**Copper-Chitosan Nanocomposite Hydrogels Against Aflatoxigenic** *Aspergillus flavus* **from Dairy Cattle Feed**

**Kamel A. Abd-Elsalam 1,\* , Mousa A. Alghuthaymi <sup>2</sup> , Ashwag Shami 3,\* , Margarita S. Rubina <sup>4</sup> , Sergey S. Abramchuk <sup>4</sup> , Eleonora V. Shtykova <sup>5</sup> and Alexander Yu. Vasil'kov <sup>4</sup>**


Received: 29 June 2020; Accepted: 16 July 2020; Published: 21 July 2020

**Abstract:** The integration of copper nanoparticles as antifungal agents in polymeric matrices to produce copper polymer nanocomposites has shown excellent results in preventing the growth of a wide variety of toxigenic fungi. Copper-chitosan nanocomposite-based chitosan hydrogels (Cu-Chit/NCs hydrogel) were prepared using a metal vapor synthesis (MVS) and the resulting samples were described by transmission electron microscopy (TEM), X-ray fluorescence analysis (XRF), and small-angle X-ray scattering (SAXS). Aflatoxin-producing medium and VICAM aflatoxins tests were applied to evaluate their ability to produce aflatoxins through various strains of *Aspergillus flavus* associated with peanut meal and cotton seeds. Aflatoxin production capacity in four fungal media outlets revealed that 13 tested isolates were capable of producing both aflatoxin B1 and B2. Only 2 *A. flavus* isolates (Af11 and Af 20) fluoresced under UV light in the *A. flavus* and *parasiticus* Agar (AFPA) medium. PCR was completed using two specific primers targeting aflP and *aflA* genes involved in the synthetic track of aflatoxin. Nevertheless, the existence of *aflP* and *aflA* genes indicated some correlation with the development of aflatoxin. A unique DNA fragment of the expected 236 bp and 412 bp bands for *aflP* and *aflA* genes in *A. flavus* isolates, although non-PCR fragments have been observed in many other Aspergillus species. This study shows the antifungal activity of Cu-Chit/NCs hydrogels against aflatoxigenic strains of *A. flavus*. Our results reveal that the antifungal activity of nanocomposites in vitro can be effective depending on the type of fungal strain and nanocomposite concentration. SDS-PAGE and native proteins explain the apparent response of cellular proteins in the presence of Cu-Chit/NCs hydrogels. *A. flavus* treated with a high concentration of Cu-Chit/NCs hydrogels that can decrease or produce certain types of proteins. Cu-Chit/NCs hydrogel decreases the effect of G6DP isozyme while not affecting the activity of peroxidase isozymes in tested isolates. Additionally, microscopic measurements of scanning electron microscopy (SEM) showed damage to the fungal cell membranes. Cu-Chit/NC<sup>S</sup> hydrogel is an innovative nano-biopesticide produced by MVS is employed in food and feed to induce plant defense against toxigenic fungi.

**Keywords:** aflatoxins; *Aspergillus* section *Flavi*; chitosan; feeds; nanocomposites

#### **1. Introduction**

The contamination of agricultural and dairy products with aflatoxins is a major problem for economic and public health. Aflatoxins (AFs) are fungal subsidiary products mainly developed by *Aspergillus flavus* and *Aspergillus parasiticus* strains on cereals, nuts, dried fruits, dairy, and animal feed under warm and humid conditions [1,2]. The significant source of AFs spoilage is *A. flavus*, especially aflatoxin B1, which has received a lot of attention in the food and feed industry [3]. High concentrations of aflatoxin could even prompt the disease of aflatoxicosis, an infection that affects serious disease and can lead to cancer in severe cases [1,4,5]. Additionally, chronic absorption of aflatoxins causes various adverse effects, such as increased susceptibility to various pathogens, loss of production, and a decrease in milk production yield and quality in dairy cattle [6]. The nanotechnology approach seems to be an encouraging, effective, and affordable way to reduce the health problems of mycotoxins in humans and animals. There are three different approaches to reduce mycotoxin risks: effects on mold and flour retention, mycotoxin, and minimization of toxic effects by various nanomaterials [7–9]. Chitosan and self-assembled benzoic acid polymers were synthesized, and it was found that the encapsulation of CS-BA nanogels significantly enhanced the half-life and antifungal activity properties of thyme oil against *A. flavus* strains [10]. The antifungal efficacy of mycogenic silver nanoparticles hybridizing with simvastatin against three species of the *Aspergillus Flavi* group was measured. Some nano-formulations regulated the development of the toxigenic *Aspergillus* species [11]. Plant-mediated CuO NPs were synthesized from *Cissus quadrangularis* and applied as antifungal agents against *A. niger* and *A. flavus*. The produced nano-copper showed a better performance than the carbendazim fungicide [12]. In addition, hybrid nanocomposites based on organic polymeric and inorganic matrices as effective anti-aflatoxigenic strains were explored [3,4,13,14]. Chitosan-based nanocomposite film vapor assays were applied to hybrids between thyme-organo, thyme-tea tree, and thyme-peppermint EO mixtures and demonstrated strong antifungal action against some toxic fungi, including *A. flavus, A. parasiticus,* and *P. chrysogenum*, limiting their production ranged from 51 to 77% [15]. There is a direct association between the concentration of aflatoxin M1 (AFM1) in milk and aflatoxin B1 (AFB1) in dairy cattle feed which results in AFM1 being found in the milk of animals on contaminated feeds with AFB1 [16]. To our understanding, antifungal action of copper-chitosan nanocomposite-based chitosan hydrogels (Cu-Chit/NC<sup>S</sup> hydrogels) against *A. flavus* strains from animal feed samples is not previously studied. Present study aimed to: (1) recognize *alfP* and *aflA* as two essential genes that lead to development of aflatoxin in animal feed via *Aspergillus* genus. (2) determination of AFB1 and AFB2 frequency and distribution of *A. flavus* strains in relation to feed delivered to dairy cows in small farms. (3) copper-chitosan nanocomposite was produced utilizing metal vapor synthesis (MVS), the physicochemical characteristics of the nanocomposites formed were described by electron microscopy (TEM) transmission, X-ray fluorescence analysis (XRF), and X-ray scattering (SAXS) small angle. (4) The fungicidal effect of the hydrogel Cu-Chit/NCs were screened against three *A. flavus* strains. (5) Protein, isozymes, and DNA fragmentations were investigated using two electrophoresis techniques, finally, scanning electron microscope was used to assess morphological changes in NCs-treated fungi.

### **2. Materials and Methods**
