Green Synthesis of Ag NPs Using Ustilago maydis as Reducing and Stabilizing Agent

Ustilago maydis (UM) is a fungus that grows naturally on Zea mays ; it reduces the corn yields, and thus, it represents huge economic loss; however, it can be used as an exotic food, and in the present work, it is successfully used as a reducing and stabilizing agent for the preparation of silver nanoparticles (Ag NPs) due to its content of amino acids and biosurfactants. The effects of the concentration of UM aqueous extract, pH, and sunlight on the particle size, surface plasmon resonance, stability, and morphology of Ag NPs obtained by green synthesis were evaluated. A green reduction was observed only in presence of UM, and colloidal Ag NPs were obtained with or without the presence of sunlight; nevertheless, continuous sunlight exposure greatly increased the reaction rate. Ag NPs tend to increase in size from 153nm to 1400nm at a higher pH and a greater amount of UM, and also, UM tends to stabilize the Ag NPs preventing their agglomeration according to measurement of zeta potential ( − 10.75 0.84mV) and SEM observation; furthermore, surface plasmon resonances were more intense between 400 and 480nm of wavelength adding greater amount of UM. This study concludes that UM not only reduces AgNO 3 but also acts as stabilizer of Ag NPs.

eir properties depend on their size, surface structure and shape. Ag NPs can be obtained via photochemical [15] and chemical reduction [16][17][18]. e use of natural sources as reducing agents has been considered a part of green synthesis and has drawn attention because it is considered safe, nontoxic, cost-e ective, and environment friendly. A high diversity of plant extracts, such as Eugenia jambolana, Saraca asoca, Rhynchotechum ellipticum, Malus domestica, no precipitation within a year because of the amino acids, vitamins, and carbohydrates that supply reducing and capping agents [22]. Sunlight and artificial light are photoinductors in the reduction of AgNO 3 as a precursor for Ag NPs, and it is difficult to establish the exact mechanism of how the natural extracts induce the reduction and structural conformation of Ag NPs [23][24][25].
On the other hand, Ustilago maydis (UM) is a smut fungus that belongs to Basidiomycetes; it infects maize (Zea mays) and represents crop loss, except in Mexico, where it is called "huitlacoche," and it is an exotic food with a unique appearance and flavor and a valuable nutritional source [26]. e UM contains amino acids, fatty acids [27], vitamins, phenolic compounds [28], and biosurfactants [29] different from those contained in maize and together have the potential to reduce and stabilize the Ag NPs, as this study suggests. e aim of this work was to obtain and characterize the Ag NPs obtained using U. maydis as a reducing bioagent and to evaluate the effect of its concentration, pH, and sunlight on the Ag NPs formation since there is lack of information in using U. maydis as a stabilizing agent nor the quantification of sunlight effect in the Ag NPs synthesis using a green reductor.

Materials and Reactants.
UM was purchased at a local market in Santa María del Monte, Zinacantepec, Mexico. AR-grade AgNO 3 was purchased from Sigma Aldrich. e pH was adjusted to 0.1 N with NaOH and potentiometer Science Med with a glass electrode. Deionized water was used for the preparation of all solutions for this work (0.1 μS/ cm of electrical conductivity).

Synthesis of Ag
NPs. UM was manually separated from corn kernels and dried at 60°C in a laboratory oven for 24 h until a constant weight was reached; then, it was manually grounded and sieved before use. Extracts were prepared with 0.1, 0.5, or 0.9 g of UM powder mixed with 50 mL of deionized water and stirred at room temperature for 10 min; then, the mixture was filtrated and the pH was adjusted to 9, 10, or 11. e reduction was performed similar to [5]; it was done by mixing 10 mL of 0.01 N AgNO 3 with 10 mL of the UM aqueous extract with or without a natural or artificial source of light. By using a lux meter, it was determined that in the absence of light (inside a dark chamber), the reaction mixture was exposed to 0-20 lux, in contrast to >10000 lux, when exposed to direct midday sunlight. As a control of the reduction reaction, 10 mL of deionized water was added to the AgNO 3 solution instead of the aqueous extract of U. maydis.

Sample Characterization.
To monitor the rate of the reduction step, UV-Visible (UV-Vis) spectroscopy was performed (DR6000 spectrophotometer, HACH, USA) in a 0.2 cm path length quartz cuvette and scanning over a 240-900 nm wavelength range. e quartz cuvette was cleaned constantly with acidified water (0.2% HNO 3 ) and rinsed thoroughly with deionized water to avoid cross-interference between readings of the samples [5]. Dynamic light scattering (DLS) was done by duplicate on a laser particle size analyzer (Litesizer 500, Anton-Paar, Switzerland) with a polyethylene cuvette with 1 cm of path length, a light source of 40 mW and 658 nm in automatic measurement angle, and the 0.159 as refractive index and 4.3 m −1 as the absorption coefficient, assuming that the sample material was reduced silver and water was the solvent with a refractive index of 1.33 [5].
Zeta potential at 25°C was performed on the same particle analyzer by electrophoretic light scattering with an Omega cuvette (polycarbonate cuvette with two gold-coated electrodes located at the ends of a U-shaped capillary tube) using the Smoluchowski approximation [5]. Reaction conditions were adjusted to 0.5 g of U. maydis, at pH 10 and room temperature.

Journal of Nanotechnology
Scanning electron microscopy (SEM) was acquired from dried drops of the samples left on an aluminum disk sample holder that was mounted into the electronic microscope (JSM-6510LV, JEOL, Japan) at a high vacuum and was observed at an acceleration voltage of 5-8 kV with a secondary electron detector (SED). Energy dispersive spectrometry (EDS) analysis was performed with QUAN-TAX-EDS X-FLASH 6L 30 (BRUKER) detector, and elements were quantified with ESPRIT software [5].

Effect of Sunlight.
e sunlight effect on AgNO 3 under conditions of 0.5 g de UM at 10 pH was evaluated, and it is shown in Figures 1 and 2. e first peak of the maximum wavelength was achieved after 50 seconds of sunlight exposure, and the absorbance of the reduced AgNO 3 started to increase; a redshift on surface plasmon resonance was observed relatively proportional to the time of sunlight exposure. e well-defined absorption in the region between 400 and 480 nm is related to Ag NPs formation, the increase in absorbance is related to the number of Ag NPs formed, while the increase in maximum wavelength or redshift is related to an increase in the particle size of the Ag NPs [30][31][32]. e fact that both maximum wavelength and absorption increase is because NPs agglomeration occurs. First, the number of Ag NPs escalates and accumulates in the reaction volume, and the NPs continue to assemble with each other into clusters, as shown in Figure 2 which indicates the displacement increment of the maximum wavelength absorption of the nanoparticles during the synthesis (black curve) and the increase of absorbance intensity (red curve).
Furthermore, the experience in the dark and after 24 hours of reaction was followed. A spectrum revealing a maximum wavelength at 384 nm and 0.159 of absorbance intensity was obtained, which can be related with smaller and less abundant Ag NPs. Also, it was assessed if sunlight is required to start the reaction or if it is also necessary for the reaction to pass chemical equilibrium to reach completion. It was observed that even if the reaction occurs in the dark, changes in number and size are fewer when the reaction is kept in the dark, even with initial sunlight exposure. e vast difference between sunlight and darkness effects gave us the insight that to clearly study the effect of UM, the next reactions had to be carried out either in the absence of light for a long period of time or in sunlight for a short period of time. Hence, it was selected for 100 seconds of initial light exposure and a follow-up of up to 10 min and to 6 days to study the pH effect and the addition of UM extract to the reaction.

Effect of U. maydis and pH.
A control reaction without UM was also studied and no reaction was observed. Aqueous extracts of 0.1 and 0.9 g of UM (0.1 U and 0.9 U, respectively) were used at pH 9 and 11 with 100 seconds of light exposure (Figure 3(a)) followed by 10 min (Figure 3(b)) and 6 days (Figure 3(c)) in the absence of light to study the Ag NPs stability. In general, both amounts of UM and pH increases the number and size of Ag NPs. A 0.9 U extract at pH 11 had the highest absorbance intensity during the initial 100 seconds and the next 600 seconds in the dark. However, after 6 days of being maintained in the dark, the 0.9 U at pH 9 had the highest absorbance intensity around 420-450 nm, and thus, the higher production of dispersed Ag NPs. On the other hand, the UV-Vis spectrum of the reaction of 0.9 U at pH 11, which was the darkest of the four samples, had a wider and less defined band in the region of 600 nm that indicates that Ag NPs were agglomerated. Figure 4, the particle size distributions of Ag NPs are shown to analyze the effect that pH and UM aqueous extracts had on the colloidal solutions prepared after 100 seconds of sunlight exposure. e NPs obtained at pH 11 tend to increase in size, as was observed by UV-Vis spectroscopy. With extracts of 0.1 U, the difference of particle size greatly differs from pH 9 to 11 than when 0.9 U extracts are used at both pHs. Interestingly, in the particles obtained with the 0.1 U extract at pH 11, two distributions were found, a larger one with 1232 ± 389 nm average diameter and another of 121 ± 30 nm average diameter. No aggregation was observed at pH 9 with either 0.1 U or 0.9 U extracts, in whose peaks, the average diameter obtained for the nanoparticle populations was 153 ± 51 nm and 170 ± 67 nm, respectively. For 0.9 U at pH 11, the mean particle size increased to 256 ± 102 nm. e results are of interest because the increase of UM in the reaction had little impact on the increase of particle size, and it diminished the agglomeration of the Ag NPs obtained. DLS measurements are supported by SEM microscopy observations, as shown in Figure 5; however, comparison between techniques must be done carefully as the particle size of the Ag NPs determined DLS refers to a hydrodynamic diameter, while SEM observes a particle size in the dry state, and thus, the sizes obtained by these techniques have different interpretations. In previous SEM observations, the Ag NPs were observed as bright dots with quasi-spherical morphology and an average diameter of approximately 145 ± 47 nm for 0.1 U at pH 9, 225 ± 45 nm for 0.9 U at pH 9, 4183 ± 1263 nm for 0.1 U at pH 11, and 178 ± 78 nm for 0.9 U pH 11 (Table 1). We select some patters observed on SEM to make EDS analysis to confirm that at higher pH, Ag NPs tend to increase in size and quantity and that C atoms were found in higher presence with UM increase; for 0.1 U at pH 11, Ag NPs tend to agglomerate, and for 0.9 U at pH 11, Ag NPs tend to present as encapsulated by a biological structure that we can infer is a combination of different structures presented in UM. As can be seen, particle size determined by SEM for sample 0.1 U at pH 11 was higher than expected; it can probably be related to the sample treatment prior to the micrography that consists of sample drying at 50°C, which can induce agglomeration of the particles. In the micrography, this is observed as a bright cloud that tends to agglomerate.

Particle Size and Stability. In
In Figure 6, the zeta potential of the four conditions is shown as a box graph with the respective standard deviation. All samples presented a negative zeta potential with a significant difference (p < 0.05) for 0.9 U at pH 9 that showed the lowest negative value of −10.75 ± 0.84 mV, which is an indication of the conditions for the most stable nanoparticles obtained, which points to the fact that the increase of UM extract can confer stability to the nanoparticles, and it does not only act as a reducing agent like other green reducing agents that can be extracted from other natural sources.
Further studies must be done to identify which conditions of extraction of UM can modify its reducing and stabilizing effects on the preparation of Ag NPs via green synthesis, as well as to assess which components of the UM are responsible into confer stability.
It must be noted that the procedure presented in this work is based on Ag NPs prepared with an aqueous extract that may contain colloidal suspended solids of UM that may provide a surface interface for the cation reduction, and thus, these may work as reactive sites for stable nanoparticle growth. Also, sunlight was used for some time in the reaction, and it is composed of a wide range of wavelengths and multiple direction components that may interact differently during the reducing step of the synthesis. A direct control of the light characteristics (wavelength, incident angle, intensity, and polarization) and time exposure may result in nanoscalecontrolled particle growth and accurate assembly of the nanoparticles, as other studies have shown [33].
ough different maize varieties have significant differences on UM characteristics, it is considered to have good nutritional features as it contains essential amino acids (including lysine and tryptophan) [34], fair amount of unsaturated fatty acids, glycerol, minerals, monosaccharides and polysaccharides (including glucose and fructose as the most abundant, both reducing sugars), vitamins (including vitamin C, a potent antioxidant), and abundant phenolic compound, flavonoid compounds, and chlorophylls [26,27].
us, regarding the reduction reaction, it may be impossible to pinpoint a single specific compound that interacts with AgNO 3 and carries out the reduction to produce Ag NPs. Research studies exploring other reducing agents propose several mechanisms at alkaline conditions that can explain it, as Ag + cations can interact with hydroxide ions to form silver oxide particles that may allow for catalytic reduction of Ag + on their surface [35,36]. Also, it has been demonstrated that Ag + cations can be reduced by aldehydes [37][38][39] and alkoxides [40,41] which can be formed in organic matter containing alcohols, ketones, and aldehydes in the presence of dissolved oxygen. Even at neutral pH, it has been proposed that conversion of hydroxyls groups into aldehydes and later to carboxylic acids may be a reduction mechanism for Ag + [42]. Moreover, dehydration of UM may have initially produced esterification of various components that were reversibly reconverted to monosaccharides, alcohols, and fatty acids in the presence of water and then again oxidized by Ag + . e stabilization of nanoparticles prevents their agglomeration and sedimentation; therefore, it allows to adjust particle sizes and shapes; for this purpose, surfactants are typically added to the reaction. It has been identified that under low nitrogen concentrations, UM produces large amounts of ustiliagic acid and ustilipid acid, two biosurfactants derived from glycolipids [29]. Furthermore, lignin, a complex polymer present in plant support tissues including corn and likely also present in UM, has been used successfully as a reducing and capping agent in Ag PNs synthesis [36] and may be part of the active compounds in UM extracts. is is relevant since not only reducing agents are of interest but also amphipathic biochemical molecules with surface active properties have several advantages in green synthesis, compared to synthetic surfactants, because the former are biodegradable and have low toxicity, and they work effectively with controlled activity at specific pH and temperatures.

Conclusions
Aqueous extracts of Ustilago maydis, natural smut of Zea mays, induce the reduction of AgNO 3 to obtain Ag NPs within sizes between 100 and 5000 nm, depending on the  Figure 6: Zeta potential of Ag NPs obtained under 0.1 g or 0.9 g of U. maydis at pH 9 and pH 11 after 100 s of sunlight exposure at room temperature. reaction conditions. e main factors that affect the size and stability of Ag NPs are the presence of sunlight, pH, and quantity of UM aqueous extract which not only acts as a reducing agent but also confers stability to the metallic nanostructures. e analysis of this green synthesis method confirms that components in UM are effective in reducing silver nitrate and in stabilizing Ag NPs. Further studies are necessary to know which components of UM act as reducer and stabilizer.

Data Availability
e data used to support this study are included within the article.
Disclosure e authors are part of SNI-Conacyt.

Conflicts of Interest
e authors declare that they have no conflicts of interest.