Mycotoxins represent a heterogeneous group of low molecular weight chemical compounds, which present a biological activity, produced in the secondary metabolism by some fungal species, which mainly belong to Aspergillus
genera. These secondary metabolites have a cytotoxic, mutagenic and carcinogenic activity in both man and animals and, usually, they can be found as common contaminants into feed and food chains. The development of toxigenic fungi and subsequently the synthesis of mycotoxins may occur in each of the step of these chains, starting from field cultivation up to consumption, passing through storage and preservation. Some mycotoxins can be carcinogenic (fumonisins, FB, Group 2B: possible carcinogen for humans; IARC), carcinogenic and teratogenic (ochratoxin A, OTA, Group 2B, IARC), carcinogenic, mutagenic and teratogenic (aflatoxin B1, AF, Group 1; aflatoxin M1, Group 2B; IARC), [1
]. Mycotoxins contamination in cereals intended for human and animal consumption, is a serious food safety issue regarding productions from all over the world. In particular, maize and by-products could be contaminated by different class of mycotoxins including one of the most dangerous to human health and animal found in nature, aflatoxin B1 (IARC) [1
The major part of the methods for controlling mycotoxin contamination into feed- and foodstuffs has been performed by using chemicals that are hazardous for humans and the environment. The growing interest and awareness toward environmental pollution have addressed the research on the possibility to adopt “green” approaches in the control of fungal contamination and to prevent or detoxify mycotoxins. Due to high economic losses and health hazard issues as consequence of AF contamination, several strategies have been studied and applied to reduce the risk in maize [2
]. These strategies can be divided into: (1) stopping the infection process (host plant resistance, biocontrol); (2) pre-harvest crop management practices (good agricultural practices) and (3) post-harvest management strategies (timely-harvesting, properly drying). The former consists in finding the source of resistance in maize germplasm with natural resistance to A. flavus
infection (pre-harvest resistance). As a result, many new sources of resistance were identified or released, such as Mp420, Mp313E, Mp715, Mp717 and GT-MAS:gk, CI2, MI82, and Tex6. These lines tend to possess undesirable agronomic characteristics [3
], and cannot be used directly to breed for commercial aflatoxin resistant maize varieties. Recently, -omic approaches boosted the findings of novel gene target for selecting maize resistance to A. flavus
and/or aflatoxins [4
] even if a concrete result has not been yet achieved. Breeding programs exist to develop new varieties and to replace varieties that have lost their resistance, but the maintenance cost of this system is high. Agricultural scientists recognized the potential negative consequences of planting large areas to single, uniform crop cultivars as early as the 1930s. Thus, an Integrated Pest Management (IPM) approach is a widely recognized ecosystem approach to crop production and protection that combines different management strategies and practices to grow healthy crops and minimize the use of pesticides with considerable success. The same concept could be easily transferred to aflatoxin management, in which complementary, low cost and efficient strategies can be combined to control toxin contamination in maize. In relation to this, several sustainable strategies have been/are currently established for answering to this demand. Notably, biocontrol agents and predictive models have been recently developed for controlling aflatoxin in the pre-harvest [6
Disease management programs to risk use predictive modeling tools-stratify members in order to optimize the utilization of available pest management resources. Focusing the research on a time scale of one decade—by taking into account environmental and monitoring data from past research—may lead to create and apply predictive models that can forecast the spread of main pests and diseases of maize. These models integrate natural and social systems because examine a variety of coupled interactions (insects, maize and fungal pathogens) and feedbacks among relevant systems (food safety and production yields) [8
]. These types of mechanistic models have been successfully applied to prevent A. flavus
contamination in maize [6
Over recent years, biocontrol agents able to challenge specific pathogens in maize have been selected [7
]. Some of them pose cross-contamination risk (i.e
., introducing a microorganism safe in a determinate environment but unsafe for another) and some other are not stable in the wild (sexual recombination amongst vegetative incompatible selected strains), [11
]. Alternatively, and more safely, bioactive compounds from biocontrol agents may be used for enhancing plants defences and/or for inhibiting pathogen growth and/or toxin synthesis [12
In this paper, we propose two valid, low cost methods for safely containing aflatoxin contamination into maize kernels at large scale. The former concerns the optimization and large-scale production of an exo-polysaccharides mixture originated by Trametes versicolor while the second regards the attempt to use detoxifying agents, namely lignin degrading enzymes from T. versicolor, in feed composition for abating AFs content.
3. Materials and Methods
3.1. Fungal Strains
Trametes versicolor strains CF 117, CF 294 were obtained from the collection of the Laboratory of Plant Pathology, Department of Environmental Biology, University “La Sapienza” of Rome. The strains were cultured on Potato Dextrose Agar (PDA, Biolife, Milan, Italy) in Petri dishes incubated at 25 °C for 7 days. Ten-day liquid cultures of Potato Dextrose Broth (PDB, Himedia, Mumbai, India) of different isolates were prepared from Petri dishes and used as inoculum.
Aspergillus flavus (NRRL 3357), producer of aflatoxin B1, was provided by the laboratory of Payne, G.A. of North Carolina State University (Raleigh, NC, USA). The isolate was cultured on PDA at 30 °C for 7 days and a suspension of 1 × 106 conidia per mL of sterilized distilled water was used as inoculum.
3.2. Assays of T. versicolor Culture Filtrates on Aflatoxin Production: “In vitro Experiments”
A solution of molasses (from sugar beet processing, kindly provided by COPROB, Minerbio, Italy) and yeast extract (Biolife, Milan, Italy) in a concentration of 30 g/L and 2 g/L respectively, was inoculated with 10% v/v of homogenized mycelia of T. versicolor CF117 isolate. The fungal cultures were incubated at 25 °C in rotary shaken conditions (150 rpm) for 7 and 10 days. The mycelium was separated from the culture medium by filtration through 0.45 µm filters (Sartorius, Goettingen, Germany) and the filtrate was lyophilized (lyophilized filtrate, LF). The LF was added (final concentration 0.5% and 1% w/v) to 1mL of PDB in 10 mL tubes flasks. The tubes were inoculated with A. flavus conidia, as reported above and incubated at 30 °C for 5 days. After incubation, aflatoxin B1 were monitored in the culture filtrates by Agilent 1200 HPLC-DAD (Agilent, Waldbronn, Germany).
3.3. Assays of T. versicolor Solid Substrate on Aflatoxin Production: “In vitro Experiments”
Ground beet pulp (from sugar beet processing, kindly provided by COPROB, Minerbio, Italy) was used as non-inert solid support for growing T. versicolor
. One Kg of sugar beet pulp (BP) was autoclaved at 120 °C for 20 min in a plastic bag with filter strips (Mycelia BVBA, Nevele, Belgium) and inoculated with 1 L of T. versicolor
CF117 suspension, grown in molasses solution (see above) for 10 days. The bags were incubated at 25 °C for 10 days and the solid substrate was lyophilized (lyophilized solid, BPTV). BPTV was added (final concentration 0.1%, 0.5% and 1% w
) to 1 mL of PDB in 10 mL tubes flasks. The tubes were inoculated with A. flavus
conidia, as reported above and incubated at 30 °C for 7 and 10 days. At 7 and 10 days after inoculation (dpi), aflatoxin B1 were monitored in the culture filtrates by HPLC-DAD (Agilent, Santa Clara, CA, USA) as previously described [21
] with modifications (described below).
3.4. Assays of T. versicolor Culture Filtrates on Aflatoxin Degradation: “In vitro Experiments”
Thirty-five mL of the following culture medium, glucose, 5 g/L; asparagine, 1 g/L; K2
, 1 g/L; yeast extract, 0.5 g/L; MgSO4
O, 0.5 g/L; KCl, 0.5 g/L; FeSO4
, 1 g/L; MnSO4
, 0.008 g/L; ZnSO4
, 0.003 g/L; CuSO4
O, 0.03 g/L; NH4
, 1 g/L was inoculated with T. versicolor
CF294 5% v
and incubated at 25 °C. After 3 days, the entire biomass was transferred into 3 L of the same culture medium without glucose, with the addition of 2,5-xylidine 50 µM. The addition of xylidine and the absence of glucose in the growth substrate, create conditions that are conductive for the production of laccase [22
]. The culture were incubated for further 7 days at 25 °C. The mycelium was separated from the culture medium by filtration through 0.45 µm filters and the filtrate was stored at 4 °C. 300 ng of aflatoxin B1 (Sigma, Milan, Italy) was added to 500 µL of culture filtrate with different laccase activity (adjusted by diluting culture filtrate with distilled water) and incubated for 6, 24, 48, 72, 144 h. Control was represented by aflatoxin B1 dissolved in PBD solution. Aflatoxin B1 content wad determined by HPLC-DAD (Agilent, Santa Clara, CA, USA) as previously described [21
] with modifications (described below).
3.5. Experiments on Maize and Seeds
Preparation of a stock of contaminated maize with A. flavus
: 100g of sterilized maize seeds were inoculated with 1 × 106
conidia of A. flavus
. The samples were incubated at 30 °C for 7 days. Three grams of A. flavus
inoculated maize were added to 27 g of sterilized maize and treated with 3 g of BPTV (see above). The samples were incubated at 30 °C for 7, 14 and 21 days. Aflatoxin B1 content was determined by HPLC-DAD as previously described [21
] with modifications (described below).
3.6. Assays of T. versicolor Culture Filtrates on Aflatoxin Degradation: “In vivo Experiments”
At lab scale, 100 g of maize seeds (see above) were placed on plastic sheets and were treated, via
nebulizer, with 1 or 2 mL of T. versicolor
CF294 culture filtrate having laccase activity of 3.5 U/mL. The plastic sheets were then closed, leaving an opening for the exchange of air with the outside. The sheets were incubated at 30 °C for 96 h. At large scale, 30 Kg of maize seeds and 30 Kg of milled maize seed, were treated with T. versicolor
CF294 culture filtrate having laccase activity of 3.5 U/mL. A nebulizer both on the seeds and on the flour provided 300 mL of culture filtrate per 30 Kg of seeds. The samples were kept at room temperature for 7 and 14 days. Aflatoxin B1 content was determined by HPLC-DAD as previously described [21
] with modifications (described below).
3.7. Cell Viability Assays
For testing if the AFB1 fragments originated following 72 h-incubation at 25 °C with 3.5 U/mL of CF294 culture filtrate having laccase activity affect cell viability, human OCI-AML3 and U937 (0.3 × 106/mL) cells were seeded into 24-well plates and incubated for 48 h with the different samples following which 100 µL of each condition were transferred in triplicate to a 96-well plate (500 μL/well). The cells were incubated in presence of 50 μL of buffer BES (25 mM BES; 250 mM NaCl; pH 6.0), or of buffer containing CF294 culture filtrate having laccase activity of 3.5 U/mL, or of buffer containing CF294 culture filtrate having laccase activity of 3.5 U/mL which have reacted for 72 h with 150 ng of AFB1 at 25 °C, or of buffer containing 150 ng of AFB1. Then, 10 µL of MTT [3-4,5-dimethylthythiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma-Aldrich, Milan, Italy) were added to each well and plates were incubated for 3 h at 37 °C with 5% CO2. After 3 h, MTT was dissolved with 100 µL of solubilization solution and absorbance was measured at 570 nM with a spectrophotometer. All the experiments were performed at least three times and results were expressed as mean ± standard deviation (SD).
3.8. Analytical Determination
One hundred milliliters of a solution of methanol-water 80:20 v/v, were added to 50 g of maize seeds and 5 g of NaCl. The solution was blended at high speed for 1 min and 10 mL of the extract were diluted with 40 mL of water. Ten milliliters of the diluted solution were filtered through 0.45 μm filter and purified by immunoaffinity column (Aflatest, Vicam, Boston, MA, USA). Aflatoxin B1 was eluted with 1mL of methanol (Sigma, Milan, Italy), dried and the residue was dissolved with 200 μL of methanol. Aflatoxin B1 content was determined by HPLC-DAD equipped with a reversed phase Zorbax-Aq C18 column (150 × 2.0 mm I.D, 3.5 μm) thermostated at 30 °C. The separations were performed by gradient elution of increasing concentration of acetonitrile (Romil, Cambridge, UK) in water (Romil, Cambridge, UK), both acidified with 1% v/v of formic acid, at a flow rate of 0.2 mL/min. Detection was performed at 363 nm. For the quantification of aflatoxin B1 in different matrices, a calibration curve was constructed using standard aflatoxin B1 at different concentrations. The identification and quantification of aflatoxin B1 were performed respectively based on its retention times and spectroscopic spectrum and by the external standard method using a six point regression graph of the UV-visible absorption data collected at 363 nm.
3.9. Laccase Activity
The enzymatic activity of laccase was assayed by the method described by Harkin and Obst [23
]. To 2.5 mL of a solution of 50 mM sodium acetate at pH 4.5, were added 0.4 mL of a 0.5 M solution of syringaldazine (Sigma, Milan, Italy) in ethanol; 100 µL T. versicolor
CF 294 culture filtrate were added to the solution and the reaction is monitored by UV-vis spectrophotometer (Beckman DU530 UV/VIS). Oxidation of syringaldazine was monitored through absorbance increase at 525 nm (ε = 65,000 M−1
). One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μM of syringaldazine per min−1
3.10. Statistical Analysis
The results obtained in the present investigations were subjected to statistical analysis using SPSS package 12.0 version. We computed statistical parameters mean, S.D, S.E and t-value are interpreted at α = 0.05 level of significance.