INFLUENCE OF CHOSEN MICROBES AND SOME CHEMICAL SUBSTANCES ON THE PRODUCTION OF AFLATOXINS

Aflatoxins are produced as secondary metabolites by A. flavus, A. parasiticus, A. nomius and A. tamarii. The aflatoxin biosynthetic pathway involves several enzymatic steps and genes (apa-2, ver-1) that appear to be regulated by the aflR gene in these fungi. The aim of this work was the detection of aflatoxins by the HPLC method and the ascertainment of factors influencing their production. A. parasiticus CCM F-108, A. parasiticus CCF 141, A. parasiticus CCF 3137 and two isolates A. flavus were used. These toxigenic isolates were recovered from spice (strain 1) and wraps (strain 2). The gene for the production of aflatoxin B1 for each species of fungi was detected using an optimized PCR method. Rhodotorula spp., Lactococcus lactis subsp. lactis CCM 1881, Flavobacterium spp. and fungal strain Pythium oligandrum were tested for inhibition of aflatoxins production and fungal growth. Having used the HPLC detection, various preservatives (propionic acid, citric acid, potassium sorbate) were tested from the viewpoint of their influence on the growth of aflatoxigenic fungi followed by the production of aflatoxins. The growth of A. flavus and A. parasiticus and aflatoxin production in Potato Dextrose Agar supplemented with propionic acid (1000-2000-3000 mg/kg), citric acid (2000-3000-4000 mg/kg) and potassium sorbate (500-800-1000 mg/kg) was tested by Agar Dilution Method. After 72 h of incubation was evaluated growth of fungi, all samples were frozen for later extraction and aflatoxins quantification by HPLC. Effect of peptone and sucrose additions were studied in yeast extract (2%) supplemented with peptone (5-10-15%) or sucrose (15%). Growth inhibition of Aspergillus by Pythium oligandrum was tested on wood surface. As shown, the highest inhibition effect on the aflatoxins production was obtained when propionic acid was applied in concentrations since 1000 mg/kg. A total inhibition of the fungi growth and aflatoxins production was observed in all samples containing peptone in the concentration range tested. Significant limitation of the growth and production of aflatoxins was also observed in the presence of other microorganisms such like Pythium oligandrum and Rhodotorula spp.


INTRODUCTION
Microscopic filamentous fungi present a worldwide problem.Fungal physiology refers to the nutrition, metabolism, growth and reproduction of fungal cells.It also generally relates to interaction of fungi with their biotic and abiotic environment, including cellular response to stress.The dynamics of fungal activities are central to the efficiency of forestry and agricultural operations, in all three forms: mutualistic symbionts, pathogens and saprophytes as they mobilize nutrients and affect the physical-chemical environment (Gqaleni et al., 1997;Chourasia, 1993;Paster, 1999).
Fungi are also responsible for the organic pollutants detoxification and for the bioremediation of heavy metals in the environment.
The fungal negative effect on humans, animals and plants is the main reason for monitoring the contamination of food and feed (Gunterus et al. 2007, Huang et al. 2009).Some fungi colonize plant seeds and thereby cause loss of stored harvests and reduce seed germination rates, they can also produce mycotoxins which have mutagenic properties.Some fungi are pathogens of plants or animals (and even other fungi).Fungi can also cause diseases in humans, many of them as so-called opportunistic pathogens that attack people whose immune systems are weakened.
Aspergillus species compete with Fusarium and Penicillium species for the dominance among the world's fungal flora (Nesci et al., 2003).
Aflatoxins are secondary metabolites produced by Aspergillus flavus, Aspergillus parasiticus, Aspergillus nomius and the newly described Aspergillus tamarii and have the highest toxicity among mycotoxins (hepatotoxigenic, hepatocarcinogenic and immunosuppressive effects).Due to their toxicity including the carcinogenic activity, aflatoxins affect not only the health of humans and animals but also the economics of agriculture and food.For the production of aflatoxins it is particularly important where their biosynthetic pathway stops.
Aflatoxinogenic fungi are important contaminants of certain foods and animal feeds because of their ability to produce aflatoxins.When these fungi invade and grow in commodities such as peanuts, corn and cottonseed, Volume 9 10 No. 1/2015 the resulting contamination with aflatoxins often makes the commodities unfit for consumption.Historically, identification of filamentous fungal (mould) species has been based on morphological characteristics, both macroscopic and microscopic.These methods may often be time-consuming and inaccurate, which necessitates the development of identification protocols that are rapid, sensitive, and precise.The polymerase chain reaction (PCR) has shown a great promise in its ability to identify and quantify individual organisms from a mixed culture environment; however, the cost effectiveness of single organism PCR reactions is quickly becoming an issue (Nigam and Singh, 2000;Bennett and Papa, 1988;Weidenbörner, 2001).
The aim of this work was the determination of aflatoxins by the HPLC method and the ascertainment of factors influencing their production.

Bacterial strains, growth medium and cultural conditions
Six fungal, two bacterial and one yeast strains were used: A. parasiticus CCM F-108, A. parasiticus CCF 141, A. parasiticus CCF 3137 and two isolates A. flavus.These toxigenic isolates were recovered from spice (strain 1) and wraps (strain 2).Rhodotorula spp.* , Lactococcus lactis subsp.lactis CCM 1881, Flavobacterium spp.and fungal strain Pythium oligandrum * were tested for inhibition of aflatoxins production and fungal growth.Strains of CCM were provided by Czech Collection of Microorganisms (Brno, Czech Republic).Strains of CCF were provided by Department of Botany (Faculty of Science, Charles University, Praha, Czech Republic), the others ( * ) were provided by Department of Biological and Biochemical Sciences (Faculty of Chemical technology, University of Pardubice, Pardubice, Czech Republic).A suspension of bacterial and yeast or fungal strain was prepared from freshly grown colonies on MRS for Lactococcus lactis, Malt agar for yeast 24-48 h incubation at optimum temperature.Concentration of bacterial and yeast cells was adjusted to 10 8 cfu/mL using 0.5 McFarland turbidity scale.Suspensions of fungal spores were prepared from 7days cultures grown on Malt agar´s slant at 24 °C.The density of spores was adjusted to 10 6 spores/mL using Bürker counting cell.All the nutrient media used throughout the study were purchased from HiMedia, India.The growth of A. flavus and A. parasiticus and aflatoxin production was tested by Agar Dilution Method.Control Petri dishs were kept without bacteria and yeast in similar way.After 7 days of incubation was evaluated growth of fungi, all samples were frozen for later extraction and aflatoxins quantification by HPLC (Malir et al., 2006).
After incubation (25 °C, 72 h) was evaluated growth of fungi on Malt agar, production of aflatoxins was quantification by HPLC (Malir et al., 2006).
Growth inhibition of Aspergillus by Pythium oligandrum was tested on wood surface (5x5 cm).

Extraction of aflatoxins
The sample was agitated with 50 mL of a mixture of 60% methanol with 0,8 g NaCl for 3 min at room temperature.5 mL of solution was diluted by 10 mL deionized water.Extraction of aflatoxins was performed by Afla B affinity colum (VICAM, USA).After filtration of the substrate was added 1mL to developer (8 mL deionized water, 1mL trifluoroacetic acid, 1 mL acetic acid).Then the amount of aflatoxins were detected by HPLC.

Production of aflatoxins in potato dextrose broth
The optimized PCR reaction selectivity was tested on collection cultures and it was proven that both PCR methods are highly specific for the determination of aflatoxinogenic strains of A. flavus and A. parasiticus isolated from food and feed.When verifying the PCR selectivity to detect the regulatory gene important for the aflatoxin B1's production, a positive reaction was observed also in the non-aflatoxinogenic strain of A. versicolor CCM F-585.The aflatoxin B1's biosynthetic production pathway renders various intermediate products, one of which is versicolorin A, the final metabolite of the A. versicolor CCM F-585 fungus (Zachová et al., 2003).

The production of aflatoxins was demonstrated only by
Aspergillus parasiticus CCF 141, Aspergillus parasiticus CCF 3137 and Aspergillus flavus CCM-F 108 (Table 1).

Fungal growth inhibition and the production of aflatoxins by a conserving substance
Growth of selected strains of fungi was monitored in the presence of the preservative on the soil potato agar at 25 °C for 72 hours.Agar Dilution Method was used to determine the inhibition of mold growth.Size of colony was compared with the size of colonies on the medium without preservatives.Citric acid inhibited at least growth of Aspergillus flavus CCM F-108 and the most growth of Aspergillus parasiticus CCF 3137 (Table 3).Propionic acid inhibited growth of at least Aspergillus parasiticus CCF 3137 and the most inhibited Aspergillus flavus CCM F-108, whose growth was completely suppressed.Growth of Aspergillus parasiticus CCF-141 was inhibited completely also (Table 3).Suppression of growth of Aspergillus flavus detected by the agar diluted method on Sabouraud agar at 21°C for 5 days provides Čonková et al. (1993), namely even lower concentrations of propionic acid than was used for our experiment (Table 3).Potassium sorbate inhibited the growth of most Aspergillus flavus CCM F-108 and a minimum growth of Aspergillus parasiticus CCF 3137.This strain was isolated from environment of the rainforest in Malaysia, and is therefore probably more resistant than the strains of Aspergillus parasiticus CCF 141 and Aspergillus flavus CCM F-108, obtained from the Czech Republic.Significant inhibition of growth of Aspergillus flavus CCM F-108 is already at a concentration of potassium sorbate and 500 mg/kg (Table 3).The concentration of potassium sorbate (higher than 1500 mg/kg) is needed for pronounced inhibition of growth for Aspergillus parasiticus, so there, as confirmed the authors Liewen and Marth (1984) in their study.Propionic acid inhibits growth of Aspergillus the most of preservatives (Table 3).
Aflatoxins production in the presence of preservatives Conserving substance factors were tested in potato dextrose broth (30 °C, 72 h).All the preservatives inhibited the production of aflatoxins.When aflatoxins were created, aflatoxins were produced in quantities less than the detection limit determination. A. parasiticus CCM F-108 and Aspergillus parasiticus CCF 141 were the most sensitive to preservatives.Same results are featured by Chourasia (1993).
Similar to our findings also Čonková et al. (1993) feature the fungicidal efficiency of propionic acid resulting from the experiment at 88.9%.In their study, Tsai et al. (1984) determined the production of aflatoxins by strain Aspergillus parasiticus NRRL 2999 in the presence of 0.2 -0.4% propionic acid.
The largest production of aflatoxins was determined in the presence of citric acid.Production of aflatoxin B1 and G1 decreased with increasing concentration of citric acid (Figure 1, Table 4 and Table 5).
In the presence of potassium sorbate were Aspergillus parasiticus CCF 3137 produced only aflatoxins B1 and G1, at a concentration of 500 mg/kg (B1 0,29 ng/mL, G1 0,036 ng/mL).Information on the aflatoxin production inhibition by organic acids and potassium sorbate are rather sparse, some data can be found in Tsai et al. (1984).

The reduction of aflatoxins by peptone and sucrose
The aflatoxin pathway gene expression analysis was carried out in inducing (YES medium) and non-inducing (YEP medium) conditions (Sweeney et al., 2000; Scherm and Palomba, 2004; Scherm et al., 2005).In our experiment, peptone and sucrose inhibited most of the aflatoxin production in all of the concentrations.
The growth of aflatoxigenic fungi was not particularly influenced by the presence of peptone and sucrose.

Pythium oligandrum on wood block
The growth of neither Aspergillus nor Pythium oligandrum was visible on wood block after the incubation period, therefore swabbing by sterile swab was carried out.Swab cotton was then shaken out to physiological solution and the surface of the potato dextrose agar was inoculated with 0.1 mL of the homogenate and incubation was allowed for 72 h at 25 °C.
After the incubation period, the Pythium oligandrum growth was quite prominent, while the growth of A. flavus was strongly inhibited.
Without the presence of Pythium oligandrum spores of A. flavus survive on wooden plate.

Pythium oligandrum in liquid medium
This section of the experiment proved a major growth inhibition of fungi, in some strains up to four orders; hence also the aflatoxins production was reduced by Pythium oligandrum.
Pythium oligandrum Dresch., has been identified as one of the most numerous species observed in agricultural soils.In certain areas, it has proven its ability to induce plant disease suppression (Floch et al., 2009).Inoculation with P. oligandrum or its elicitors can sensitive plants and make them respond more quickly and efficiently to pathogen attacks and trigger systemic resistance.Additionally, P. oligandrum has been reported to promote plant growth according to a recent study this phenomenon is mediated by tryptamine, an auxin produced by P. oligandrum.Interactions between host fungi and P. oligandrum involve a complex set of events (Rey et al., 2005).In our study also showed that P. oligandrum not only inhibited of growth of Aspergillus, but inhibited production of aflatoxins as well.The size of fungal colony was measured on a medium for selected microorganisms (Malt, MRS, BHI) (Table 6).After incubation, an analysis of aflatoxins was carried out using HPLC.
After microaerophile cultivation on MRS at 30 °C, Aspergillus parasiticus CCF 141 and Aspergillus parasiticus CCF 3137 occurred for the production of aflatoxins, which was probably caused by low pH.The fungi were stressed and resisted the production of aflatoxins in this medium, as well as his work indicates Nesci et al. (2003).Author tested the effect of pH on aflatoxin production of Aspergillus parasiticus RCT 1920 and Aspergillus parasiticus RCD 106 on corn MALT agar at 25 °C for 5 days.
Ehrlich et al. ( 2005) also examined the production of aflatoxins B1 and G1 in the presence of different acidic pH.Found that gene expression for the production of aflatoxin B1 with decreasing pH increases and is the largest production of aflatoxin B1, production of aflatoxin G1 is stopped.
In our case, stress of the A. parasiticus probably contributed microaerophilic environment of cultivation and production of nisin by Lactococcus.Lactococcus and Rhodotorula inhibited growth all strains of A. parasiticus (Figure 2 and Figure 3).tested the inhibition of aflatoxin production by protein extracts of Flavobacterium.Aspergillus flavus and Aspergillus parasiticus isolated from spices with protein extract of Flavobacterium in BHI broth at 30º C for 5 days were cultivated.Inhibition of aflatoxin production by the bacteria, according to these authors 80% (Smiley and Draughon, 2000).
In the presence of Lactococcus and Rhodotorula, others the strains do not produced of aflatoxins.Only Rhodotorula strain in density of 10 4 cfu/mL inhibited of production of aflatoxins at least.0,329 ng/mL of AFB1 and 0,293 ng/mL of AFG1 were produced by A. parasiticus CCF 3137 (Figure 4).
The aflatoxin production by Aspergillus sp. in the presence of peptone and sucrose was observed by the HPLC method.In all applied peptone concentrations, the strains were considerably inhibited.
Results revealed that Pythium oligandrum caused a total inhibition of growth and aflatoxin biosynthesis in the toxigenic Aspergillus sp.A strong effect on limiting the growth of aflatoxigenic fungi and aflatoxin biosynthesis was also observed when using the Rhodotorula.
Also Lactococcus an inhibiting effect, although only in its highest concentration (10 8 cfu/mL).

CONCLUSION
This work also aimed to detect aflatoxins by the HPLC method and to determine factors influencing their production.
The collection cultures' aflatoxin production was quantitatively determined by the optimized HPLC method.
Chemical factors influencing growth and production of aflatoxins were tested.Tested preservatives (propionic acid, citric acid and potassium sorbate) showed different effect on different strains.
The most sensitive to preservatives was A. flavus CCM F-108.The most efficient growth inhibition of fungi and Table 6 The growth rate of fungi A. parasiticus in the presence of different microorganisms after 72 hours.

Figure 1
Figure1The dependence of the aflatoxin production on the citric acid concentration in Aspergillus parasiticus CCF 3137 (potato dextrose broth, 25 ˚C, 72 h).

Figure 4
Figure 4The dependence of aflatoxin production by A. parasiticus CCF 3137 on the number of Rhodotorula cell (MALT, 25 °C, 72 h).
).By contrast,  Tsai et al. (1984)states in YES broth at 25 °C for 72 hours difference in the production of aflatoxin B1 Aspergillus Strain of Aspergillus flavus CCM F-108 produced by aflatoxin B1, in the amount of 331.5 ng/mL and aflatoxin B2 in the amount of 2.44 ng/mL (Table1).

Table 5
Aflatoxin production by A. flavus from spices for chicken (Strain 1) in potato dextrose broth with preservatives (25 °C, 72 hours).The dependence of the fungal growth rate on the number of Flavobacterium cells (BHI, 30 °C, 72 h).The dependence of the fungal growth rate on the number of Rhodotorula cells (MALT, 25 °C, 72 h).

Aflatoxins production inhibition by different microorganisms In
the presence of Lactococcus, no tested fungi produced any aflatoxins.The authors Smiley