**6. Current EU regulations concerning mycotoxins**

Since the discovery of aflatoxins in the 1960s, regulations have been established in many countries to protect consumers from harmful mycotoxins that can contaminate foods. Maxi‐ mum levels of mycotoxins have been established by the European Commission after consul‐ tations with the Scientific Committee for Food, based on the analysis of scientific data collected by EFSA and the Codex Alimentarius.

These data include [73, 96]:

Fumonisin B1 was classified by the IARC as a group 2B carcinogen (possibly carcinogenic for humans) [44]. Fumonisins, which inhibit the absorption of folic acid through the foliate re‐ ceptor, have also been implicated in the high incidence of neural tube defects in the rural population known to consume contaminated corn, such as the former Transkei region of

Trichothecenes have been proposed as potential biological warfare agents. In the years 1975-1981, T-2 toxin was implicated as a chemical agent "yellow rain" used against the Lao Peoples Democratic Republic. A study conducted from 1978 to 1981 in Cambodia revealed the presence of T-2 toxin, DON, ZEA, and nivalenol in water and leaf samples taken from the affected areas [75, 84]. Clinical symptoms proceeding to death included vomiting, diar‐ rhoea, bleeding, and difficulty with breathing, pain, blisters, headache, fatigue and dizzi‐ ness. There also occurred necrosis of the mucosa of the stomach as well as the small intestine, lungs and liver [85]. One disease outbreak was recorded in China and was associ‐ ated with the consumption of scabby wheat containing 1000-40000 ppb of DON. The disease is characterized by gastrointestinal symptoms. Also, in India there took place a reported in‐ fection associated with the consumption of bread made from contaminated wheat (DON 350-8300 ppb, acetyldeoxynivalenol 640-2490 ppb, NIV 30-100 ppb and T-2 toxin 500-800 ppb). The disease is characterized by gastrointestinal symptoms and throat irritation, which developed within 15 minutes to one hour after ingestion of the contaminated bread [81].

Animals may show varied symptoms upon contact with mycotoxins, depending on the ge‐ netic factors (species, breed, and strain), physiological factors (age, nutrition) and environ‐ mental factors (climatic conditions, rearing and management). The natural contamination with mycotoxins in animal feed usually does not occur at the levels that may cause acute or overt mycotoxicosis, such as hepatitis, bleeding, nephritis and necrosis of the oral and enter‐ ic epithelium, and even death. It is often difficult to observe and diagnose the symptoms of the disease, but it certainly is the most common form of mycotoxicosis in farm animals, af‐ fecting such parameters as productivity, growth and reproductive performance, feed effi‐

The negative effects of mycotoxins on the performance of poultry have been shown in nu‐ merous studies. For example, feeding the broilers with feed containing an AFs mixture (79% AFB1, 16% AFG1, AFB2 4% and 1% AFG2) in the concentration of 3.5 ppm decreased their body weight and increased their liver and kidney weight [75, 86]. Feeding OTA (0.3-1 ppm) to broilers reduced glycogenolysis and dose-dependent accumulation of glycogen in the liv‐ er. These negative metabolic reactions were attributed to inhibition of cyclic adenosine 3',5' monophosphate-dependent protein kinase, and were reflected in reduced efficiency of feed

*Fusarium* mycotoxins proved to be harmful to poultry. In addition to reduced feed intake and weight gain, sore mouth, cheeks and plaque formation was observed after 7-day-old chicks were exposed to T-2 toxin (4 or 16 ppm) [75, 87]. Pigs are among the most sensitive species to mycotoxins. In the study by [88], pigs in response to AFs (2 ppm), OTA (2 ppm),

South Africa and some areas of Northern China [75, 83].

202 Soybean - Pest Resistance

**5.2. Negative effects of mycotoxins on animal**

utilization and teratogenic malformations [75].

ciency, milk and egg production.


The first two factors provide the information necessary for risk assessment and exposure as‐ sessment, respectively. Risk assessment is the scientific evaluation of the likelihood of known or potential adverse health effects resulting from human exposure to food-borne hazards. It is a fundamental scientific basis for the notification of regulations. The third and fourth factors are important factors in enabling the practical enforcement of mycotoxins, through appropriate procedures as regards sampling and analysis. The last factor is the only one economic in nature, but it is equally important in decision-making to establish reasona‐ ble rules and restrictions for mycotoxins in foods and feeds [96].

homogenisation, for different matrix types have been regulated. The EU Commission Regu‐ lation (EC) 401/2006 established the methods of sampling and analysis for the official control of mycotoxins in foodstuffs [106]. Official sampling plans for aflatoxins in dry figs, ground‐ nuts, peanuts, oilseeds, apricot kernels and tree nuts and for ochratoxins in coffee and liquo‐ rice root are provided in the Commission Regulation (EU) No 178/2010 [107 ]. The sampling frequency and the method of sampling for cereals and cereal products for lots >50 tonnes and <50 tonnes, as well as for retail packed products were presented. Moreover, the proce‐ dures of subdivision of lots into sublots depending on the product and lot weight were also

According to the current regulations where no specific methods for the determination of mycotoxin levels in food are required by the EU regulations, laboratories may select any method provided that they meet the relevant criteria presented in [106, 107]. These criteria are different in relation to individual mycotoxins, and the limit of detection, precision, and recovery depends on the concentration range. The analytical results must be submitted corrected or uncorrected for recovery and the level of recovery expressed in % must be re‐

The main analytical procedures for the determination of the major mycotoxins from com‐ plex biological matrices consist of the following steps: sampling, extraction, purification, de‐ tection, quantification, and finally confirmation. The current development in mycotoxin

**Regulation Matrix Maximum levels [ppb]**

Processed cereal-based foods for infants and

Bread (including small bakery wares), pastries, biscuits, cereal snacks and breakfast cereals

Maize-based breakfast cereals and maize-based

consumption, cereal flour, bran and germ as an end product marketed for direct human

Unprocessed maize with the exception of unprocessed maize intended to be processed by

Cereals intended for direct human

**FOOD**

All cereals and all products derived from cereals 2.0 - - - - Maize and rice 5.0 - - - -

Unprocessed cereals - - 1250 100 - Unprocessed durum wheat and oats - - 1750 - - Pasta (dry) - - 750 - -

**AFB1 OTA DON ZEA F**

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0.10 - - - -





summarised [106, 107].

estimation was reviewed by [108-110].

young children

snacks

wet milling

consumption

ported too.

Commission Regulation (EU) 165/2010

Commission Regulation (EC) 1126/2007

According to the Commission Regulations, the maximum levels should be set at a strict lev‐ el, which is reasonably achievable by following good agricultural and manufacturing practi‐ ces and taking into account the risk related to the consumption of food. Health protection of infants and young children requires establishing the lowest maximum levels, which is ach‐ ievable through the selection of raw materials used for the manufacturing of foods for this vulnerable group of consumers. Development of international trade, progress in research fo‐ cused on mycotoxin food contamination and their toxicological properties cause changes in the mycotoxin-related legislationacross the European Union. The Commission Regulation 466/2001 [97] setting the maximum levels for certain contaminants in foodstuffs has been substantially amended many times. Te current maximum levels for mycotoxins in food are specified by the Commission Regulation EU 1881/2006 and the Commission Regulation EU 105/2010 as regards OTA, the Commission Regulation EU 165/2010 as regards aflatoxins, and the Commission Regulation EU 1126/2007 as regards *Fusarium* toxins [62, 65, 98, 99]. There have also been established maximum levels for aflatoxins, ochratoxin A, patulin, and *Fusarium* toxin (fumonisin, deoxynivalenol, zearalenone) in different products: nuts, cereals, dried fruit, unprocessed cereals, processed cereal-based food, coffee, wine, spices, and liquo‐ rices [62, 65, 97-99].

The number of countries that have regulations concerning mycotoxins is continuously in‐ creasing, and at least 100 countries are known to have founded specific limits for different combinations of mycotoxins and commodities, often accompanied by the prescribed or rec‐ ommended procedures for sampling and analysis [100]. Specific regulations for food in dif‐ ferent world regions were summarized by [101].

As for feeds, the legal situation is somewhat different and only aflatoxin B1 is regulated by the Directive 2002/32/EC on undesirable substances in animal food amended by the Com‐ mission Directive (EC) 100/2003 [102, 103]. For other mycotoxins, such as deoxynivalenol, zearalenone, ochratoxin A and fumonisin B1 and B2 - only non-binding recommendation val‐ ues in the Commission Recommendation 2006/57/EC [104] are determined for feeds (Table 6). This results from the fact that with the exception of aflatoxin-contaminated feed which either directly or indirectly affects human health, there is only a slight transfer to animal products [104, 105].

Table 5 presents the current maximum levels of mycotoxin content as regards cereals and cereal-based foods and feeds.

Mycotoxins in agricultural commodities are distributed heterogeneously. Therefore, sam‐ pling plays a crucial role in making the estimation of the levels of mycotoxin presence more precise. In order to obtain representative samples, sampling procedures, and particularly homogenisation, for different matrix types have been regulated. The EU Commission Regu‐ lation (EC) 401/2006 established the methods of sampling and analysis for the official control of mycotoxins in foodstuffs [106]. Official sampling plans for aflatoxins in dry figs, ground‐ nuts, peanuts, oilseeds, apricot kernels and tree nuts and for ochratoxins in coffee and liquo‐ rice root are provided in the Commission Regulation (EU) No 178/2010 [107 ]. The sampling frequency and the method of sampling for cereals and cereal products for lots >50 tonnes and <50 tonnes, as well as for retail packed products were presented. Moreover, the proce‐ dures of subdivision of lots into sublots depending on the product and lot weight were also summarised [106, 107].

known or potential adverse health effects resulting from human exposure to food-borne hazards. It is a fundamental scientific basis for the notification of regulations. The third and fourth factors are important factors in enabling the practical enforcement of mycotoxins, through appropriate procedures as regards sampling and analysis. The last factor is the only one economic in nature, but it is equally important in decision-making to establish reasona‐

According to the Commission Regulations, the maximum levels should be set at a strict lev‐ el, which is reasonably achievable by following good agricultural and manufacturing practi‐ ces and taking into account the risk related to the consumption of food. Health protection of infants and young children requires establishing the lowest maximum levels, which is ach‐ ievable through the selection of raw materials used for the manufacturing of foods for this vulnerable group of consumers. Development of international trade, progress in research fo‐ cused on mycotoxin food contamination and their toxicological properties cause changes in the mycotoxin-related legislationacross the European Union. The Commission Regulation 466/2001 [97] setting the maximum levels for certain contaminants in foodstuffs has been substantially amended many times. Te current maximum levels for mycotoxins in food are specified by the Commission Regulation EU 1881/2006 and the Commission Regulation EU 105/2010 as regards OTA, the Commission Regulation EU 165/2010 as regards aflatoxins, and the Commission Regulation EU 1126/2007 as regards *Fusarium* toxins [62, 65, 98, 99]. There have also been established maximum levels for aflatoxins, ochratoxin A, patulin, and *Fusarium* toxin (fumonisin, deoxynivalenol, zearalenone) in different products: nuts, cereals, dried fruit, unprocessed cereals, processed cereal-based food, coffee, wine, spices, and liquo‐

The number of countries that have regulations concerning mycotoxins is continuously in‐ creasing, and at least 100 countries are known to have founded specific limits for different combinations of mycotoxins and commodities, often accompanied by the prescribed or rec‐ ommended procedures for sampling and analysis [100]. Specific regulations for food in dif‐

As for feeds, the legal situation is somewhat different and only aflatoxin B1 is regulated by the Directive 2002/32/EC on undesirable substances in animal food amended by the Com‐ mission Directive (EC) 100/2003 [102, 103]. For other mycotoxins, such as deoxynivalenol, zearalenone, ochratoxin A and fumonisin B1 and B2 - only non-binding recommendation val‐ ues in the Commission Recommendation 2006/57/EC [104] are determined for feeds (Table 6). This results from the fact that with the exception of aflatoxin-contaminated feed which either directly or indirectly affects human health, there is only a slight transfer to animal

Table 5 presents the current maximum levels of mycotoxin content as regards cereals and

Mycotoxins in agricultural commodities are distributed heterogeneously. Therefore, sam‐ pling plays a crucial role in making the estimation of the levels of mycotoxin presence more precise. In order to obtain representative samples, sampling procedures, and particularly

ble rules and restrictions for mycotoxins in foods and feeds [96].

rices [62, 65, 97-99].

204 Soybean - Pest Resistance

products [104, 105].

cereal-based foods and feeds.

ferent world regions were summarized by [101].

According to the current regulations where no specific methods for the determination of mycotoxin levels in food are required by the EU regulations, laboratories may select any method provided that they meet the relevant criteria presented in [106, 107]. These criteria are different in relation to individual mycotoxins, and the limit of detection, precision, and recovery depends on the concentration range. The analytical results must be submitted corrected or uncorrected for recovery and the level of recovery expressed in % must be re‐ ported too.

The main analytical procedures for the determination of the major mycotoxins from com‐ plex biological matrices consist of the following steps: sampling, extraction, purification, de‐ tection, quantification, and finally confirmation. The current development in mycotoxin estimation was reviewed by [108-110].



mate, and agronomic practices, before attempting to implement the provisions specified in the Code. The recommendations for the reduction of various mycotoxins in cereals are div‐ ided into two parts: recommended practices based on Good Agricultural Practice (GAP) and Good Manufacturing Practice (GMP); a complementary management system to consider in

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Recommendations to be taken into account before the harvest in order to reduce the risk of

**•** avoid nutrient stress – apply the appropriate amount of organic or inorganic fertiliser;

The main mycotoxin hazards associated with pre-harvest in Europe are the toxins that are produced by fungi belonging to the genus *Fusarium* in the growing crops. It is important to note that although *Fusarium* infection is generally considered to be a pre-harvest problem, it is certainly possible for poor drying practices to lead to crops' susceptibility in storage and mycotoxin contamination [113]. This part of the book will discuss some pre-harvest strat‐ egies appropriate to reduce the prevalence of fungi belonging to the genus *Fusarium* and

There are inherent differences in the susceptibility of cereal species to *Fusarium* infections. The differences between crop species appear to vary between countries. This is probably due to the differences in the genetic pool within each country's breeding program and the diverse environmental and agronomic conditions in which crops are cultivated [114, 115]. It was observed that oats had higher levels of DON than barley and wheat in Norway from 1996 to 1999, whereas the DON levels in wheat, barley and oats were similar when grown

Numerous studies have shown that fumonisins or DON contamination in wheat is affected by the previous crop. It was shown that a higher incidence of Fs occurred in wheat after

the future is the use of Hazard Analysis Critical Control Point (HACCP) [111].

mould contamination and mycotoxin production include [112]: **•** use certified seed or ensure it is free from fungal infections;

**•** sow the seed as early as possible, so that crop matures early;

**•** when practising minimum or zero tillage, remove crop residues;

under the same field conditions in Western Canada in 2001 [116].

**•** avoid drought stress – irrigate if possible;

**•** plant resistant varieties where these are available

**•** weed regularly;

**•** rotate crops;

their mycotoxins.

**7.1. Resistance**

**7.2. Field management**

Crop rotation

**•** control insect and bird pests;

**Table 5.** Legislation on mycotoxins as regards cereals and cereal-based foods and feeds

### **7. Prevention strategies of exposure to mycotoxins**

Several codes of practice have been developed by Codex Alimentarius for the prevention and reduction of mycotoxins in cereals, peanuts, apple products, and other raw materials. In order for this practice to be effective, it will be necessary for the producers in each country to consider the general principles given in the Code, taking into account their local crops, cli‐ mate, and agronomic practices, before attempting to implement the provisions specified in the Code. The recommendations for the reduction of various mycotoxins in cereals are div‐ ided into two parts: recommended practices based on Good Agricultural Practice (GAP) and Good Manufacturing Practice (GMP); a complementary management system to consider in the future is the use of Hazard Analysis Critical Control Point (HACCP) [111].

Recommendations to be taken into account before the harvest in order to reduce the risk of mould contamination and mycotoxin production include [112]:


**Regulation Matrix Maximum levels [ppb]**

Unprocessed cereals - 5.0 - - -

Maize by-products - - 12000 3000 -

All feed materials 20 - - - - Complete feedingstuffs for dairy animals 5 - - - - Complete feedingstuffs for calves and lambs 10 - - - -

**FEED**

(-) limit not established; AFB1 – aflatoxin B1; OTA – ochratoxin A; ZEA – zearalenone; DON – deoxynivalenol; F – fumoni‐

Several codes of practice have been developed by Codex Alimentarius for the prevention and reduction of mycotoxins in cereals, peanuts, apple products, and other raw materials. In order for this practice to be effective, it will be necessary for the producers in each country to consider the general principles given in the Code, taking into account their local crops, cli‐

Milling fractions of maize and milling products with particle size "/> 500 micron not used for

Milling fractions of maize and maize milling products with particle size ≤ 500 micron not used for direct human consumption

Processed cereal-based foods for infants and

Processed maize-based foods for infants and

All products derived from unprocessed cereals, including processed cereal products and cereals intended for direct human consumption

Processed cereal-based foods for infants and

Cereals and cereal products with the exception

Complementary and complete feedingstuffs for

Complementary and complete feedingstuffs for

Complementary and complete feedingstuffs for

Complete feedingstuffs for pigs, poultry, cattle,

**Table 5.** Legislation on mycotoxins as regards cereals and cereal-based foods and feeds

**7. Prevention strategies of exposure to mycotoxins**

direct human consumption

young children

young children

young children

pigs

poultry

of maize by-products

calves, lambs and kids

sheep and goats

Commission Regulation (EC) 1881/2006

206 Soybean - Pest Resistance

Commission Recommendation (EC) 576/2006

Commission Directive (EC) 100/2003

sins

**AFB1 OTA DON ZEA F**











20 - - - -


The main mycotoxin hazards associated with pre-harvest in Europe are the toxins that are produced by fungi belonging to the genus *Fusarium* in the growing crops. It is important to note that although *Fusarium* infection is generally considered to be a pre-harvest problem, it is certainly possible for poor drying practices to lead to crops' susceptibility in storage and mycotoxin contamination [113]. This part of the book will discuss some pre-harvest strat‐ egies appropriate to reduce the prevalence of fungi belonging to the genus *Fusarium* and their mycotoxins.

#### **7.1. Resistance**

There are inherent differences in the susceptibility of cereal species to *Fusarium* infections. The differences between crop species appear to vary between countries. This is probably due to the differences in the genetic pool within each country's breeding program and the diverse environmental and agronomic conditions in which crops are cultivated [114, 115]. It was observed that oats had higher levels of DON than barley and wheat in Norway from 1996 to 1999, whereas the DON levels in wheat, barley and oats were similar when grown under the same field conditions in Western Canada in 2001 [116].

#### **7.2. Field management**

#### Crop rotation

Numerous studies have shown that fumonisins or DON contamination in wheat is affected by the previous crop. It was shown that a higher incidence of Fs occurred in wheat after maize and, in particular, in wheat after a succession of two maize crops and in wheat fol‐ lowing grain maize compared to silage maize. In Ontario, Canada, in 1983, the fields where maize was the previous crop had a significantly higher incidence of fumonisins than the fields where the previous crop was a small grain cereal or soybean [117]. In a repeated study, the following year, the fields where maize was the previous crop had a 10-fold DON content than the fields following a crop other than maize [118]. The research of [119] found higher levels of fumonisins in wheat following wheat rather than wheat following fallow.

barley and triticale. Similarly, in the studies of [127], a significant increase in fumonisins and deoxynivalenol contamination in the grain of wheat and kernels was observed with increas‐ ing N fertilizer from 0 to 80 kg/ha. That research concluded that in practical crop husbandry, Fs cannot be sufficiently controlled by only manipulating the N input [111]. The study of [128] showed that the use of six different combinations of agricultural practices (sowing time, plant density, N fertilization and European corn borer (ECB) control with insecticide) can effectively lead to good control of fumonisins and deoxynivalenol in maize kernels.

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209

In accordance with the guidelines contained in the Codex Alimentarius [122], farmers should minimize insect damage and fungal infections of the crop by proper use of registered insecticides, fungicides and other appropriate practices within an integrated pest manage‐

Some studies have been conducted to examine the effectiveness of the fungicides which are applied during flowering can reduce *Fusarium* infections and subsequent DON in the har‐ vested grains. The results of [129] provided that azoles, tebuconazole, metconazole and pro‐ thioconazole significantly reduced the *Fusarium* disease symptoms and *Fusarium* mycotoxin concentrations. The greatest reduction in the DON concentration occurred with prothioco‐ nazole (10-fold). Azoxystrobin had little impact on the mycotoxin concentration in the har‐ vested grain infected by *Fusarium* species, but could increasing the mycotoxin concentration in grains when *F. nivale* was the predominant species present [130, 131]. Fungicide mixtures of azoxystrobin and azole resulted in a lower reduction of DON, compared to azole alone [120, 132]. A number of trials in Germany have indicated that some strobilurin fungicides applied before anthesis can also result in increased DON compared to unsprayed plots [133]. Reductions in DON observed in field experiments using fungicides against natural in‐ fections of *Fusarium* are lower and inconsistent [134]. This is probably due to the fact that

Alternatively, a limited number of biocompetitive microorganisms have been shown useful for the management of *Fusarium* infections [111]. Research has demonstrated the successful use of bacteria in biocontrol of mycotoxigenic fungi. One bacterium, *Enterobacter cloacae* was discovered as an endophytic symbiont of corn [135]. Corn plants with roots endophytically colonized by these bacteria were observed to be fungus-free and *in vitro* control of *F.verticil‐ lioides* and other fungi with this bacterium was demonstrated. An endophytic bacterium, *Ba‐ cillus subtilis* showed promising for reducing the mycotoxin contamination with *F.verticillioides* during the endophytic growth phase [136]. Yeast antagonists such as *Crypto‐ coccus nodaensis* were isolated from wheat anthers. The antagonists reduced *Fusarium* head blight severity by up to 93% in greenhouse and by 56% in field trials when sprayed onto flowering wheat heads [137]. The most successful antagonists reduced the DON content of

Actions to be taken during harvest in order to reduce the risk of mould contamination and

during a natural infection, the infection occurs over a longer period of time.

grain more than 10-fold in greenhouse studies [138].

mycotoxin production include [112]:

**7.4. Use of chemical and biological agents**

ment program.

An observational study performed using commercial fields in Canada [120] identified signif‐ icantly lower DON content in wheat following soybean or wheat, compared to wheat fol‐ lowing maize. In New Zealand, an observational study determined that higher levels of DON occurred in wheat grown after maize (mean = 600 ppb) and after grass (mean = 250 ppb), compared to small grain cereals (mean = 90 ppb) and other crops (mean = 70 ppb). The highest levels were recorded in wheat-maize rotations [121].

Codex recommends that crops such as potatoes, other vegetables, clover and alfalfa that are not hosts to *Fusarium* species should be used in rotation to reduce the inoculum in the field [122].

#### **7.3. Soil cultivation**

Soil cultivation can be divided into ploughing, where the top 10-30 cm of soil are inverted; min‐ imum tillage, where the crop debris is mixed with the top 10-20 cm of soil; and no till, where seed is directly drilled into the previous crop stubble with minimum disturbance to the soil structure [111]. In the 1990s, a large observational study of Fs and DON was conducted in Ger‐ many (n=1600). The DON concentration of wheat crops after maize was ten-times higher in the field that was min-tilled compared to the ploughed one [123]. In wheat the DON concentra‐ tion after min-till was 1300 ppb, after no-till it was 700 ppb and after ploughing it was 500 ppb [120]. Studies in France have determined that crop debris management can have a large im‐ pact on the DON concentration at harvest, particularly after maize. The highest DON concen‐ tration was found after no-till, followed by min-till, whereas the lowest DON levels were recorded after ploughing. The reduction in DON has been linked to the reduction in crop resi‐ due on the soil surface [124]. Large replicated field trials in Germany identified that there was a significant interaction between the previous crop and the cultivation technique [125]. Follow‐ ing sugar beet, there was no significant difference in the DON concentration between wheat plots receiving different methods of cultivation; however, following a wheat crop without straw removal, direct drilled wheat had a significantly higher DON level compared to wheat from plots which were either ploughed or min-tilled [125].

In accordance with the guidelines contained in the Codex Alimentarius, soil should be tested to determine if there is need to apply a fertilizer and/or soil conditioners to assure adequate soil pH and plant nutrition to avoid plant stress, especially during seed devel‐ opment [122].

Research of [126] showed that supplementary nitrogen and a plant growth regulator in‐ creased, by up to 125%, the incidence of infection by *Fusarium* species in the seed of wheat, barley and triticale. Similarly, in the studies of [127], a significant increase in fumonisins and deoxynivalenol contamination in the grain of wheat and kernels was observed with increas‐ ing N fertilizer from 0 to 80 kg/ha. That research concluded that in practical crop husbandry, Fs cannot be sufficiently controlled by only manipulating the N input [111]. The study of [128] showed that the use of six different combinations of agricultural practices (sowing time, plant density, N fertilization and European corn borer (ECB) control with insecticide) can effectively lead to good control of fumonisins and deoxynivalenol in maize kernels.

#### **7.4. Use of chemical and biological agents**

maize and, in particular, in wheat after a succession of two maize crops and in wheat fol‐ lowing grain maize compared to silage maize. In Ontario, Canada, in 1983, the fields where maize was the previous crop had a significantly higher incidence of fumonisins than the fields where the previous crop was a small grain cereal or soybean [117]. In a repeated study, the following year, the fields where maize was the previous crop had a 10-fold DON content than the fields following a crop other than maize [118]. The research of [119] found higher levels of fumonisins in wheat following wheat rather than wheat following fallow.

An observational study performed using commercial fields in Canada [120] identified signif‐ icantly lower DON content in wheat following soybean or wheat, compared to wheat fol‐ lowing maize. In New Zealand, an observational study determined that higher levels of DON occurred in wheat grown after maize (mean = 600 ppb) and after grass (mean = 250 ppb), compared to small grain cereals (mean = 90 ppb) and other crops (mean = 70 ppb). The

Codex recommends that crops such as potatoes, other vegetables, clover and alfalfa that are not hosts to *Fusarium* species should be used in rotation to reduce the inoculum in

Soil cultivation can be divided into ploughing, where the top 10-30 cm of soil are inverted; min‐ imum tillage, where the crop debris is mixed with the top 10-20 cm of soil; and no till, where seed is directly drilled into the previous crop stubble with minimum disturbance to the soil structure [111]. In the 1990s, a large observational study of Fs and DON was conducted in Ger‐ many (n=1600). The DON concentration of wheat crops after maize was ten-times higher in the field that was min-tilled compared to the ploughed one [123]. In wheat the DON concentra‐ tion after min-till was 1300 ppb, after no-till it was 700 ppb and after ploughing it was 500 ppb [120]. Studies in France have determined that crop debris management can have a large im‐ pact on the DON concentration at harvest, particularly after maize. The highest DON concen‐ tration was found after no-till, followed by min-till, whereas the lowest DON levels were recorded after ploughing. The reduction in DON has been linked to the reduction in crop resi‐ due on the soil surface [124]. Large replicated field trials in Germany identified that there was a significant interaction between the previous crop and the cultivation technique [125]. Follow‐ ing sugar beet, there was no significant difference in the DON concentration between wheat plots receiving different methods of cultivation; however, following a wheat crop without straw removal, direct drilled wheat had a significantly higher DON level compared to wheat

In accordance with the guidelines contained in the Codex Alimentarius, soil should be tested to determine if there is need to apply a fertilizer and/or soil conditioners to assure adequate soil pH and plant nutrition to avoid plant stress, especially during seed devel‐

Research of [126] showed that supplementary nitrogen and a plant growth regulator in‐ creased, by up to 125%, the incidence of infection by *Fusarium* species in the seed of wheat,

highest levels were recorded in wheat-maize rotations [121].

from plots which were either ploughed or min-tilled [125].

the field [122].

208 Soybean - Pest Resistance

opment [122].

**7.3. Soil cultivation**

In accordance with the guidelines contained in the Codex Alimentarius [122], farmers should minimize insect damage and fungal infections of the crop by proper use of registered insecticides, fungicides and other appropriate practices within an integrated pest manage‐ ment program.

Some studies have been conducted to examine the effectiveness of the fungicides which are applied during flowering can reduce *Fusarium* infections and subsequent DON in the har‐ vested grains. The results of [129] provided that azoles, tebuconazole, metconazole and pro‐ thioconazole significantly reduced the *Fusarium* disease symptoms and *Fusarium* mycotoxin concentrations. The greatest reduction in the DON concentration occurred with prothioco‐ nazole (10-fold). Azoxystrobin had little impact on the mycotoxin concentration in the har‐ vested grain infected by *Fusarium* species, but could increasing the mycotoxin concentration in grains when *F. nivale* was the predominant species present [130, 131]. Fungicide mixtures of azoxystrobin and azole resulted in a lower reduction of DON, compared to azole alone [120, 132]. A number of trials in Germany have indicated that some strobilurin fungicides applied before anthesis can also result in increased DON compared to unsprayed plots [133]. Reductions in DON observed in field experiments using fungicides against natural in‐ fections of *Fusarium* are lower and inconsistent [134]. This is probably due to the fact that during a natural infection, the infection occurs over a longer period of time.

Alternatively, a limited number of biocompetitive microorganisms have been shown useful for the management of *Fusarium* infections [111]. Research has demonstrated the successful use of bacteria in biocontrol of mycotoxigenic fungi. One bacterium, *Enterobacter cloacae* was discovered as an endophytic symbiont of corn [135]. Corn plants with roots endophytically colonized by these bacteria were observed to be fungus-free and *in vitro* control of *F.verticil‐ lioides* and other fungi with this bacterium was demonstrated. An endophytic bacterium, *Ba‐ cillus subtilis* showed promising for reducing the mycotoxin contamination with *F.verticillioides* during the endophytic growth phase [136]. Yeast antagonists such as *Crypto‐ coccus nodaensis* were isolated from wheat anthers. The antagonists reduced *Fusarium* head blight severity by up to 93% in greenhouse and by 56% in field trials when sprayed onto flowering wheat heads [137]. The most successful antagonists reduced the DON content of grain more than 10-fold in greenhouse studies [138].

Actions to be taken during harvest in order to reduce the risk of mould contamination and mycotoxin production include [112]:


nation [141, 142]. Chemical decontamination is very effective, but these methods are expensive and affect the feedstuff quality. Among the chemical methods, only peroxide and ammonia are mostly used for aflatoxin removal from feed. Ammoniation works by irreversi‐ bly converting AFB1 to less toxic products such as AFD1 [143]. Data show that treatment of maize contaminated with 1000 or 2000 ppb aflatoxins with 1% of aqueous ammonia for 48 h removed 98% of the aflatoxins. There was no significant change in the dietary intake, body weight gain, and feed conversion ratio in chickens fed with ammonia-treated aflatoxin-con‐ taminated maize, whereas these parameters were suppressed in birds fed with aflatoxincontaining diet [142]. Atmospheric ammoniation of corn does not appear to be an effective method for the detoxification of *F.moniliforme–*contaminated material. In the research of [144], the levels of fumonisin B1 in naturally contaminated corn were reduced by about 45% due to the ammonia treatment. Despite this, the toxicity of the culture material in rats was

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A recent and promising approach to protect animals against the harmful effects of mycotox‐ in-contaminated feed is the use of mycotoxin binders (MB). They are added to the diet in order to reduce the absorption of mycotoxins from the gastrointestinal tract and their distri‐ bution to blood and target organs. These feed additives may act either by binding mycotox‐ ins to their surface (adsorption), or by degrading or transforming them into less toxic metabolites (biotransformation). Various inorganic adsorbents, such as hydrated sodium calcium aluminosilicate, zeolites, bentonites, clays, and activated carbons, have been used as mycotoxin binders. The use of mycotoxin binders is discussed in some review articles [145-147]. The best aflatoxin adsorbent seems to be HSCAS (hydrated sodium calcium alu‐ minosilicate), which rapidly and preferentially binds aflatoxins in the gastrointestinal tract [148-150]. The prevention of aflatoxicosis in broiler folders was examined by [150]. HSCAS and activated charcoal were incorporated into the diets for broilers containing purified afla‐ toxin B1 (7.5 ppm), or natural aflatoxin produced by *Aspergillus parasiticus* on rice (5 ppm). The authors showed that HSCAS significantly decreased the growth-inhibitory effects of AFB1 or AFs on the growing chicks, namely by 50 to 67%. The authors suggest that HSCAS can modulate the toxicity of aflatoxins in chickens; however, adding activated charcoal to

the diet did not appear to have protective properties against mycotoxicosis [150].

nately, few of these findings have practical application.

Physical and chemical methods have a lot of disadvantages; in many cases they do not meet the FAO requirements. Therefore, the use of other methods is considered. Biological meth‐ ods, involving decontamination with microorganisms or enzymes, give promising results. Recently, an increase in the research connected with mycotoxin detoxification by microor‐ ganisms has been observed. Several studies have shown that some bacteria, moulds and yeasts such as *Flavobacterium auriantiacum*, *Corynebacterium rubrum*, lactic acid bacteria (*Lac‐ tobacillus acidophilus, L.rhamnosus, L.bulgaricus*), *Aspergillus niger, Rhizopus nigricans*, *Candida sp*., *Kluyveromyces sp.*, etc. are able to conduct detoxification of mycotoxins (Tab. 6). Unfortu‐

Already in 1966, a review of microorganisms was conducted by [151] as for their capability of degrading aflatoxins. It was found that yeasts, actinomycetes and algae did not show this trait, but some moulds, such as *Aspergillus niger*, *A. parasiticus, A. terreus, A. luchuensis,* and

not altered by ammoniation.

**•** bundles of stover should also be placed on platforms to dry and not left lying on the soil

The post-harvest strategies include improving the drying and storage conditions together with the use of chemical, physical or biological methods.

### **8. Methods of removing mycotoxins from cereals**

When mycotoxin prevention is not satisfactory, some decontamination methods are needed. The use of detoxification methods is allowed only in the case of feed and feed components. Foodstuffs containing contaminants exceeding the maximum levels should not be placed on the market either as such, in the form of a mixture with other foodstuffs or used as an ingre‐ dient in other foods. Food contaminated with mycotoxins is not safe for consumers and no decontamination methods can be used.

According to FAO [111, 139, 140] the feed decontamination process must:


There are some physical methods of decontamination of feed components such as sort‐ ing grains, washing procedures, gamma radiation and UV treatment and also extraction with organic solvents. These methods are summarized by [140]. Physical removal of damaged, mouldy or discoloured kernels significantly decreased the concentration of AF in peanuts. Sorting is not effective for maize and cottonseed. Washing with water or so‐ dium carbonate solutions could decrease the concentration of DON, ZEA and fumonisins in wheat and maize.

High temperature is not used for decontamination of agricultural products, due to thermo‐ stability of mycotoxins. Different types of radiation were tested for mycotoxin detoxifica‐ tion, but the results were not effective enough.

Chemical compounds such as organic acids, ammonium, sodium hydroxide, hydrogen per‐ oxide, ozone, chloride and bisulphite were tested for their efficacy in mycotoxin decontami‐ nation [141, 142]. Chemical decontamination is very effective, but these methods are expensive and affect the feedstuff quality. Among the chemical methods, only peroxide and ammonia are mostly used for aflatoxin removal from feed. Ammoniation works by irreversi‐ bly converting AFB1 to less toxic products such as AFD1 [143]. Data show that treatment of maize contaminated with 1000 or 2000 ppb aflatoxins with 1% of aqueous ammonia for 48 h removed 98% of the aflatoxins. There was no significant change in the dietary intake, body weight gain, and feed conversion ratio in chickens fed with ammonia-treated aflatoxin-con‐ taminated maize, whereas these parameters were suppressed in birds fed with aflatoxincontaining diet [142]. Atmospheric ammoniation of corn does not appear to be an effective method for the detoxification of *F.moniliforme–*contaminated material. In the research of [144], the levels of fumonisin B1 in naturally contaminated corn were reduced by about 45% due to the ammonia treatment. Despite this, the toxicity of the culture material in rats was not altered by ammoniation.

**•** harvest as quickly as possible

**•** transport the crop to the homestead as soon as possible

field on a platform or cut and tied into stooks) to dry

with the use of chemical, physical or biological methods.

decontamination methods can be used.

**•** destroy, inactivate or remove mycotoxins

**•** destroy all fungal morphological forms

in wheat and maize.

**•** not significantly increase the cost of production

tion, but the results were not effective enough.

**8. Methods of removing mycotoxins from cereals**

According to FAO [111, 139, 140] the feed decontamination process must:

**•** not decrease the nutritive value and organoleptic properties

**•** if lack of labour force or time prevents removal from the field, then dry the crops on plat‐ forms raised above ground (if climate is hot and the drying crop can be left to stay on the

**•** bundles of stover should also be placed on platforms to dry and not left lying on the soil The post-harvest strategies include improving the drying and storage conditions together

When mycotoxin prevention is not satisfactory, some decontamination methods are needed. The use of detoxification methods is allowed only in the case of feed and feed components. Foodstuffs containing contaminants exceeding the maximum levels should not be placed on the market either as such, in the form of a mixture with other foodstuffs or used as an ingre‐ dient in other foods. Food contaminated with mycotoxins is not safe for consumers and no

**•** not produce toxic, carcinogenic or mutagenic residues in decontaminated final products

There are some physical methods of decontamination of feed components such as sort‐ ing grains, washing procedures, gamma radiation and UV treatment and also extraction with organic solvents. These methods are summarized by [140]. Physical removal of damaged, mouldy or discoloured kernels significantly decreased the concentration of AF in peanuts. Sorting is not effective for maize and cottonseed. Washing with water or so‐ dium carbonate solutions could decrease the concentration of DON, ZEA and fumonisins

High temperature is not used for decontamination of agricultural products, due to thermo‐ stability of mycotoxins. Different types of radiation were tested for mycotoxin detoxifica‐

Chemical compounds such as organic acids, ammonium, sodium hydroxide, hydrogen per‐ oxide, ozone, chloride and bisulphite were tested for their efficacy in mycotoxin decontami‐

**•** avoid field drying

210 Soybean - Pest Resistance

A recent and promising approach to protect animals against the harmful effects of mycotox‐ in-contaminated feed is the use of mycotoxin binders (MB). They are added to the diet in order to reduce the absorption of mycotoxins from the gastrointestinal tract and their distri‐ bution to blood and target organs. These feed additives may act either by binding mycotox‐ ins to their surface (adsorption), or by degrading or transforming them into less toxic metabolites (biotransformation). Various inorganic adsorbents, such as hydrated sodium calcium aluminosilicate, zeolites, bentonites, clays, and activated carbons, have been used as mycotoxin binders. The use of mycotoxin binders is discussed in some review articles [145-147]. The best aflatoxin adsorbent seems to be HSCAS (hydrated sodium calcium alu‐ minosilicate), which rapidly and preferentially binds aflatoxins in the gastrointestinal tract [148-150]. The prevention of aflatoxicosis in broiler folders was examined by [150]. HSCAS and activated charcoal were incorporated into the diets for broilers containing purified afla‐ toxin B1 (7.5 ppm), or natural aflatoxin produced by *Aspergillus parasiticus* on rice (5 ppm). The authors showed that HSCAS significantly decreased the growth-inhibitory effects of AFB1 or AFs on the growing chicks, namely by 50 to 67%. The authors suggest that HSCAS can modulate the toxicity of aflatoxins in chickens; however, adding activated charcoal to the diet did not appear to have protective properties against mycotoxicosis [150].

Physical and chemical methods have a lot of disadvantages; in many cases they do not meet the FAO requirements. Therefore, the use of other methods is considered. Biological meth‐ ods, involving decontamination with microorganisms or enzymes, give promising results. Recently, an increase in the research connected with mycotoxin detoxification by microor‐ ganisms has been observed. Several studies have shown that some bacteria, moulds and yeasts such as *Flavobacterium auriantiacum*, *Corynebacterium rubrum*, lactic acid bacteria (*Lac‐ tobacillus acidophilus, L.rhamnosus, L.bulgaricus*), *Aspergillus niger, Rhizopus nigricans*, *Candida sp*., *Kluyveromyces sp.*, etc. are able to conduct detoxification of mycotoxins (Tab. 6). Unfortu‐ nately, few of these findings have practical application.

Already in 1966, a review of microorganisms was conducted by [151] as for their capability of degrading aflatoxins. It was found that yeasts, actinomycetes and algae did not show this trait, but some moulds, such as *Aspergillus niger*, *A. parasiticus, A. terreus, A. luchuensis,* and *Penicillium reistrickii,* partially transformed aflatoxin B1 to a new product. Among them, only the bacteria *Flavobacterium aurantiacum* (now *Nocardia corynebacterioides*) is able to remove aflatoxin, both from the media and from the natural environments such as milk, oil, cocoa butter and grain. It was shown that to obtain the apparent loss of the toxin, it was necessary to use the bacterial population with the density of more than 1010 CFU/ml [154, 188].

thors selected two out of 70 isolates of the *Aspergillus* species - *Aspergillus fumigatus* and *As‐ pergillus niger*, which transformed ochratoxin A to ochratoxin α and phenylalanine within 7

Mycotoxins in Cereal and Soybean-Based Food and Feed

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213

In vitro studies conducted by [186] demonstrated the degradation of 12 trichothecene myco‐ toxins conducted by bacteria isolated from the digestive tract of chickens. The transforma‐ tion of the toxin led to their partial or total deacylation and de-epoxidation. Similarly, it was shown, that the strains of anaerobic bacteria - isolated from the rumen, Gram positive, preclassified to the genus *Eubacterium* - are able to perform the transformation of type A tricho‐

The above-presented examples of microbial activity aimed at removal of mycotoxins are mainly of scientific nature, allowing for a better understanding of the strains, their proper‐ ties and the mechanisms of the processes. Their limited practical application made that re‐ search turned in the direction of such organisms, which can be used in biotechnological processes during production, such as fermented food production, where the raw material may be contaminated with mycotoxins. The most important among them are lactic acid bac‐

Literature data indicate the existence of strains of lactic acid bacteria with different abilities to remove mycotoxins, as demonstrated both in *in vitro* and *in vivo* studies conducted by various authors with the use of some strains of probiotic *Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium bifidum*, *B. longum,* and *Streptococcus* spp., *Lactococcus salivarius*, *Lactobacillus delbrueckii* subsp. *bulgaricus* [155, 156, 158, 160, 169, 189, 190]. According to [191], the decontamination process is very fast; after 4h the toxin concentration was reduced from 50 to 77%. It was observed that heat-inactivated cells were more effective than living cells, which results from the changes in the surface properties of cells, which occur under high temperature [191]. The capacity to reduce the content of ochratoxin A in milk by lactic acid bacteria belonging to the species *Lactococcus salivarius*, *Lactobacillus delbrueckii* subsp. *bulgari‐ cus* and *Bifidobacterium bifidum* was confirmed in [167]. The content of patulin in the medium decreased in the level from 10 to 82% under the influence of bacteria belonging to the genus *Lactobacillus* and *Bifidobacterium*. The decontamination process depends on the inoculum density, pH and the concentration of toxins. Among the studied strains, *L.acidophilus*, re‐

moves up to 96% of the toxin added to the medium in an amount of 1ppm [166].

Our *in vivo* experiments indicate that the use of probiotics as feed additives limited the ef‐ fects of mycotoxins in animals, as well as reduced the accumulation of toxins in the tissues, thus reducing the contamination of food of animal origin with the toxins [192]. It was shown that *Lactobacillus rhamnosus* bacteria limited by 75% the adsorption of aflatoxin B1 in the di‐

The second group of organisms with a potential application in detoxification is constituted by *Saccharomyces cerevisiae* yeasts. Our own research demonstrated that these organisms are capa‐ ble of eliminating ochratoxin A from the plant raw material during fermentation and chroma‐ tographic analysis did not show any products of OTA metabolism, which proves that it was not the case of biodegradation. The amount of ochratoxin A removed by bakery yeasts after 24-

days of incubation on both liquid and solid media.

thecenes to non-toxic forms [185].

gestive tract of chickens [189].

teria and yeasts *Saccharomyces cerevisiae* [163].


**Table 6.** Decontamination abilities of microorganisms

It was observed that cultures of toxinogenic *Aspergillus flavus* and *Aspergillus parasiticus* were able to reduce aflatoxin contamination. Aflatoxins were degraded by the strains that pro‐ duce them, but only after the fragmentation of the mycelium. The cause of this phenomenon was absorption into the cell wall of mycelium [165]. In the research of [176], 10 yeast strains of the *Saccharomyces*, *Kluyveromyces* and *Rhodotorula* genera were studied for their ability to perform biodegradation of fumonisin B1, ochratoxin A and trichothecenes. Significant differ‐ ences were demonstrated between the strains, but there were no preferences as to the types of mycotoxins. Fumonisins were removed by the majority of the strains in 100%, the remov‐ al rate for deoxynivalenol ranged from 63 to 100%, and for ochratoxin A from 69 to 100%. The possibility of using moulds to remove ochratoxin A was studied by [179, 182]. The au‐ thors selected two out of 70 isolates of the *Aspergillus* species - *Aspergillus fumigatus* and *As‐ pergillus niger*, which transformed ochratoxin A to ochratoxin α and phenylalanine within 7 days of incubation on both liquid and solid media.

*Penicillium reistrickii,* partially transformed aflatoxin B1 to a new product. Among them, only the bacteria *Flavobacterium aurantiacum* (now *Nocardia corynebacterioides*) is able to remove aflatoxin, both from the media and from the natural environments such as milk, oil, cocoa butter and grain. It was shown that to obtain the apparent loss of the toxin, it was necessary

**Mycotoxin Microorganism References**

*Lactobacillus acidophilus, L.johnsonii, L.salivarius, L.crispatus, L.gasseri, L.rhamnosus, Lactococcus lactis, Bifidobacterium longum, B.lactis, Mycobacterium luoranthenivorans, Rhodococcus erythropolis, Bacillus megaterium, Corynebacterium rubrum, Kluyveromyces marxianus, Saccharomyces cerevisiae, Aspergillus niger, A. terreus, A.luchuensis, Penicillium reistrickii, Trichoderma viride*

*subsp. Bulgaricus, L. acidophilus, Bifidobacterium animalis, B. bifidum, Lactobacillus plantarum, L. brevis, L. sanfranciscensis, L.acidophilus, Acinetobacter calcoaceticus, Rhodococcus erythropolis, Oenococcus oeni, Saccharomyces cerevisiae, Kluyveromyces marxianus, Rhodotorula rubra, Phaffia rhodozyna, Xanthophyllomyces dendrorhous, Metschnikowia pulcherrima, Pichia guilliermondii, Trichosporon mycotoxinivorans, Rhizopus sp., Aureobasidium pullulans, Aspergillus niger, A.carbonarius, A. fumigatus, A. versicolor*

*Saccharomyces cerevisiae, Kluyveromyces marxianus, Rhodotorula rubra*

*Saccharomyces cerevisiae, Kluyveromyces marxianus, Rhodotorula rubra*

*Trichosporon mycotoxinivorans*

It was observed that cultures of toxinogenic *Aspergillus flavus* and *Aspergillus parasiticus* were able to reduce aflatoxin contamination. Aflatoxins were degraded by the strains that pro‐ duce them, but only after the fragmentation of the mycelium. The cause of this phenomenon was absorption into the cell wall of mycelium [165]. In the research of [176], 10 yeast strains of the *Saccharomyces*, *Kluyveromyces* and *Rhodotorula* genera were studied for their ability to perform biodegradation of fumonisin B1, ochratoxin A and trichothecenes. Significant differ‐ ences were demonstrated between the strains, but there were no preferences as to the types of mycotoxins. Fumonisins were removed by the majority of the strains in 100%, the remov‐ al rate for deoxynivalenol ranged from 63 to 100%, and for ochratoxin A from 69 to 100%. The possibility of using moulds to remove ochratoxin A was studied by [179, 182]. The au‐

[151-165]

[166-183]

[176, 184]

[176, 185, 186]

[179, 183, 187]

to use the bacterial population with the density of more than 1010 CFU/ml [154, 188].

Aflatoxin B1 *Flavobacterium aurantiacum (Nocardia corynebacterioides),*

Ochratoxin A *Lactococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii*

Fumonisin B1 *Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides,*

Trichotecenes Ruminant bacteria, chicken intestinal microflora,

**Table 6.** Decontamination abilities of microorganisms

212 Soybean - Pest Resistance

Zearalenone Soil bacteria, *Propionibacterium fraudenreichii, Rhizopus sp.,*

In vitro studies conducted by [186] demonstrated the degradation of 12 trichothecene myco‐ toxins conducted by bacteria isolated from the digestive tract of chickens. The transforma‐ tion of the toxin led to their partial or total deacylation and de-epoxidation. Similarly, it was shown, that the strains of anaerobic bacteria - isolated from the rumen, Gram positive, preclassified to the genus *Eubacterium* - are able to perform the transformation of type A tricho‐ thecenes to non-toxic forms [185].

The above-presented examples of microbial activity aimed at removal of mycotoxins are mainly of scientific nature, allowing for a better understanding of the strains, their proper‐ ties and the mechanisms of the processes. Their limited practical application made that re‐ search turned in the direction of such organisms, which can be used in biotechnological processes during production, such as fermented food production, where the raw material may be contaminated with mycotoxins. The most important among them are lactic acid bac‐ teria and yeasts *Saccharomyces cerevisiae* [163].

Literature data indicate the existence of strains of lactic acid bacteria with different abilities to remove mycotoxins, as demonstrated both in *in vitro* and *in vivo* studies conducted by various authors with the use of some strains of probiotic *Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium bifidum*, *B. longum,* and *Streptococcus* spp., *Lactococcus salivarius*, *Lactobacillus delbrueckii* subsp. *bulgaricus* [155, 156, 158, 160, 169, 189, 190]. According to [191], the decontamination process is very fast; after 4h the toxin concentration was reduced from 50 to 77%. It was observed that heat-inactivated cells were more effective than living cells, which results from the changes in the surface properties of cells, which occur under high temperature [191]. The capacity to reduce the content of ochratoxin A in milk by lactic acid bacteria belonging to the species *Lactococcus salivarius*, *Lactobacillus delbrueckii* subsp. *bulgari‐ cus* and *Bifidobacterium bifidum* was confirmed in [167]. The content of patulin in the medium decreased in the level from 10 to 82% under the influence of bacteria belonging to the genus *Lactobacillus* and *Bifidobacterium*. The decontamination process depends on the inoculum density, pH and the concentration of toxins. Among the studied strains, *L.acidophilus*, re‐ moves up to 96% of the toxin added to the medium in an amount of 1ppm [166].

Our *in vivo* experiments indicate that the use of probiotics as feed additives limited the ef‐ fects of mycotoxins in animals, as well as reduced the accumulation of toxins in the tissues, thus reducing the contamination of food of animal origin with the toxins [192]. It was shown that *Lactobacillus rhamnosus* bacteria limited by 75% the adsorption of aflatoxin B1 in the di‐ gestive tract of chickens [189].

The second group of organisms with a potential application in detoxification is constituted by *Saccharomyces cerevisiae* yeasts. Our own research demonstrated that these organisms are capa‐ ble of eliminating ochratoxin A from the plant raw material during fermentation and chroma‐ tographic analysis did not show any products of OTA metabolism, which proves that it was not the case of biodegradation. The amount of ochratoxin A removed by bakery yeasts after 24hour contact equalled from 29% to 75% for 5 mg d.m/ml and 50 mg d.m./ml, respectively. The process of adsorption proved to be very fast; immediately after mixing the cells with the toxin its amount significantly decreased, and lengthening the contact up to 24 hours did not bring further notable changes. The presence of physiologically active cells is not necessary in order to remove the toxin; the dead biomass also removed OTA from the buffer and the amount of the toxin removed was much bigger than in the case of the active biomass. In the case of the 5 mg/ml density, 54% of the toxin was adsorbed, i.e. twice more than in the case of the active bio‐ mass [171]. The reason for OTA removal was adsorption of the toxin to the yeast cell wall. This mechanism was independent of the type of toxin, as demonstrated in relation to aflatoxin B1, zearalenone and T-2 toxin and patulin. The compounds of the cell wall that are involved in the binding process are probably β-D-glucan and its esterified form [193, 194]. Yeasts and their cell wall components are also used as feed additives for animals, and as adsorbents, which effec‐ tively limits mycotoxicosis in farm animals [195, 196].

**References**

Inc; 2000. p. 759-783.

1020475411125

ica 2005;8 51-56.

0000020591.71894.48.

DOI:10.1007/s11259-011-9483-9.

343-350.

Food Microbiology 1996;33 85-102.

DOI:10.1023/A:1022941304447

Journal of Stored Product Research 1995;31 1-16.

Canadian Journal of Botany 1982;60(12) 2716-2723.

and Food Chemistry 2003;51 7079-7085. DOI:10.1021/jf030228g.

[1] Legan JD. Cereals and cereal products. In: Lund BM, Baird-Parker TC, Gould GW. (eds.) The microbiological safety and quality of food. Gaithersburg: Aspen Publishers

Mycotoxins in Cereal and Soybean-Based Food and Feed

http://dx.doi.org/10.5772/54470

215

[2] Miller JD. Fungi and mycotoxins in grain: implications for stored product research.

[3] Jeleń HH, Majcher M, Zawirska-Wojtasik R, Wiewiórowska M, Wąsowicz E. Deter‐ mination of geosmin, 2-methylisoborneol, and a musty-earthy odor in wheat grain by SPME-GC-MS, profiling volatiles, and sensory analysis. Journal of Agriculture

[4] Bhattacharya K, Raha S. Deteriorative changes of maize, groundnut and soybean seeds by fungi in storage. Mycopathologia 2002;155 135–141. DOI:10.1023/A:

[5] Filtenborg O, Frisvad JC, Thrane U. Moulds in food spoilage. International Journal of

[6] Pacin AM, Gonzalez HHL, Etcheverry M, Resnik SL, Vivas L, Espin S. Fungi associ‐ ated with food and feed commodities from Ecuador. Mycopathologia 2002;156 87-92.

[7] Tabuc C, Stefan G. Assessment of mycologic and mycotoxicologic contamination of soybean, sunflower and rape seeds and meals during 2002 – 2004. Archiva Zootechn‐

[8] Miller W A, Roy KW. Mycoflora of soybean leaves, pods, and seeds in Mississippi.

[9] Villarroel DA, Baird RE, Trevathan LE, Watson CE, Scruggs ML. Pod and seed myco‐ flora on transgenic and conventional soybean [*Glycine max* (L.) Merrill] cultivars in Mississippi. Mycopathologia 2004;157 207-215. DOI:10.1023/B:MYCO.

[10] Leslie JF, Pearson CAS, Nelson PE, Toussoun TA. *Fusarium* spp. from corn, sorghum, and soybean fields in the central and eastern United States. Phytopathology 1990;80

[11] Ivić D, Domijan A-M, Peraica M, Miličević T, Cvjetković B. *Fusarium* spp. contamina‐ tion of wheat, maize, soybean, and pea in Croatia. Archives of Industrial Hygiene

[12] Pereyra CM, Cavaglieri LR, Chiacchiera SM, Dalcero AM. Mycobiota and mycotox‐ ins contamination in raw materials and finished feed intended for fattening pigs pro‐ duction in eastern Argentina. Veterinary Research Communication 2011;35 367-379.

and Toxicology 2009;60 435-442. DOI:10.2478/10004-1254-60-2009-196.

The potential application of yeasts as adsorbents for foods and feeds depends on the stabili‐ ty of the toxin binding to the cells in the conditions of the gastrointestinal tract. According to [194], zearalenone adsorption is most effective at a pH close to neutral and acidic, and there‐ fore those which prevail in some regions of the gastrointestinal tract. The result of the use of yeasts to remove ochratoxin A is detoxification of the environment, as demonstrated in the cytotoxicity and genotoxicity tests using pig kidney cell lines [197]. Some yeasts also exhibit features of probiotic activity, which is an additional argument for the use of these organisms

The use of microorganisms or their cell components for decontamination of foods and feeds has raised high hopes, but also the controversy from the perspective of the con‐ sumer. There are no legal regulations devoted to this issue, and the data referring to the stability of the microorganism-toxin connection in the gastrointestinal tract, as well as toxicological data are still incomplete. The only group of microorganisms, which in addi‐ tion to other advantageous features of health promotion has the ability to remove toxins, is probably that of probiotic lactic acid bacteria. Also, *Saccharomyces cerevisiae* yeast and its cell wall component - glucan can be used for this purpose. These factors can be ap‐ plied both as human dietary supplements and ingredients in animal nutrition, as well as during biotechnological processes.
