**2. Conditions affecting mycotoxin production**

Cereals in the field are exposed to fungi from the soil, birds, animals, insects, organic fertil‐ izers, and from other plants in the field. Mechanical damage of raw material or food due to insects and pests is a disturbing problem mainly in tropical regions, particularly as food contaminants are present in the field more abundantly than in the storage. Many different insects, e.g. European corn borer and sap beetles have the capability of promoting infections of various crops with mycotoxigenic fungi [25].

Mycotoxin production is determined by genetic capability related to strain and environmen‐ tal factors including the substrate and its nutritious content. Toxin production is dependent on physical (temperature, moisture, light), chemical (pH value, nutrients, oxygen content, preservatives), and biological factors (competitive microbiota). Each fungus requires special conditions for its growth and other conditions for its toxin production.

#### **2.1. Physical factors**

The most important factor governing colonisation of grains and mycotoxin production is the availability of water which on the field comes mainly with rainfall. The second important fac‐ tor is temperature. The moisture and temperature effects on mycotoxin production often dif‐ fer from those on germination and growth. Table 2 presents the moisture and temperature requirements of most common toxigenic fungi for their growth and mycotoxin production.

It was found that optimal temperature for *F.graminearum* growth on soybean contained in the range 15-20o C (in isothermal temperature) and 15/25o C (in cycling temperature). The op‐ timal temperature for mycotoxin production on soybean was 20o C for deoxynivalenol (DON) and 15o C for zearalenone (ZEA). After 15 days of incubation, the maximum levels 39 ppm and 1040 ppm for ZEA and DON, respectively, were detected. Fumonisins were pro‐ duced by *Fusarium graminearum* only the on culture medium at 30o C; on soybean no fumoni‐ sins were detected [31].

unclear. A relationship between mycotoxin production and sporulation has been document‐ ed in several toxigenic fungi. For example, chemical substances that inhibit sporulation of *Aspergillus parasiticus* have also been shown to inhibit the production of aflatoxin [33]. Chemical preservatives such as organic acids (sorbic, propionic, acetic, benzoic) or fungi‐ cides have been used to restrict the growth of mycotoxigenic fungi. It was found that pro‐ pionic acid at the concentration of up to 0.05% inhibited the growth and ochratoxin production by *Penicillium auriantogriseum*. A more effective result in higher temperature was observed [34]. Inhibiting fungal growth and toxigenic properties by organic acids is connect‐ ed with lowering the pH value. It was found that ammonium and sodium bicarbonate at the concentration of 2% fully inhibited the development of the cultures of *Aspergillus ochraceus*, *Fusarium graminearum* and *Penicillium griseofulvum* inoculated into corn. The production of ochratoxin A by *Aspergillus ochraceus* was reduced from 26 ppm in untreated corn to 0.26

*Aspergillus parasiticus* 10-43 32-33 0.84 12-40 25-30 0.87 [28, 29] *Aspergillus flavus* 6 – 45 35 – 37 0.78 12-40 30 0.82 [25] *Aspergillus versicolor* 4 – 39 25 – 30 0.75 15-30 23 – 29 "/>0.76 [25, 30]

**For growth For mycotoxin production Temperature [ Ref. oC] Minimal a <sup>w</sup> Temperature [oC] Minimal a <sup>w</sup>**

0.90 TeA

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Mycotoxins in Cereal and Soybean-Based Food and Feed

0.88 PA

[25, 26, 28]

191

[28, 29]

**Range Optimum Range Range Optimum Range**

*Alternaria alternata* 0 – 35 20 – 25 0.88 5-30 20-25 0.95-1.0 AOH

*Aspergillus ochraceus* 8-37 24-30 0.76-0.83 12-37 25-31 0.85 OTA

OTA – ochratoxin A; PA – penicillic acid AOH – alternariol, TeA – tenuazonic acid, ND – no data

**Table 2.** Environmental requirements for growth and mycotoxin production

*Fusarium culmorum* <0 – 31 21 0.89 11-30 25-26 Nd [25] *Fusarium graminearum* Nd 24 – 26 0.89 Nd 24-26 Nd [25] *Fusarium sporotrichoides* -2 – 35 22 – 28 0.88 6-20 Nd Nd [25] *Penicillium verrucosum* 0-31 20 0.81-0.83 4-31 20 0.86 [28, 29] *Penicillium expansum* -6 – 35 25 – 26 0.82 – 0.85 0-31 25 0.95 [25]

The simultaneous presence of different microorganisms, such as bacteria or other fungi, could disturb fungal growth and the production of mycotoxins. For instance, *Alternaria* and *Fusarium* are antagonistic, and *Alternaria* was less abundant in grain with a high incidence

ppm in bicarbonate-treated corn samples [35].

rate of *F.culmorum*. *Epicoccum* is a strong antagonist too [25].

**2.3. Biological factors**

**Species**

Most fungi need at least 1-2% of O2 for their growth. The influence of high carbon dioxide and low oxygen concentrations on the growth and mycotoxin production by the foodborne fungal species was investigated by [32]. Three groups of species were distinguished: first, which did not grow in 20% CO2 <0.5% O2 (*Penicillium commune, Eurotium chevalieri* and *Xero‐ myces bisporus*); second, which grew in 20% CO2 <0.5% O2, but not 40% CO2 <0.5% O2 (*Peni‐ cillium roqueforti* and *Aspergillus flavus*); and third, which grew in 20%, 40% and 60% CO2 <0.5% O2 (*Mucor plumbeus*, *Fusarium oxysporum*, *F.moniliforme*, *Byssochlamys fulva* and *B.ni‐ vea*). The production of aflatoxin, patulin, and roquefortine C was greatly reduced under all of the atmospheres tested. For example, aflatoxin was not produced by *A. flavus* during growth under 20% CO2 for 30 days. Patulin was produced by *B.nivea* in the atmospheres of 20% and 40% CO2, but only at low levels [32].

#### **2.2. Chemical factors**

Nutritional factors such as carbonohydrate and nitrogen sources and microelements (cop‐ per, zinc, cobalt) affect mycotoxin production, but the mechanisms of this impact are still


OTA – ochratoxin A; PA – penicillic acid AOH – alternariol, TeA – tenuazonic acid, ND – no data

**Table 2.** Environmental requirements for growth and mycotoxin production

unclear. A relationship between mycotoxin production and sporulation has been document‐ ed in several toxigenic fungi. For example, chemical substances that inhibit sporulation of *Aspergillus parasiticus* have also been shown to inhibit the production of aflatoxin [33]. Chemical preservatives such as organic acids (sorbic, propionic, acetic, benzoic) or fungi‐ cides have been used to restrict the growth of mycotoxigenic fungi. It was found that pro‐ pionic acid at the concentration of up to 0.05% inhibited the growth and ochratoxin production by *Penicillium auriantogriseum*. A more effective result in higher temperature was observed [34]. Inhibiting fungal growth and toxigenic properties by organic acids is connect‐ ed with lowering the pH value. It was found that ammonium and sodium bicarbonate at the concentration of 2% fully inhibited the development of the cultures of *Aspergillus ochraceus*, *Fusarium graminearum* and *Penicillium griseofulvum* inoculated into corn. The production of ochratoxin A by *Aspergillus ochraceus* was reduced from 26 ppm in untreated corn to 0.26 ppm in bicarbonate-treated corn samples [35].

#### **2.3. Biological factors**

**2. Conditions affecting mycotoxin production**

of various crops with mycotoxigenic fungi [25].

**2.1. Physical factors**

190 Soybean - Pest Resistance

the range 15-20o

(DON) and 15o

sins were detected [31].

**2.2. Chemical factors**

Cereals in the field are exposed to fungi from the soil, birds, animals, insects, organic fertil‐ izers, and from other plants in the field. Mechanical damage of raw material or food due to insects and pests is a disturbing problem mainly in tropical regions, particularly as food contaminants are present in the field more abundantly than in the storage. Many different insects, e.g. European corn borer and sap beetles have the capability of promoting infections

Mycotoxin production is determined by genetic capability related to strain and environmen‐ tal factors including the substrate and its nutritious content. Toxin production is dependent on physical (temperature, moisture, light), chemical (pH value, nutrients, oxygen content, preservatives), and biological factors (competitive microbiota). Each fungus requires special

The most important factor governing colonisation of grains and mycotoxin production is the availability of water which on the field comes mainly with rainfall. The second important fac‐ tor is temperature. The moisture and temperature effects on mycotoxin production often dif‐ fer from those on germination and growth. Table 2 presents the moisture and temperature requirements of most common toxigenic fungi for their growth and mycotoxin production. It was found that optimal temperature for *F.graminearum* growth on soybean contained in

ppm and 1040 ppm for ZEA and DON, respectively, were detected. Fumonisins were pro‐

Most fungi need at least 1-2% of O2 for their growth. The influence of high carbon dioxide and low oxygen concentrations on the growth and mycotoxin production by the foodborne fungal species was investigated by [32]. Three groups of species were distinguished: first, which did not grow in 20% CO2 <0.5% O2 (*Penicillium commune, Eurotium chevalieri* and *Xero‐ myces bisporus*); second, which grew in 20% CO2 <0.5% O2, but not 40% CO2 <0.5% O2 (*Peni‐ cillium roqueforti* and *Aspergillus flavus*); and third, which grew in 20%, 40% and 60% CO2 <0.5% O2 (*Mucor plumbeus*, *Fusarium oxysporum*, *F.moniliforme*, *Byssochlamys fulva* and *B.ni‐ vea*). The production of aflatoxin, patulin, and roquefortine C was greatly reduced under all of the atmospheres tested. For example, aflatoxin was not produced by *A. flavus* during growth under 20% CO2 for 30 days. Patulin was produced by *B.nivea* in the atmospheres of

Nutritional factors such as carbonohydrate and nitrogen sources and microelements (cop‐ per, zinc, cobalt) affect mycotoxin production, but the mechanisms of this impact are still

C for zearalenone (ZEA). After 15 days of incubation, the maximum levels 39

C (in cycling temperature). The op‐

C for deoxynivalenol

C; on soybean no fumoni‐

conditions for its growth and other conditions for its toxin production.

C (in isothermal temperature) and 15/25o

timal temperature for mycotoxin production on soybean was 20o

duced by *Fusarium graminearum* only the on culture medium at 30o

20% and 40% CO2, but only at low levels [32].

The simultaneous presence of different microorganisms, such as bacteria or other fungi, could disturb fungal growth and the production of mycotoxins. For instance, *Alternaria* and *Fusarium* are antagonistic, and *Alternaria* was less abundant in grain with a high incidence rate of *F.culmorum*. *Epicoccum* is a strong antagonist too [25].

At 30o C, the ochratoxin production by *Aspergillus ochraceus* was inhibited by *A.candidus*, *A.flavus*, and *A.niger* in 0.995 aw. At 18o C and 0.995 aw, the interaction between *Aspergillus ochraceus* and *Alternaria alternata* resulted in a significant stimulation of ochratoxin A pro‐ duction [36]. Therefore, several microorganisms were reported as effective biocontrol agents against several fungal plant pathogens [37]. It was determined that *Trichoderma harzianum* produces a lytic enzyme, chitinase, which manifests antifungal activity against a wide range of fungal strains. It was found that non-toxigenic *T.harzianum* isolates significantly reduce the production of six types of A trichothecenes in cereals [38].

can be colonised by *A.flavus* and related species in the field. Out of the other grains, rice is an important dietary source of aflatoxins in tropical and subtropical areas. In regions with moderate climate, the problem is connected with imported commodities or the local crops that are wet or stored in improper conditions [45]. The carcinogenicity, mutagenicity and acute toxicology of AFB1 have been well documented. The IARC determined it to be a hu‐

Ochratoxin A is a chlorinated isocumarin derivative, which contains a chlorinated isocoumar‐ in moiety linked through a carboxyl group to L-phenylalanine via an amide bond. It is colour‐ less, crystalline, and soluble in polar organic solvents compounds. This toxin is more stable in the environment than AFs. The studies of [45] reported that thermal destruction of OTA oc‐

*riantiogriseum*, *P.nordicum*, *P.palitans*, *P. commune*, *P.variabile* and by *Aspergillus* species e.g. *A.ochraceus*, *A.melleus, A.ostanius*, as well as the aspergilli species of section *Nigri*. In moderate climates, the main producers of OTA are *Penicillium* species, while *Aspergillus* species domi‐ nate in tropical and subtropical climates. Ochratoxin A is often found with citrinin produced by *Penicillium aurantiogriseum*, *P.citrinum*, and *P.expansum* [48]. Significant human exposure comes from the consumption of grape juice, wine, coffee, spices, dried fruits and cereal-based products, e.g. whole-grain breads, and in addition to this from products of animal origin, e.g. pork and pig blood-based products. The Scientific Panel on Contaminants in the Food Chain of the European Food Safety Authority (EFSA) has derived an OTA tolerable weekly intake (TWI) on the level of 120ng/kg b.w. The IARC [49] determined it to be a possible human carci‐ nogen (group 2B). Ochratoxins are the cause of urinary tract cancers and kidney damage. In ru‐

minants, ochratoxin A is divided to non-toxic ochratoxin alfa and phenylalanine [44].

Citrinin is a polyketide nephrotoxin produced by several species of the genera *Aspergillus*, *Penicillium* and *Monascus*. Some of the citrinin-producing fungi are also able to produce ochratoxin A or patulin. Citrinin is insoluble in cold water, but soluble in aqueous sodium hydroxide, sodium carbonate, or sodium acetate; in methanol, acetonitrile, ethanol, and most other polar organic solvents. Thermal decomposition of citrinin occurs at >175 °C un‐ der dry conditions, and at > 100 °C in the presence of water. The known decomposition products include citrinin H2 which did not show significant cytotoxicity, whereas the de‐ composition product citrinin H1 showed an increase in cytotoxicity as compared to the pa‐ rent compound [50].The most commonly contaminated commodities are barley, oats, and corn, but contamination can also occur in case of other products of plant origin e.g. beans, fruits, fruit and vegetable juices, herbs and spices, and also in spoiled dairy products [50].

Fumonisins are a group of diester compounds with different tricarboxylic acids and polyhy‐ dric alcohols and primary amine moiety. There are several fumonisins, but only fumonisins

C. OTA is produced by *Penicillium* species such as *P.verrucosum*, *P.au‐*

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193

man carcinogen (group 1A).

**3.2. Ochratoxin A (OTA)**

curs after exceeding 250o

**3.3. Citrinin**

**3.4. Fumonisins (Fs)**

According to [39], soybean is not a favourable medium for ZEA production since it possess‐ es some features that limit the production of this toxin by *Fusarium* isolates. Similarly, the production of aflatoxin B1 by *Aspergillus flavus* was suppressed by soybean phytoalexin – glyceollin [40].
