Eco-friendly high-rate formation of silver nanoparticles in agave inulin and its bactericidal effect against Escherichia coli

Four samples (S1A, S2A, S3A, S4A) with different agave inulin concentrations and the same AgNO₃ concentration, the same temperature, and the same pH were prepared. Table 1 shows the synthesis parameters and the corresponding values used. These samples were analyzed by UV-Vis spectroscopy 23 days after the preparation.

Three samples (S1B, S2B, S3B) with different AgNO₃ concentrations and the same agave inulin concentration, value of temperature, and pH were prepared. Table 2 shows the parameters and their corresponding values used in the synthesis. The samples were analyzed by UV-Vis spectroscopy 22 days after the preparation.

Four samples (S1C, S2C, S3C, S4C) with different temperature values and the same agave inulin concentration, AgNO₃ concentration, and pH were synthesized. Table 3 shows the parameters and their corresponding values used for the synthesis. The samples were analyzed by UV-Vis spectroscopy 11 days after the preparation. The sample obtained with a temperature of 75°C was analyzed by TEM.

Five samples (S1D, S2D, S3D, S4D, S5D) with different pH values and the same agave inulin concentration, AgNO₃ concentration, and temperature were prepared. Table 4 shows the parameters and the corresponding values used in the synthesis. The samples were analyzed by UV-Vis spectroscopy 40 min after the preparation. The samples S3D (pH = 10) and S2D (pH = 11) were characterized by TEM.

The bactericidal effect of silver nanoparticles was determined using a strain of E. coli. This bacterium was extracted from Ameca’s river (located in Jalisco, Mexico), and a bacterial culture was characterized in a chromogenic agar (CHRO-MOgar) and by PCR to confirm its presence by malB gene amplification. Subsequently, the bacterium was exposed to two different nanoparticle concentrations labeled S1_bac and S2_bac. The first sample of nanoparticles (S1_bac) was synthesized using a 10 mM of AgNO₃ in solution with agave inulin at a concentration of 60 mg/mL. The second sample (S2_bac) was synthesized using 1 mM of AgNO₃ in solution with agave inulin at a concentration of 20 g/mL. Both samples were prepared with a pH of 12.

To determine the antibacterial activity of the nanoparticles, the micro dilution method was employed [26]. Table 5 shows the content of each of the test tubes. The six tubes each contained 5 mL of Luria Bertani (LB) broth. Tubes 2–6 contained 100 μL of the bacterial culture at a concentration of 10⁸ CFU/mL. In tube 3, 100 μL of agave inulin was added to reach a concentration of 60 mg/mL, and in tube 6 100 μL of agave inulin was added to the broth to reach a concentration of 20 mg/mL to verify the absence of an antibacterial effect by agave inulin. Finally, instead of agave inulin solution, tubes 4 and 5 received 100 μL of silver nanoparticles corresponding to the samples S1_bac and S2_bac, respectively. The tubes were incubated at 37°C for 18–24 h, and the antibacterial effect was detected by the unaided eye.

Figure 2 presents UV-Vis absorption spectra of the samples prepared with different agave inulin concentration. Details of the mixing proportions are shown in Table 1 (in the experimental section), 23 days after the preparation. The samples S2A, S3A, and S4A exhibited an absorption peak at 442, 440, and 439 nm, respectively, and this indicates that quasi-spherical AgNPs were formed [27]. Nevertheless, for the sample S1A, with less agave inulin, the absorption peak did not appear, suggesting insufficient or null nanoparticle formation. This occurred even though S1A had the same molar concentration of AgNO₃ as the rest of these samples; therefore, we can say that the concentration of agave inulin molecules plays a key role in nanoparticle formation.

Fig. 2.

The hydrolysis of agave inulin is relatively stable at room temperature and neutral pH, so upon the addition of water, the inulin chain undergoes cleavage of the glycosidic bonds and finally breaks down into its monosaccharides [14]. This leads to hydroxyl ion formation, favoring the reduction of Ag⁺ ions [28]. The natural polysaccharide has been considered to contribute in the reduction and stabilization of AgNPs [29].

In addition, as can be seen in Figure 2, there are some differences in the spectra. The nanoparticles obtained using a higher concentration of agave inulin influenced the amplification of the absorption peak. In agreement with the Lambert-Beer relation, Equation (1), this can be associated with an increase in nanoparticle concentration. Furthermore, the absorption peak shifts slightly toward shorter wavelengths, suggesting a slight decrease in nanoparticle size.

Figure 3 shows the UV-Vis absorption spectra of the samples prepared with different AgNO₃ concentrations. Details of the mixing proportions are shown in Table 2 (in the experimental section), after measuring the absorbance 22 days after the preparation. Similarly, AgNO₃ concentrations had an effect on the intensification and in shifting the absorption peak to shorter wavelengths. This can be explained by the effect of the concentration and by a decrease in the nanoparticle size [9].

Fig. 3.

Figure 4 presents the UV-Vis absorption spectra corresponding to the samples obtained with different temperatures. Details of the mixing proportions are shown in Table 3 (in the experimental section) measuring the absorbance 11 days after the preparation. Samples S1C and S2C show less absorption around 400 nm; hence, there is little formation of AgNPs.

Fig. 4.

By contrast, sample S3C, obtained at 60°C, shows a more intense absorption peak. However, at 75°C, the sample S4C presents an absorption peak that is still more intense. Its absorbance value is the highest in comparison with the results presented in Figures 2 and 3. Hence, temperature influenced the acceleration of the nanoparticle formation.

In addition, Figure 5 presents the corresponding size distribution fitted with the log-normal model and micrographs of nanoparticles obtained with a temperature of 75^°C (sample S4C). In this analysis, 354 particles were considered, where most of the nanoparticles exhibit a relatively small size of <10 nm with an average size of 7.99 nm. In agreement with the micrographs, the nanoparticles presented a quasi-spherical shape. This result reinforces the interpretation inferred from the profile of the absorption on the basis of the nanoparticle shape.

Fig. 5.

Figure 6 presents UV-Vis absorption spectra of the samples obtained with different pHs; details of the mixing proportions are shown in Table 4 (in the experimental section). The absorption peak became more intense as pH increased. In fact, within the first forty minutes, the sample with a pH of 11 showed an absorbance of 1.7, whereas the sample with the highest pH had an absorption peak so intense that it exceeded the measurable range and its center could not be observed. In comparation with the other parameters used here, this indicates that a pH of 12 has more of an effect on the acceleration of nanoparticle formation. This result also correlates with the sample’s dark color and the resulting difficulty in measuring light transmission.

Fig. 6.

Regarding the non-definition of this absorption peak, it was monitored while diluting the sample with distilled water with the following dilutions of distilled water to silver nanoparticles: 1:1, 2:1, 3:1, 4:1 and 5:1, where the first number is milliliters of distilled water diluting 1 milliliter of silver nanoparticles solution (the second number) in every case. Figure 7(a) shows the UV-Vis spectra of this monitoring. The absorbance decreases with the dilution with distilled water, showing that, despite the impossibility of observing its absorption peak, it is possible to recover the profile of the absorption peak generated by the diluted silver nanoparticles and thus identifying the center of its absorbance maximum, which is 402 nm. In this way, it can be observed that the absorption peak shifted towards a shorter wavelength (from 432 to 402) by means of increasing the pH (see Figures 6 and 7a), suggesting that the nanoparticle size decreases with the pH.

Fig. 7.

To understand the scope of the maximum absorbance of the original sample, absorbance monitoring allowed us to generate a calibration line, as seen in Figure 7(b). In this linear regression plot, an absorbance of 11.9 was calculated. Furthermore, since the sample was diluted into 3:1, 4:1, and 5:1, the profile of its absorption peak is similar. On the other hand, the linear regression predicts with an acceptable precision the absorbance monitoring by UV-Vis spectroscopy.

In addition, Figure 8 presents a size distribution of the sample obtained using a pH of 11 (Sample S2D), there being 282 nanoparticles considered in the statistic, resulting in an average size of 6.04 nm. On the other hand, Figure 9 presents the size distribution of the sample obtained using a pH of 10 (Sample S3D), with 311 nanoparticles considered, resulting in an average size of 8.03 nm. The size distribution of nanoparticles obtained with a pH of 11 is narrower and presents the smallest nanoparticles. This result is similar to the work in which the authors published the effect of pH on the synthesis of AgNPs using citric and malic acids and reporting that the average size of the quasi-spherical nanoparticles decreases in a pH of 10 to 11 [30].

Fig. 8.

Fig. 9.

NaOH plays an important role in the intensification of the rate of nanoparticle formation. NaOH is a strong base that dissociates completely in solution to form hydroxyl ions [31]. Then, a large amount of these ions in an aqueous solution of inulin leads to an increment in its pH, which directly influences a notable increase in the speed of breaking the glycosidic bonds that join the fructose units that make up inulin [14]. Subsequently, as there is a greater number of fructose units, in consequence, there is a greater number of reducing molecules that immediately tend to give electrons to the Ag⁺ ions being reduced to Ag⁰ and consequently there is a rapid formation of silver nanoparticles.

A second important factor is the temperature. Nanoparticle preparation can be simpler without adjusting the pH and its temperature, and only using ingredients such as agave inulin, AgNO₃, and distilled water. Although it is very important to consider the effect of storage time, because a sample used immediately will employ a smaller concentration than expected.

It has been reported that bactericidal properties depend on nanoparticle size; for instance, if the silver nanoparticles have a diameter of ≈1–10 nm, they present a direct interaction with the bacteria [32]. In addition, the concentration of this type of nanoparticle is another characteristic that affects bacterial growth [33, 34].

In this study, two samples, S1_bac and S2_bac, were prepared with a molar concentration of 10 mM and 1 mM of AgNO₃ in dissolution with 60 mg/mL and 20 mg/mL of agave inulin, respectively. Each one was fixed to a pH of 12 and were prepared at the same temperature (see Section 2.5 for details of the preparation). Considering that the sample S1_bac showed an antibacterial effect, consequently, the next theoretical consideration was made to determine a nanoparticle concentration (nanoparticles/mL).

For this, it is necessary to know the average size of the nanoparticle, which can be estimated from analysis of the micrographs taken by TEM. Figure 10 presents the size distribution and three micrographs of the sample S1_bac. Several nanoparticles smaller than 10 nm can be observed. If the average size of D_p = 8.71 nm and assuming that the nanoparticle has a spherical shape, its mass can be written as mp=π6DP3ρAg,$${{\rm{m}}_{\rm{p}}} = {\pi \over 6}{\rm{D}}_P^3{\rho _{Ag}},$$ where ρ_Ag represents the silver density (10.49 g·cm^-3). Substituting the density and the particle diameter, m_p = 3.6×10^-15mg is obtained.

Fig. 10.

The concentration of silver in the sample is given by C_Ag = MW_AgC_AgNO3, where MW_Ag is the atomic weight of silver (107.8682 g·mol^-1) and C_AgNO3 is the concentration of AgNO₃ (10 mM). Substituting as seen above, C_Ag = 1.07 mg·mL^-1 is obtained. Furthermore, dividing this concentration by the average mass, the nanoparticle concentration of ≈ 2.9×10¹⁴ nanoparticles/mL is obtained.

With this size distribution (Fig. 10) in comparison with the others, the procedure for obtaining the number of nanoparticles is more precise, because the nanoparticles present a narrower size distribution.

Figure 11 presents test tubes with dilutions to study the bactericidal effect according with the order shown in Table 5 (in the experimental section). Tube 1, containing only LB, represents the sterility group or the positive control, and tubes 2, 3, 5, and 6 show the bacterial growth. A solution of 100 μL of agave inulin was added to tubes 3, and 6 to verify the absence of the antibacterial effect by agave inulin. In addition, 100 μL of S1_bac and S2_bac were added to tubes 4 and 5, respectively. Tube 4 showed a clear bactericidal effect on the E. coli bacterium because there was not observed turbidity. This indicated that the sample S1_bac caused the damage to the bacterium where this sample was prepared with the highest concentration of AgNO₃; hence, the concentration of silver nanoparticles had an effect against the E. coli. In consequence, sample S1_bac is going to be used for further analysis against the E. coli bacterium.

Fig. 11.

Although the bactericidal mechanisms of silver ions are not well understood, it is inferred that they include binding and interaction mechanisms of silver nanoparticles with the cell wall and membrane [35]. It has been shown that there is electrostatic attraction between the negatively charged cell membrane of the bacteria and the positively charged silver nanoparticles, so the bactericidal activity is directly related to the positive charge of the silver ions [36, 37].

The bactericidal activity of AgNPs has been reported on Gram-negative bacteria such as E. coli. In this case, the concentration of silver nanoparticles is related to the formation of “holes” in the bacterial cell wall, and the accumulation of AgNPs in the cell membrane is related to the increase in the permeability of the bacterium, which triggers its death [38]. Another mechanism suggests that silver depletion could trigger the progressive release of lipopolysaccharides and cell membrane proteins, leading to the formation of “holes” [39]. It has also been proposed that silver ions released from silver nanoparticles can inactivate DNA replication and cause inhibition of enzyme functions as a result of their interaction with phosphorus moieties in DNA [40].