Antimicrobial bentonite by the addition of geranyl acetate for aflatoxin B1 adsorption

A nanocomposite composed of organophilic bentonite (OB) with geranyl acetate (GA) was prepared on a shaker for 1 hours, 180 rpm and room temperature, being subsequently dried an oven at 90 oC±2 oC during 48 hours. This material was then characterized for its antibacterial against Staphylococcus aureus and Escherichia coli and antifungal activity against Aspergillus flavus and Aspergillus niger, respectively. Moreover, the adsorption capacity of OB and OB/GA was evaluated using Aflatoxin B1 in the concentration of 107 μg∙L. After 17 h, the composite removed a significant amount of mycotoxin, being below 20 μg∙L. The results showed that this composite has a good adsorption capacity, can be effective in the removal of mycotoxin in aqueous media and excellent antibacterial and antifungal activity.


Introduction
Animal nutrition usually includes a mix of foods that are listed in order to meet the nutritional needs of animals, also to provide what they need to maintain their health, well-being and production, and all this linked to the lowest possible cost (Pereira et al., 2019;Liu, et al., 2021). About 80% of the raw materials used to manufacture feed revolve around the use of corn, soybeans and their derivatives. However, due to climate change and other prevailing factors, many of these crops are subject to contamination by mycotoxins, among which stand out aflatoxins, produced by fungi of the genus Aspergillus that present an imminent risk to human and animal health and are related to several diseases and pathologies, in addition to causing great economic damage to the food industry (Zain, 2011;Nones et al., 2014;Raiola, et al., 2015;Oplatowska-Stachowiaket, al., 2016;Li, et al., 2018).
Several studies have shown the ability of adsorption of mycotoxins promoted by bentonites, recognized as a promising and effective food additive due to its cost-effectiveness and absence of significant side effects (Magnoli, et al., 2008;Carraro, et al., 2014;Gan, et al., 2019;Liu, et al., 2021). In order to maintain or enhance the adsorptive effect and still add other properties to bentonite, the insertion of organic compounds can be explored, which is still little explored, opening the way for the study of geranyl acetate, an important ester widely used in industries food, pharmaceutical and cosmetics (Gonçalves, et al., 2012;Gupta, et al., 2013;Zeferino, et al., 2021;Liu, et al., 2021).
The objective of this work was to obtain a composite with antimicrobial and adsorbent activity, using organophilized bentonite and geranil acetate. By associating the adsorption properties of bentonite with the antifungal and antibacterial activity of geranyl acetate it is possible to obtain a compound capable of eliminating or controlling the growth of bacteria and fungi and, simultaneously, adsorb mycotoxins, which is not yet available on the market according to a bibliographic survey.

Obtaining the composite
The reaction mixtures were stirred vigorously at constant temperature for 1 hour. Synthesis conditions were performed, considering the temperature of 60±5 ºC and concentration of surfactant the 85 mmol•100 g -1 . After intercalation, all ODA-bentonite samples were washed, and separated by filtration, all samples were dried in an oven at 80 ºC for 24 h. The organophilization step was based on the methodology suggested by according to Macuvele, et al. (2017). After organophilization, a composite was prepared in the following proportions: 2.5 g organophilic bentonite: 40 mL acetone: 0.75 g geranyl acetate (Bent/ODA/0.75 GA). The mixture was kept under agitation for 24 h using a shaker at 180 rpm, at room temperature. After this mixing period, the samples were dried in an oven at 50±2 ºC for 24 h.

Antimicrobial analysis for bacteria
The antimicrobial activity was evaluated for gram-positive bacteria Staphylococcus aureus (ATCC 6538) and gramnegative bacteria Escherichia coli (ATCC 35218), according to methodologies previously described by Clinical and Laboratory Standards Institute (CLSI, 2012). For inoculum preparation, some bacterial colonies were selected and transferred to a sterile 0.9% saline. The solution turbidity was adjusted using a Spectrophotometer with a wavelength reading of 619 nm, yielding the equivalent concentration of about 10 4 UFC•mL -1 .
In this technique, we seeded the microorganism of interest in Petri dishes with a culture medium Plate Count Agar (PCA) using a swab. Three equidistant holes were made in each plate, with 8.0 mm diameter approximately. In each hole was deposited a composite and incubated at 36±1°C for 24 h. This antimicrobial analysis was performed in triplicate and the diameters of the inhibition halos were measured and associated with the antimicrobial actions according Eq. 1, in methodology proposed by Fiori, et al. (2009), the inhibition halos diameter measurements were subjected to the Tukey test in order to assess the significant differences existence at the 5% significance level (p <0.05).

Dbac = Dex -Din
(1) where Dbac is the sum of the bactericide diameters and represents the bactericide action of the materials, Dex is the inhibition halo diameter of the microorganism and the Din is the diameter occupied by the composite.

Antimicrobial analysis for fungi
The For the construction of the calibration curve, standard solutions of AFB1 were prepared in ultrapure water at concentrations of 0.02, 0.10, 0.20, 1.00 and 2.00 mg•L -1 , and the calibration curve for AFB1 was determined based on the concentrations versus the peak area. Analyzes were performed using an Agilent chromatograph coupled to a mass detector. A 3.0x100 mm 2.7 Poroshell Sb C18 column (Agilent, PN 685975-302) was used. The mobile phase used was composed of methanol:ultrapure water with 0.1% formic acid (50:50), which was pumped without a ramp in isocratic mode, with a flow of 0.850 mL•min -1 , with an oven temperature 40 °C.

Antibacterial activity
To evaluate the organo-bentonites and Bent/ODA/0.75 g GA, diffusion tests were carried out in solid medium. Figure   1 show tests performed images with the bacteria Staphylococcus aureus and Escherichia coli, respectively. The inhibition halos diameter values are shown in Table 1.
It is possible to observe through the Figures above that for the organophilized bentonite there was no formation of an inhibition halo for both tested bacteria, which once again reinforces that the bentonite does not present antimicrobial activity by itself and that the organophilization process does not changed this feature. After the insertion of geranil acetate to the

Antifungal activity
After organophilization and insertion of geranil acetate, the composite was tested for its antifungal activity. Figure 2 shows the antifungal activity of organophilized bentonite and Bent/ODA/0.75 g of GA against fungi producing Aflatoxin B1, Aspergillus flavus and Aspergillus niger at a concentration of 10 5 spores•mL -1 .  Figure 2 shows the results obtained for the antifungal analysis for organophilized bentonite and Bent/ODA/0.75 g of AG, where once again it was found that organophilized bentonite does not have the ability to inhibit the growth or kill microorganisms in test, fungal growth when evaluated this material occurred abundantly, receiving the maximum rating on the growth scale, completely filling the surface of the plate. After the insertion of the antimicrobial agent geranyl acetate, there was a marked decrease in cell density. When the composite Bent/ODA/0.75 g of AG was evaluated against Aspergillus flavus it was noticed the presence of growth traces and for Aspergillus niger no growth was observed. The classification of each of the tested composites can be seen in Table 2.

Adsorption Experiments
Through the analyzes carried out in LCMS it was verified that the initial concentration of Aflatoxin B1 was 107 µg•L -1 and that after 17 hours of contact with organo-bentonites and Bent/ODA/0.75 g of GA this concentration was below the limit of quantification of the equipment, which is 20 µg•L -1 , thus ensuring that the final concentration of Aflatoxin B1 is below 50 µg•Kg -1 , which is what the Brazilian feed legislation provides (Brasil, 1988).

Conclusion
The results obtained indicate that composite based bentonite and geranyl acetate is antibacterial, antifungal and Aflatoxin B1 adsorbent, which can be used concomitantly in the elimination of fungi that produce Aflatoxins, as well as adsorbing them if they are present. This result is very interesting, as it allows the use of this material for applications in animal feed. Future works suggest the study of in vivo toxicity, release of geranyl acetate present in bentonite and incorporation of the material developed in the feed.