg/L CaCO3)	4150 ± 185.71
Density (kg/m3)	1010.14 ± 10.11
Electrical conductivity (mS/cm)	4.85 ± 0.40
Total phenols (mg/L)	839.64 ± 37.96
Reducing sugars (mg/L)	48.48 ± 8.11
COD (mg/L)	30,500 ± 786.63
BOD5 (mg/L)	3000 ± 443.84
SST (mg/L)	11,400 ± 469.19
SSED (mL/L)	315 ± 51.79
ST (mg/L)	21,500 ± 885.77
Chlorides (mg/mL)	0.02272 ± 0.0029
Carbonates (mg/mL)	0.05040 ± 0.0047
Bicarbonates (mg/mL)	0.16714 ± 0.0124

shows the results of the physicochemical analysis of the Nej used in this study. The high levels of TSS and SSED in the sample are mainly due to the pericarp fragments and other organic particles that are shed from the corn during the nixtamalization process, which contributes to the turbidity and density of the Nej sample. Additionally, it has been noted that the primary components of Nej include various corn constituents, such as AX and coumaric compounds, along with dissolved substances like total phenols, reducing sugars, and carotenoids, which impart the characteristic color (yellow) of this effluent. These factors contribute to its significant pollution potential [16].

According to data reported in other studies [7,9], the typical COD values found in Nej samples are between 10,000 and 28,000 mg/L. This wide range can be attributed to organic matter, which includes carbohydrates, proteins, lipids, and phenolic compounds that contribute to environmental pollution when this effluent is discharged into distinct water bodies. Similarly, the high level of BOD₅ indicates that Nej significantly damages the quality of the aquatic medium because the dissolved oxygen is quickly consumed by microorganisms when decomposing the organic matter of this wastewater effluent [7,9].

3.3. Quantification of FA in Nej

Figure 1 shows the HPLC analysis of FA obtained from the standard solution and the Nej. From this figure, the presence of FA in the Nej sample can be confirmed since the retention time of the Nej sample coincides with that of the FA standard solution (3.79 min). This finding suggests the presence of FA in all the samples under study. The calibration curve was obtained by plotting the peak areas from standard solutions against the FA concentration and peak areas from both the standard (40 µg/mL) and Nej extract were used to determine the FA content in the last sample. The additional signals found in the Nej extract chromatogram are related to other phenolic compounds with similar chemical structures and retention times close to those of FA [23,24], and even to some other organic acids present in Nej. These results are consistent with other studies reported in the literature [25,26].

Table 3. FA concentration in Nej samples.

Sample	Concentration, FA mg/mL of Nej
M1	0.81607
M2	0.34481
M3	0.55771
M4	0.05181
M5	0.33595
M6	0.69290

shows the concentrations of FA obtained in the different Nej samples. As can be seen, a wide variation in the FA content is noticeable. This is because the concentration of this compound depends on the operating conditions applied in the nixtamalization process (i.e., temperature, cook time, the amount of water and Ca(OH)₂, and the type of corn); these values are within the range reported in the literature [23,24,27]. Therefore, the Nej samples obtained can effectively support the synthesis of AgNPs due to their FA content. The same applies for some other bioactive compounds that can be used as reducing or stabilizing agents.

Figure S1 shows the calibration curve for FA, using HPLC for quantitative determination. These results support the application of this chromatographic method to investigate, qualitatively and quantitatively, the presence of FA in the samples under study. The calibration curve runs from 1 to 40 μg/mL, denoting the proper sensitivity of the technique for this compound. The calibration curve shows a linear behavior in the range of concentrations from 1 to 40 μg/mL and an adequate linear regression with a high correlation coefficient (as indicated in Figure S1). The standard deviation values are low, confirming that the developed method is precise and accurate for the quantification of FA. The HPLC analysis is also specific, showing a clear, well-defined peak for this phenolic substance, with the same retention time observed in standard (laboratory-prepared) and environmental samples.

The present study aims to implement a sustainable process for the practical reuse of Nej, using it as a medium to produce metallic nanoparticles. A previous study remarks on the importance of the extraction of the FA present in Nej. FA was immobilized on chitosan to produce bioactive films with enhanced antioxidant properties [28]. Recent studies have highlighted the bioactivity of FA (as antioxidant, anti-inflammatory, antimicrobial, hepatoprotective, anti-carcinogenic, antithrombotic, antiviral). Additionally, it has been reported that FA is involved in the modulation of enzyme activity, the activation of transcriptional factors, gene expression, signal transduction, and metal chelation, supporting its applications in medicine and cosmetics [29,30,31]. The presence of FA in Nej effluent adds value to focus in developing strategies for the sustainable reuse of this effluent.

3.4. Biogenic Synthesis of AgNPs

The silver nanoparticles were successfully synthesized using Nej as a source of reducing and stabilizing agents. The formation of AgNPs was signified by a color change from yellowish to dark brown during the first minutes of the synthesis reaction. The reaction proceeds as expected since Nej acts as both a reducing agent and a coating (natural surfactant that exerts control on the size and stability of the NPs); this coincides with other reported studies where synthesis is mediated by plant extracts [3,32,33,34,35]. The synthesis process is illustrated in Figure 2. At the beginning of the reaction (Figure 2A), Ag⁺ ions dissolve and interact with reductant and surfactant agents; when the reaction mixture is heated, the rate increases (Figure 2B), which results in a color change that is characteristic of colloidal silver formation (Figure 2C) [36].

In addition, Figure S2 demonstrates the surfactant capacity of Nej by reducing AgNO₃ with FA (reagent-grade). The reduction of silver is followed by aggregation and deposition. The redox capacity of this phenolic compound is appropriate for the reduction of Ag⁺ ions (this affirmation can be supported by the formation of a silver mirror in the wall of the flask). However, it cannot control particle size, rendering a silver mirror. Furthermore, the reaction occurs at a much lower rate compared to the use of Nej for silver colloidal formation, suggesting the involvement of other reductant compounds found in Nej.

The influence of pH, AgNO₃, and Nej concentration on the synthesis of AgNPs was investigated by S/N analysis. Statistical analysis of variance (ANOVA) indicates that pH is the variable with the largest influence on the synthesis process of AgNPs. In accordance with the results of Miu and Dinischiout [37], the bioactive molecules involved in the biogenic synthesis process of AgNPs can have different levels of reductant activity depending on the pH value of the extract. In this sense, low pH values favor the protonation of some functional groups [38], hindering the interaction with Ag⁺ ions. In addition, pH has been reported to play an important role in the control of the size and shape of NPs [39] during synthesis. This is attributed to the electrostatic interactions between the particles and the charged functional groups of phytochemical compounds. Figure S3 shows the S/N analysis of the AgNP synthesis conditions, where pH and the AgNO₃ concentration were the most relevant variables impacting the formation of AgNPs (see ANOVA results in Table S1 for supporting information).

3.5. Characterization of AgNPs

3.5.1. UV-Vis Spectroscopy

UV-Vis spectroscopy is a simple and accurate technique used to monitor the formation of metallic NPs and estimate their size and size distribution. The formation of the AgNPs was followed by measuring the surface plasmon resonance (SPR) over 300–700 nm (Figure 3). AgNPs have a single-peak plasmon resonance centered on 434 nm, which is characteristic of this metal [37,38]. Likewise, the AgNP spectra depict the effect of temperature on the synthesis mediated by Nej. Figure 3 illustrates how increasing temperature favors the reduction reaction of Ag⁺ ions, resulting in the formation of more AgNPs in a shorter time [38,39]. In addition, the absorption peak of the particles produced at 80 °C is narrow, denoting lower dispersion in the small size of AgNPs.

3.5.2. XRD

The crystalline nature of AgNPs was confirmed by XRD. Figure 4 shows the XRD pattern where Bragg’s reflections are observed at 2θ values of 38.07, 43.98, 64.72 and 77.59°, corresponding to the crystal planes (111), (200), (220), and (311), respectively. This reflects the face-centered cubic nature of AgNPs (JCPDS No. 040783) [3,40,41,42,43]. In addition, XRD analysis denotes the existence of additional diffraction signals at 2θ values of 27.96, 32.29, 46.32, 54.77, and 57.32°, corresponding to the crystals planes (111), (200), (220), (311) and (222). This reveals silver chloride (AgCl) formation [33,41,42].

The biogenic synthesis of metallic NPs renders surface-modified particles due to the extract’s organic component, employed for the reduction of metal ions [9]. In addition, the presence of chlorides (~0.02 mg/mL) and carbonates (~0.05 mg/mL) in Nej contributes to the formation of silver chloride (K_sp: 1.8 × 10⁻¹⁰) or silver carbonate (K_sp: 8 × 10⁻¹²) [9]. In the same manner, the NPs have a crystallite size of 25.65 nm according to the Debye–Scherer equation.

3.5.3. TEM

TEM and HR-TEM micrographs (Figure 5A,B) show that the AgNPs predominantly exhibit a spherical morphology, with some irregular shapes and varying sizes. The SAED pattern (Figure 5C) reveals bright spots in circular rings, corresponding to the (111), (200), (220), and (311) planes of a face-centered cubic metallic silver structure [42]. Based on the interplanar distances and crystallographic data, the (111), (200), (220), (311), and (222) planes are associated with AgCl formation [44,45,46]. The particle size distribution ranges from 9 to 85 nm, with an average diameter of 20 nm and a standard deviation of 4.31 nm (Figure 5D); most nanoparticles fall within the 10–20 nm range. The presence of chlorides in Nej (~0.023 mg/mL, Table 2) results in the formation of AgCl due to its low solubility, leading to AgCl formation. AgCl formation contributes to enhanced microbicidal activity due to the controlled release of Ag⁺ ions [47,48].

EDS analysis (Figure S4) confirmed the presence of Ag, verifying nanoparticle formation with a concentration of 16.55% in atomic mass, showing the significant presence of this element. Other elements like copper (Cu) and calcium (Ca) were detected. The presence of Cu is due to the copper grid used and the presence of Ca is attributed to the Ca(OH)₂ that is added to the corn in the nixtamalization process. Major elements like oxygen and silicon likely originated from plant extracts according to [49], while boron and magnesium may be associated with impurities or organic residues from Nej. Additional elements were attributed to the natural composition of Nej, as it contains various inorganic compounds and metabolites, which may explain the presence of these elements [9,13].

3.5.4. FTIR

The FTIR analysis identifies the functional groups of the biomolecules that are present in Nej and are responsible for the reduction and stabilization of AgNPs. Figure 6 illustrates the spectral differences between Nej and Nej-functionalized AgNPs. Both spectra exhibit similar patterns, with a vibration band at 3411 cm⁻¹ attributed to OH groups from phenolic compounds, forming hydrogen bonds. The decrease in intensity of this band suggests that phenolic groups interact with AgNPs. C-H stretching is confirmed by the bands at 2921 and 2919 cm⁻¹ [32,50,51]. Bands at 1381 cm⁻¹ and 1417 cm⁻¹ correspond to C=C groups, with increased intensity post-reaction, which likely due to the interaction of C-H with hydroxyl groups [18,32]. Carbonyl (C=O) groups appear in the 1598–1615 cm⁻¹ range [41] in accordance with [24], who reported C=O and C=C bands at 1649 cm⁻¹ and 1328 cm⁻¹ in pure FA. Similar functional groups are observed in Nej samples, with the 1598 cm⁻¹ band linked to FA, as supported by FTIR data on AX [13,51]. Additionally, the band at 1042 cm⁻¹ corresponds to ether (C-O) stretching, while peaks at 824 cm⁻¹ and 558 cm⁻¹ indicate C-H stretching and C-O-C vibrations from bioactive compounds [2,49,52]. The spectral comparison confirms that functional groups are involved in nanoparticle stabilization and surface coating [3,53].

3.5.5. AFM

Figure 7 contains the AFM images of AgNPs synthesized using Nej as a reducing and stabilizing agent. AFM (height sensor) shows the presence of three-dimensional (3D) ordered structures (Figure 7(A1,A2)). They correspond to the organic and/or inorganic matter present in Nej, being responsible for the stability and control size of the particles. Figure 7(B1,B2) show a detailed 3D representation of the stabilizing structures of the Ag NP suspension. In addition, Figure 7(C1,C2) denotes the existence of Ag NPs (brown arrows) with properties (size and shape) compatible with TEM analysis. Moreover, Figure S6 illustrates the AFM analysis of Nej, exhibiting similitudes and differences to the AFM images for AgNPs. The AFM analysis of Nej depicts aggregates; however, their shape and size differ significantly from those of the one present around AgNPs. Also, Figure S5(C1,C2) does not show different phases, indicating the homogeneity of the Nej matter and the absence of AgNPs.

3.6. Evaluation of Antibacterial Activity

The bactericidal activity of AgNPs was tested by assays to determine MIC and MBC. The synthesized AgNPs were evaluated to quantify their antibacterial activity against E. coli and the results were expressed in CFUs/mL. Figure 8 shows the development of the bacterium in agar plates; the decrease in bacterial growth suggests that AgNPs possess antibacterial activity capable of inhibiting the growth of bacterial strains such as (E. coli) [51,52]. Five treatments and two controls were evaluated. The results of the antibacterial activity showed that indeed, the AgNPs synthesized from Nej and via the chemical method showed antibacterial activity against E. coli. In the same way, it is indicated that Nej did not show antibacterial activity, which can be interpreted as AgNPs being solely responsible for antibacterial activity. The results for the antibacterial testing, the MIC, and the MBC of the AgNPs are summarized in

Table 4. MIC and MBC value (μg/mL) corresponding to two synthesis routes of AgNPs.

Bacteria	Synthesis Route	MIC (μg/mL)	MBC (μg/mL)
Escherichia coli	Biogenic synthesis	0.25	0.30
	Chemical synthesis	0.75	1

.

Figure 9 shows that AgNPs synthesized from Nej exhibit higher antibacterial activity than those obtained by chemical synthesis. The AgNPs acted quickly and efficiently against E. coli; the reduction in the number of CFUs/mL was evident, with a high inhibition growth percentage at very low doses (0.5 to 1 μg/mL, chemical synthesis, and 0.15 to 0.3 μg/mL, Nej synthesis) and short exposure times (3 h). The differences in the antibacterial activity of the Nej-mediated synthesis of AgNPs may be due to the adsorption of phenolic compounds and other secondary metabolites onto their surface during the biogenic process [53,54,55]. These compounds increase the stability of the colloid and can act synergistically with silver, enhancing the nanoparticle’s ability to damage the cell walls of bacteria and promote the generation of reactive oxygen species (ROS), which are lethal to microorganisms [55]. Additionally, Nej plays a stabilizing role, facilitating the formation of small and well-dispersed NPs, which have a large active surface area in relation to their volume. This allows for more effective interaction with bacterial cells and, consequently, increases their antibacterial effectiveness [56,57,58].

Earlier studies investigated the antimicrobial activity of AgNPs synthesized from Lysinibacillus sp. culture broths against E. coli (ATCC 25922). These authors report that ionic silver (AgNO₃) is more efficient in inhibiting the growth of this bacterium; however, biogenic AgNPs act at lower doses, with MIC oscillating from 0.53 to 1.13 mg/mL, than streptomycin (16 mg/mL) [59]. The present study reports AgNPs with MICs below those previously reported, highlighting the enhancement of antimicrobial activity due to the bioactive molecules present in Nej.

Figure 9A shows the statistical analysis performed using Tukey’s test, indicating no significant difference considering the amount of CFU/mL between the PBS and Nej control groups. This finding is attributed to the fact that both controls have no noticeable antibacterial effect without AgNPs. In addition, it can be noted that as the concentration of AgNPs increases, a significant decrease in the number of CFU/mL is observed. AgNPs exert efficient antibacterial activity, decreasing bacterial growth at very low doses. This enhanced antibacterial activity results in the use of low doses of AgNPs for the treatment of bacterial infections or disinfection processes. The use of low doses of AgNPs is associated with reduced toxic effects in non-target living organisms [60,61].

3.6.1. Evaluation of the Antibacterial Activity of FA

Figure S6 and Table S2 portray the results of the antibacterial activity of FA against E. coli at different doses. Table S2 shows that the MIC of FA is 625 μg/mL, whereas the MBC is 2500 μg/mL. In addition, the FA concentration in the AgNP colloid was measured by HPLC, finding a concentration of 3.849 ± 0.087 μg/mL FA, and some other bioactive compounds contribute to the antibacterial activity of the biogenic AgNPs under study. HPLC analysis confirms the existence of FA in the colloid; however, its concentration is below the one required to exert antibacterial action; thus, AgNPs are responsible for inhibiting bacterial growth.

Previous studies demonstrated the antibacterial activity of FA and gallic acid against several pathogenic bacteria [62]. The mechanism of antibacterial activity of this compound relies on damaging the bacterial cytoplasmic membrane. In addition, other studies investigate the antioxidant activity of FA [30]. FA acts as a free radical scavenger, as an inhibitor of enzymes that catalyze free radical generation, and as an enhancer of scavenger enzyme activity. FA exerts protective action on the cells exposed to AgNPs, increasing the biocompatibility of the biogenic AgNPs.

3.6.2. Mechanism of Antibacterial Action

AgNPs have demonstrated effective antibacterial activity. However, the exact mechanism by which they inhibit cell growth has not yet been fully elucidated. In addition, the antibacterial activity of AgNPs is highly dependent on the physicochemical properties of the NMs, which are dictated by synthesis conditions. Some researchers propose that the antibacterial action of AgNPs is due to their ability to penetrate and disrupt the outer cell membrane, altering its structure [54], as shown in Figure 10. The adhesion of AgNPs to the cell membrane destabilizes and damages it, increasing its permeability and leading to the loss of cellular contents [63].

Additionally, the release of Ag⁺ ions from AgNPs enables their interaction with enzymes, resulting in their inactivation [64]. This issue can also result in the generation of ROS, which can cause damage to the cell itself. Finally, AgNPs can interact with ribosomes, promoting their disintegration and inhibiting protein synthesis [54]. This process, in turn, leads to deoxyribonucleic acid (DNA) damage, disrupting its functions and structure, and ultimately causing cell death [64,65].

3.7. Hemolysis Assay

The analysis of the hemolytic activity of NMs is used to evaluate their biocompatibility. Figure 11 shows that AgNPs exert an almost-null negative effect on erythrocytes. The AgNPs synthesized from Nej have a slightly higher percentage of hemolysis than those obtained by chemical methods, which could be attributed to their size, and to the presence of Nej metabolites adsorbed onto the AgNP surface, which can modify the physicochemical properties of the outer shell of these particles [66]. Hemolysis values are less than 1%, which is within the non-hemolytic range according to ISO 10993-4 [67], suggesting that the concentrations evaluated are safe for biomedical applications.

The hemolysis process involves the denaturation of erythrocytes due to the physicochemical interaction between the nanoparticles and the cell membrane. The surface charge of AgNPs plays a key role in this interaction. Although AgNPs encounter numerous biomedical applications, hemolytic activity is present at high doses of the NMs [68]. In this study, NMs exert antimicrobial activity at low doses; in addition, their small size favors their uptake by cells or microorganisms. The present study shows that the uptake of AgNPs by bacteria might produce different mechanisms that lead to bacterial cell death; however, in RBCs, the oxidative stress can be circumvented by the molecules present as surface coatings. Further studies will deeply investigate the nano-biointeractions between biogenic AgNPs and RBCs. Finally, this study demonstrates the biocompatibility of AgNPs at doses that exert enhanced antibacterial activity at short exposure times [66,67,68,69,70,71,72].

3.8. Toxicity and Cell Viability Assays of AgNPs

The cytotoxicity of AgNPs in human fibroblasts was assessed using the CCK-8 and LDH assays to evaluate their impact on cell viability. The results from the CCK-8 assay (Figure 12) indicated a progressive decrease in cell viability as the concentration of AgNPs increased, with significant differences observed at concentrations exceeding 0.75 μg/mL for chemical synthesis and 0.30 μg/mL for biogenic synthesis. Notably, there were no significant differences in cell viability at lower doses of AgNPs for either synthesis method. These results support the biocompatibility of the AgNPs under study at doses that efficiently exert antibacterial activity. NMs are considered non-cytotoxic when cell viability exceeds 80% [73]. As illustrated in Figure 12, the bio-viability of Ag-treated fibroblasts is higher than 80% for all the AgNP concentrations under study. Considering the lack of hemolytic and cytotoxic activity, biogenic AgNPs possess suitable properties for biomedical or food processing applications [74].

Concurrently, the LDH assay (Figure 13) revealed similar results to the CCK-8 assay, with viabilities superior to 80% (the LDH release is lower than 15%) for almost all the evaluated biogenic AgNP concentrations. The exception was the highest AgNP concentration, 30 μg/mL, which displayed an LDH release slightly higher than the viability limit for a non-cytotoxic material (23.39% of LDH release). A slight increase in lactate dehydrogenase release is observed for the cells that interact with chemically synthesized AgNPs. This increase could be due to the higher concentrations of the chemically produced AgNPs needed to completely inhibit bacterial growth. Statistical analysis confirmed the existence of significant differences among the highest concentration groups (p ≤ 0.05), supporting a dose-dependent relationship for cytotoxicity. These findings align with previous research, indicating the minimal effects of AgNPs at low doses on various cell types. For instance, ref. [75] investigated the viability of human periodontal ligament fibroblasts, exposed to different doses of AgNPs, after incorporation into dental materials and found no compromise in cell viability. Similarly, ref. [69] reported that higher doses of AgNPs significantly reduced cell viability from 75% to 7% across a concentration range of 6 to 100 μg/mL. Additionally, ref. [33] noted that biogenically synthesized AgNPs adversely affected the growth of MCF-7 cells; in this case, cytotoxic activity is desirable to destroy human breast cancer cells.

There are many advantages to using AgNPs to fight infectious diseases; however, the precise molecular mechanism of AgNP toxicity is still missing (because of its dependence on the physicochemical properties of the nanomaterials). Previous studies show that the massive uptake of AgNPs by eukaryotic cells leads to toxicity or cell death. To avoid AgNP toxicity, some researchers investigated the effect of encapsulating AgNPs in a 3D-biopolymer to limit their diffusion into eukaryotic cells. This strategy does not diminish the antibacterial activity of AgNPs since bacterial cells possess membrane proteins, with thiol groups responsible for the interaction with Ag⁺ ions. On the contrary, eukaryotic cells do not have external thiol groups; thus, AgNPs must ingress into the cell to interact with the thiol groups of intracellular molecules [76].

Nowadays, the problems associated with nanotoxicity research using traditional toxicology approaches are well-known. The optical, redox, catalytic, and adsorptive properties of nanomaterials lead to misinterpretation and hamper the suitability of the assays [77,78]. Advanced microscopy techniques have become a powerful strategy for investigating the interactions of NMs with cells. Figure 14 presents the AFM analysis of fibroblasts (control and treated AgNPs). Figure 14(A1–C2) show the images of control group cells. The micrographs exhibit cells with morphology and size characteristic of the cell line. In addition, Figure 14(D1–F2) display illustrations of AgNP-treated cells. The cells preserve their properties (size and shape). In addition, Figure 14(F1,F2) do not exhibit considerable phase changes, indicating the low diffusion of AgNPs into the cells, avoiding toxicity.

4. Conclusions

This study presents a sustainable, environmentally benign, cost-effective, and rapid biogenic method for the synthesis of silver nanoparticles (AgNPs), employing nejayote—a highly polluting aqueous effluent from the nixtamalization process—as a reducing and stabilizing agent. The findings demonstrate that nejayote is an effective medium for the green synthesis of AgNPs, offering significant advantages over conventional chemical methods. The presence of chloride ions in nejayote facilitates the concurrent formation of silver chloride (AgCl), leading to the generation of Ag/AgCl nanocomposites. These nanostructures may exhibit enhanced antibacterial activity due to the sustained and controlled release of bioactive Ag⁺ ions, a mechanism well-documented in the literature for its effectiveness against a broad spectrum of pathogenic microorganisms. Furthermore, the biogenic AgNPs synthesized in this study exhibited notable antibacterial activity and favorable biocompatibility. The latter fact may be attributed to the surface functionalization with phytochemicals and antioxidant compounds (e.g., ferulic acid), which likely play a dual role as both reducing agents and biocompatibility enhancers.

In summary, this study demonstrates (a) the reproducibility of the synthesis of AgNPs, using Nej as a reducing and stabilizing agent; (b) the enhanced antimicrobial activity due to the presence of bioactive compounds on the surface of AgNPs; (c) the biocompatibility of the AgNPs to ensure their safe application in biomedical, environmental and food processing applications; and (d) the use of Atomic Force Microscopy to investigate the entry and biocompatibility of AgNPs in healthy cells. To continue this research, the nano-biointeractions (bacterial and human cells) of AgNPs will be investigated using Holotomographic Microscopy, ensuring efficient bioactivity without deleterious effects on non-target organs.