Effect of Zinc Oxide Nanoparticles on the Growth of Mycotoxigenic Mould

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Effect of Zinc Oxide Nanoparticles on the Growth of Mycotoxigenic Mould A.A. Hassan1, M.E. Howayda1, H.H. Mahmoud2 Department of Mycology and Mycotoxins, Animal Health Research Institute (AHRI), Dokki, Giza, Egypt Central Laboratory of Elemental and Isotopic Analysis, Nuclear Research Centre, Atomic Energy Authority, Egypt

1 2

atefhassan2000@yahoo.com; 1howaydaelshafie@yahoo.com; 2hazem_h_mansour@yahoo.com

1

Abstract The nanoparticles of ZnO (ZnO-NPs) were prepared by wet reaction of zinc chloride (ZnCl2) and sodium hydroxide (NaOH) solutions. The prepared ZnO-NPs were identified and characterized by Scanning Electron Microscope (SEM), with Energy Dispersive X-Ray Spectrometer (EDX), Fourier Transmitter Infra Red (FT-IR) and UV-visible spectrometry.

From cases of dairy cattle that suffered from symptoms of toxicities as vomition, diarrhea, and loss of weight and decreased milk production, samples of milk and feed remnants were collected and tested for mycotoxigenic fungi and their toxins. The commonly isolated fungi were identified as A. flavus, A. ochraceus and A. niger.

Significant levels of aflatoxins, ochratoxin A and fumonisin B1 were detected in raw milk samples (5.9±0.98 ppb of AFM1; 6.5±0.35 ppb of OA and 0.0 level of FB1 ) and cattle feeds (21±1.5 and 30±3.1 ppb of AFB1; 18.5±0.55 ppb of OA and 3.1± 1.89 ppb of Fumonisins). The effect of prepared ZnO- NPs on the growth and mycotoxins production by mycotoxigenic moulds were evaluated which was concentration dependent. The levels of produced mycotoxins were decreased when the concentration of ZnONPs increased. The growth of aflatoxigenic moulds and aflatoxins production were inhibited by addition of 8 µg/ml of ZnO-NPs; while that of ochratoxin A and fumonisin B1 producing moulds and mycotoxins production were inhibited by addition of 10 μg/ml of ZnO-NPs to tested medium. When the treated mycotoixigenic fungal cells were subjected to SEM, the damage and rupture of fungal cell wall were detected in the area of surrounding growth media.

Therefore, it is suggested that the application of ZnO-NPs in food systems is effective in inhibiting the growth of fungi and mycotoxins production.

Keywords Zno-Nanoparticles (Zno-Nps); Mycotoxigenic Moul; Mycotoxins; Aflatoxins; Ochratoxin A; Fumonisins

Introduction Nanoparticles are used nowadays in all sciences in area of chemistry, physics, medicine and biology.

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Because of their high flexibility and strength, nanotechnologies have huge potential for solving numerous problems in increasing the treatment efficiency and diagnosis of various diseases to economize materials and energy resources. Metalbased nanoparticles are derived from iron, titanium and a variety of other metals and their oxides ( Mahdi et al., 2012). The nano-scale of metals play roles in understanding the ability to manipulate biological processes which will be the central theme for present biomedical and biological issues that need a nanoscience or nanotechnology approach (Whitesides, 2003). The development of this technology in nanomedicine is clearly obvious with the possibility to diagnose diseases. The properties of small size of nano-particles when compared with the bulk material were that the nano-particles have a large surface area related to their volume. In addition, nanotechnology has the potential to impact many aspects of food and agricultural systems, food security, disease treatment delivery methods, new tools for molecular and cellular biology, new materials for pathogen detection (Weiss et al., 2006). However, nanoparticles have been reported for the application in nano-sensor and nanotracer (Moraru et al., 2003).

Mycotoxins are formed by certain fungal species, whenever environmental factors are conductive.

During the growth of these frequently occurring mycomycetes on food stuffs and animal feeds, the process takes place as a secondary metabolism. Unlike many bacterial toxins, mycotoxins are in general highly stable in face of conventional heating process of foodstuffs for human consumption. These fungi and mycotoxins have serious effects upon the growth rate and health of human being as well as animals,Ciegler and Vesonder (1983); FDA, 2000 and Bahtnager and Ehrlich(2002). Some mycotoxins as aflatoxin, ochratoxin, patulin and zearalenone have

Studies in Chemical Process Technology (SCPT) Volume 1 Issue 4, November 2013

been recorded to cause a public health hazard and found to be carcinogenic, tremorgenic, haemorrhagic and dermatitis (Beuchat, 1978; Wray, 1981 and Bahtnager and Ehrlich (2002).

In recent years, the use of inorganic antimicrobial agents in nonfood applications has attracted interest for the control of microbes (Wilczynski, 2000 and Awad et al., 2011). Among these agents, nanoparticles of MgO, CaO, and ZnO exhibit strong antibacterial activity (Sawai, 2003). Metal oxides nanoparticles are characterized with very high surface area to volume; particularly ZnO–NPs is of fewer hazards to the environment. ZnO is 1 of 5 zinc compounds that are currently listed as generally recognized as safety (GRAS) by the U.S. Food and Drug Administration (FDA), and Zinc salt has been used for the treatment of zinc deficiency (Lopes de Romana et al., 2002).

However, there are few data that has demonstrated antimicrobial efficacy of ZnO in foods. The large increase in the number and occurrence of antibioticresistant bacterial and fungal strains has prompted a renewed interest in the use of metals as an antibacterial and antifungal agent (Navarro et al., 2008). The activities of NPs of metal oxide (ZnO, MgO, and CaO) powders against Staphylococcus aureus, E.coli, or fungi were quantitatively evaluated in culture media (Sawai, and Yoshikawa, 2004).

Therefore, the present work was undertaken to detect the prevalence of mycotoxigenic mould in feed and food and evaluate the antifungal effect of ZnO-NPs against growth of mycotoxigenic moulds and mycotoxins production in food.

Materials and Methods Feed and Milk Samples One hundred samples of cattle feeds and raw milk of dairy cows (50 of each) were collected from animal and poultry farms with symptoms of toxicities as vomition, nausea, loss of weight gains and decreased milk production in diseased cows.

Mycotoxigenic Strains The strains of A.flavus, A. ochraceus and A.niger which were recovered from the collected feed and milk samples screened for mycotoxins production were used for evaluation on the efficacy of ZnO nanoparticles in inhibition of fungal growth and mycotoxins production.

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Standards and Immunoaffinity Columns Aflatoxins Ochratoxin a and Fumonisins B1

of

Standards of Aflatoxins M1, B1, B2, G1 and G2, Ochratoxin A and Fumonisin B1 were purchased from Sigma Chemical Company (USA).

Isolation and Identification of Fungi from Feed and Milk Samples The samples of feeds and milk were examined for fungal contamination according to the technique of (Pitt and Hocking, 2009).

Screening of Isolated Strains of a. Flavus, a.

Ochraceus and A.Niger for Production Respective Mycotoxins Using Synthetic Medium (Gabal Et Al., 1994) The spore suspension of some isolated strains of A. flavus, A.ochraceus and A.niger recovered from cattle feeds and raw milk samples were prepared and adjusted to be approximately (5x106 spore/ml) by haemocytometer. 1 ml of spore suspension was inoculated into each flask containing (25 ml) of sterile Y.E.S. (2% yeast extract, 20% sucrose for A. flavus) and (4% yeast extract, 15% sucrose for A.ochraceus and A.niger), at room temperature (20-22°C) in the dark for 20 days. After incubation, the extraction and measurement of mycotoxins were implemented as below (See, 8) Synthesis and Characterization of Zno-Nps (Gong Et Al., 2007) ZnO-NPs was synthesized by chemical reaction of zinc chloride (ZnCl2) solution with sodium hydroxide (NaOH) solution according to the following equations: -ZnCl2+2NaOH=Zn(OH)2+NaCl and Zn(OH)2= ZnO+H2O. The precipitate of Zn(OH)2 was washed five times by 18Ω ohm double deionized water to eliminate sodium chloride (NaCl) and then diluted by isopropanol and centrifuge at 4000 rpm for 15 min. at room temperature, after that, the supernatant was decant and the precipitate was then dried for 5 hours at 250ºC. The obtained ZnO-NPs were subjected to optical measurements which were carried out by using a UV-Vis spectrophotometer (Lamda-25; PerkinElmer; Waltham, Massachusetts) and scanning the spectra between 250 and 800 nm at the resolution of 1 nm. In Fourier transform infrared (FTIR) analysis, the FTIR spectrum of the dried sample was recorded on a thermo 1600 instrument in the range from 450 to 4000 cm–1 at a resolution of 4 cm–1. Energy-dispersive 67

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x-ray (EDX) spectroscopy analysis on the confirmation of elemental ZnO was carried out. A scanning electron microscope (JEOL 6380A; Tokyo, Japan) was used to record the micrograph images of synthesized ZnO-NPs. Evaluation the Effect of Zno-Nps on the Growth of Toxigenic Moulds and Mycotoxins Production, ( Hili Et Al. , 1997 ) Gradual concentrations of ZnO-NPs were added to a set of flasks containing YES broth (0, 2 , 4, 6, 8, and 10 μg/ml of ZnO-NPs). The flasks were autoclaved for 20 minuts at 120 psi, cooled at room temperature and inoculated with spore suspention of toxignic strains.

These procedures were repeated for each kind of toxigenic mould (A. flavus, A.ochraceus and A.niger).

A separate flasks was treated with addition of 3% HSCA silicat instead of nanoparticles and kept as control. Then, all the inoculated flasks were incubated at room temperature (22-25oC) for 20 days. After the end of incubation period, the content of each flask was filtrated, where the filtrates were subjected for measurement of the levels of mycotoxins, whereas, the mycelial cells were used for further detection on the effect of ZnO-NPs using scanning electron microscopy.

Quantitative Estimation of Aflatoxins, Ochratoxins, and Fumonisins B1 by A Fluorometeric Method (AOAC (1990) At the end of the inocubation period, (25 ml) of fungal culture filtrate were extracted three times with (50 ml) chloroform. The chloroform extract was collected and evaporated in a rotator flask evaporator. The residue of chloroform extract was dissolved in (100 ml) methanol: water (80:20 V/V) and filtered through fluted filter paper. Then (10 ml) of filtrate were diluted with (40 ml) distilled water. The diluted extract was filtered through microfiber filter paper. 4 ml of filtered diluted extract were completely passed through FGis Afla test, Ochra test or Fumi test-affinity column at a rate of (2 drops/second) until air come through column which was washed twice with (10 ml) distilled water at a rate of (2 drops/second). The affinity column was eluted by passing (1 ml) HPLC grade methanol through column at a rate of (1-2 drops/second) and the sample elute was collected in a glass cuvette. 1 ml of afla test developer was added to elution in the cuvette and measured. The cuvette was placed in a calibrated fluorometer and aflatoxin B1 68

concentration was read after (60 seconds).

Scanning Electron Microscopy (SEM) (Patil et al., 2010) The mycelial cells obtained after filtration of YES were subjected to a scanning electron microscope (SEM), where the morphological changes of mycotoxigenic isolates by ZnO-NPs were observed. Strains were prepared by cutting the mycelia of the fungus, fixed for a minimum of 3 h in 2.5% (v/v) glutaraldehyde (100 mM phosphate buffer solution, pH 7.2), and then fixed in1% (w/v) osmium tetra oxide for 1 h. The agar blocks were dehydrated through a graded series of ethanol (30, 50, 60, 70, 80, 90, 95, and 100%; each level was applied twice for 15 min each time) and ethanol: isoamyl acetate (3:1, 1:1, 1:3, and 100% isoamyl acetate twice for 30 min). The fungal cells were dried using liquid CO2 and coated with gold-coater for 5 min. The coated samples were observed under JSM-5600 LV with accelerating voltage of 10 kV. Statistical Analysis The results were statistically analyzed by one-way classification throughout analysis of variance (ANOVA) using (SPSS 14, 2006).

Results and Discussion Nanotechnology has been noticeably developed with potential effects on science specially by using nanoscale chemical elements. Among nano material ZnO has gained more attention due to its special properties and less hazard to environment, however, like most of nanoparticles, it is toxic to organisms, which can be used as antibacterial, antifungal and antiviral agents (Brigger, 2002 and Brewer, 2007).

In the present work, one hundred samples of animal feeds and raw milk of dairy cows (50 of each) were collected from farms of diseased dairy animals and screened for suspected mycotoxigenic moulds. The animals in these farms suffered from signs of toxicities as vomiting, nausea, loss of weight gains and milk production in diseased cows. Aspergillus flavus, A.parasiticus, A.ochraceus and A.niger were the most commonly isolated fungi.

As shown in Table 1, most of isolated aspergilli from dairy feeds were toxin producers, where 60% of Aspergillus flavus, 100% of A.parasiticus, 50% of A.ochraceus and 60% of A.niger produced mean levels of mycotoxins at (30±3.1 (AFB1); 21±1.5(AFB1);

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18.5±0.55(OA) and 3.1± 1.89 (Fs), respectively.

Whereas, 30% of Aspergillus flavus and 25 % of A.ochraceus that recovered from milk produced mean levels of mycotoxins at 5.9±0.98 ppb (AFM1) and 6.5±0.35 ppb of (OA), respectively. On the other hand, A.niger and A.parasiticus isolated from milk failed to produce any toxins (Table 1).

TABLE 1 PREVALENCE OF MYCOTOXIGENIC MOULDS IN FEEDS AND RAW MILK OF DAIRY COWS

Tested Aspergillus species isolates.

A. flavus.

A. parasiticus.

A. ochraceus.

A. niger.

A. flavus.

A. parasiticus.

A. ochraceus.

A. niger.

+ve. Samples Source of isolates.

Feeds of dairy cows.

Raw milk.

No.

%

12/20 2/2 10/20 6/10 3/10 0/1 1/4 -

60% 100% 50% 60% 30% 0 25% -

Mean levels of mycotoxins ± S.E.(ppb).

30±3.1(AFB1) 21±1.5(AFB1) 18.5±0.55(OA) 3.1± 1.89 (Fs) 5.9±0.98(AFM1) 0 6.5±0.35 (OA) -

AFB1=Aflatoxin B1-OA=Ochratoxin A FB1=Fumonisin B1-AFM1=Aflatoxin M1

The mycotoxigenic members of Aspergillus species as A. flavus; A.ochraceus and A.niger were previously recovered in significant rates from milk and feeds in association with mycosis and mycotoxicosis in cattle (Drichuis et al., (2008) and Hassan et al., 2012 a) and chickens( Manal et al.(2012). On the other hand, many species of moulds including members of A.niger, A.flavus and A.ochraceus occurred regularly in different cheese and milk samples (Sampayo et al., 1995 and Aly, 1999). Whereas, A. flavus was recovered from milk and milk products (30% of milk, 25% of fresh Damietta cheese and 15% of fresh kareish cheese, followed by A. niger which was recovered at the rates of (25%, 15%, and 30%) respectively (Hassan et al., 2012 b). The detected aflatoxin B1 residues in milk and milk products reported by Hassan et al.

(2012 b) had mean levels ranging from (10- 15.67 ppb AFM) in milk and (7-8.50 ppb) in fresh Damietta cheese, however, no AFB1 was detected in fresh Kareish cheese. Mycotoxins can be traced in milk from food animals, while the amount of detectable residues depends on the initial food contamination, duration of toxin exposure and persistence of the toxin within the animal. The latter varies with the species (Purokivi et al., 2001).

Most of detected levels of aflatoxins and other mycotoxins were over the permissible limits in food

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reported by (FDA, 2000), where it was stated that the aflatoxins must be not more than 15-20 ppb in feeds and in milk not more than 0.5 ppb AFM1). Hence, all of detected levels in the present work were health hazard. In general, moulds were recorded to cause public health hazard due to mycotoxin production such as aflatoxins, ochratoxin, patulin, Fs and zearalenone. These compounds cause some degree of acute toxicity when given in high amounts and are potential carcinogen, where in developing countries, it appears that there is a direct correlation between dietary mycotoxins intake and the incidence of liver cancer due to the impairment of protein synthesis in hepatocytes due to mycotoxicosis (Raju and Devegawda 2000); Aravind et al. 2003); Eraslan et al., 2006) and Manal et al. 2012).

Therefore, the control of fungal contamination of food and milk becomes a critical demand. In recent years, resistance to commercially available antimicrobial agents by pathogenic bacteria and fungi has been increasing at an alarming rate and has become a serious problem (Wright, 2000 and 2005).

Microorganisms, such as bacteria, moulds, yeasts and viruses, in the living environment are often pathogenic and cause severe infections in both human beings and animals. Hence, the need to search for new antimicrobial agents from natural and inorganic substances is urgently required (Kim et al., 1998 and Cho et al., 2005).

The science of nanotechnology is a recent approach that can be used in a wide range of human activity in agriculture, industry and medicine therapy. Metals NPs as ZnO nanoparticles (ZnO-NPs) have gained more attention due to its special properties and its fewer hazards to environment.

Table 2 and Fig. 1 demonstrate the synthesis of ZnoNPs using simple chemical method by wet reaction of zinc chloride and sodium hydroxide solutions. The prepared ZnO-NPs were identified and characterized

by Scanning Electron Microscope (SEM) for detection of ZnO-NPs size and as in the purity of the prepared ZnO-NPs powder was 97% by Energy Dispersive XRay Spectrometer (Japan-Jeol –LV5600) (EDX).

TABLE 2 THE CONCENTRATION (%) OF ELEMENTS AND OXIDES OF ZNONPS BY EDX

Elements.

Zn O Na Total %

Concentration % 75.12 21.64 2.24 100%

Oxides.

ZnO Na2O ------Total%

Concentration % 97.00 3.00 ------100%

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The results revealed that ZnO-NPs with spherical and granular morphology had uniform distribution.

Because of their unique properties and large number of applications, zinc oxide nanostructures are one of the main subjects of nowadays research. ZnO nanoparticles are durable, free from affecting the soil fertility in comparison to traditional antifungal agents (Violeta et al., 2011).

FIG. 1 EDX SPECTRA OF ZNO-NPS

It was reported that the characterized absorption peak of ZnO-NPs is detected at 370 nm due to electron transition from valence band to conduction band O2p↔Zn 3p. FT-IR(Fourier transform infrared) spectra of the prepared ZnO-NPs observed in the range 400-4000 cm-1, revealed the broad band at 370 cm-1 Fig. 2.

The prepared ZnO-NPs had excellent antimicrobial activities against mycotoxigenic mould of Aspergillus in comparison with the control antifungal and antimycotoxin as 3% of HSCAS (Hydrated Sodium Calcium Aluminosilicate) which caused complete inhibition of fungal growth and degradation of 100% of mycotoxins in feeds (Harvey et al., 1991 and El-Ahl et al., 2006). The production of mycotoxins by these moulds is associated with doses of ZnO-NPs in the medium used, where the aflatoxin, ochratoxin and FB1 producted by respective mould were diminished as the concentration of ZnO-NPs till no level of mycotoxins were detected when moulds were treated with 10 µg/ml of ZnO-NPs (Table 3 and Fig. 4).

TABLE 3 INFLUENCE OF DIFFERENT CONCENTRATIONS OF ZNO-NPS ON GROWTH AND MYCOTOXINS PRODUCTION BY TOXIGENIC MOULDS USING.

Mycotoxigenic Fungi

FIG. 2 FT-IR SPECTRA OF OBTAINED ZNO-NPS

This band corresponds to the E2 mode of hexagonal ZnO, whereas, measurement of its optical properties

by UV-Visible spectrometry was in the range 200 – 550 nm. (Fig. 3).

FIG. 3 THE UV-VIS ABSORBANCE SPECTRUM OF ZNO-NPS

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Aflatoxigenic fungi.

Ochratoxigenic fungi.

Fs producing fungi.

Levels of mycotoxins after treatment with different concentrations of nano particles of (ZnO)(ug/ml) 0 2 4 6 8 10 20.0 8.0± 6.0± 1.0± 0.0 0.0 ±2.0 1.7 1.5 0.5 15.0 12.0 7.0± 4.5± 2.5± 0.0 ±1.5 ±2.0 1.7 0.9 0.3 20.0 10.0 8.0± 6.0± 1.0± 0.0 ±0.8 ±3.0 2.25 0.1 0.2

Control antifungal and antimycotoxin as 3% of HSCAS (Hydrated Sodium Calcium Aluminosilicate) caused complete inhibition of fungal growth and degradation of 100%of mycotoxins in feeds (Harvey et al. (1991).

FIG. 4 INFLUENCE OF DIFFERENT CONCENTRATIONS OF ZNO-NPS ON LEVELS OF MYCOTOXINS PRODUCED BY RESPECTIVE MOULDS

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The concentration 8 µg/ml of ZnO-NPs inhibited the growth of aflatoxins producing moulds, while 10 µg/ml inhibited the growth of ochratoxins and FB1 producing moulds.

Many antimicrobial agents are limited in clinical applications, because of their cumulative and side effects on the internal organs which caused much complications as Amphotericin B and Grisofulvine as well as renal failure, fever, tremble, nausea and diarrhea seen after using these drugs and the long period of administration of the Fluconazole, liver cirrhosis, drug toxicity and stopping in testosterone synthesis (Espinel-Ingroff , 2009). Hence, the continuous investigation on new drugs with fewer complications becomes urgent requirement in hosts and is considered for knowing nanoparticles function, its ability in destroying fungal potential and membrane function (Nasrollahi1 et al., 2011).

The antifungal activities of ZnO nanoparticles against important fungal diseases caused by P. expansum subtilis, Aspergillus spp., Rhizopus spp. and yeast were investigated by (Violeta et al., 2011).

Whereas, Hosseini, et al.( 2011) reported that the MIC of ZnO against Aspergillus spp. and C.albicans was 1.013-296 μg/ml and for SDS and Fluconazole were 0.001-0.56 and 0.062-128 μg/ml, respectively. They added that the use of lower concentrations of zinc oxide particles to the activities of anti-fungal and antibacterial is the most effective. Furthermore, different studies conducted in different laboratories showed that the antibacterial activity is influenced by not only nanoparticles concentration but also the size of the ZnO particles (Violeta et al., 2011).

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(Figure 5 (1)). The effect of ZnO nanoparticles on the treated fungi at (8 μg/ml) was observed as membrane damage of Aspergillus conidial cells and some pits that have been caused in inter cellular components, leading to leakage and finally cell death. Whereas, the use of ZnO-NPs (10μg/ml), caused destruction of fungal cell with pore in their cell membrane (Fig., 5 (1, 2, 3)).

Different mechanisms were proposed to discuss the effect of ZnO-NPs on the growth of fungi. The first is the formation of H2O2 on the surface of ZnO-NPs due to the possible formation of hydrogen bond between hydroxyl group of cellulose molecules of fungi with oxygen atom of ZnO-NPs leading to inhibition of the fungi growth, while the second is the release of Zn2+ which causes damages cell membrane and interacts with intraocular contents (Moraru et al., 2003).

Several natural and engineered nanomaterials have demonstrated strong antimicrobial properties through diverse mechanisms including photocatalytic production of reactive oxygen species that damage cell components and viruses (as ZnO ), compromising the cell envelope (e.g. peptides, chitosan carboxyfullerene, carbon nanotubes, ZnO and interruption of energy transduction (Mati et al., 2010 and Violeta et al., 2011).

On the other hand, Alt et al.,(2004) and Hassan et al.(2013) evaluated the Ag-NPs capability in destroying surface membrane structure of the fungus by using SEM and detected that Ag-Nps attached to cell membrane and penetrated it in the fungal cells, then produced a site with little molecular weight in center of cells and Ag Nps attached to respiratory sequence and finally cell division stopped, leading to cell death, Ag-NPs released silver ion in fungal cell, which increased its antifungal function.

Conclusions

FIG. 5 SCANNING ELECTRON MICROSCOPY (SEM) IMAGES OF TOXIGENIC ASPERAGILLUS SPP. (X3000): (1)(L) CONTROLNORMAL CONIDIAL CELLS. (2)(M) CELLS TREATED BY 8 μg/ml ZnO-NPs. (3)(R) CELLS TREATED BY 10 μg/ml OF ZnO-NPs.

Here, the mode of action of ZnO nanoparticles on the growth of conidial Aspergillus cells and hence its abilities for mycotoxin production, was investigated

by Scanning Electron Microscopy (SEM). The normal conidial cell of aspergilli has a spherical shape and smooth cell wall and intact cell membrane of conidia

It can be concluded from this study that ZnO-NPs can inhibit the growth of mycotoxic fungi as (A. flavus, A.ochraceus and A.niger) and their production for respective mycotoxsins (AFs, OA and Fs). Therefore, the method is recommended to be used for treatment of feeds and foods as a means to reduc the hazards of mycotoxicosis in man and animals.

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