**5. Fumonisins**

Fumonisins are a group of non-fluorescent mycotoxins. They were discovered and characterized in 1988 (Bezuidenhout et al., 1988). The predominant fungus isolated from fumonisin contaminated corn, associated with the outbreak of Equine Leukoencephalomalacia (ELEM) in South Africa in 1970 and Porcine Pulmonary Edema (PPE) in Iowa, Illinois, and Georgia in 1989, was *F. verticillioides* (Yazar & Omurtag, 2008).

To date, twenty-eight fumonisins have been isolated and they can be divided into four series (A, B, C and P). FB1, FB2 and FB3 are the principal fumonisins analyzed as natural contaminants of cereals (CAST, 2003; Yazar & Omurtag, 2008). Fumonisin B1 is generally the most abundant member of this mycotoxin family; it comprises about 70 % of the total fumonisin content of *Fusarium* cultures (Reddy et al., 2010). Fumonisins have a similar structure to sphingosine, which forms the backbone of sphingolipids within the cell membrane (Sweeney & Dobson, 1998).

Fumonisins are polyketide metabolites, derived from the repetitive condensation of acetate units or other short carboxylic acids, via a similar enzymatic mechanism to that responsible for fatty acid synthesis (Huffman et al., 2010). The fumonisin biosynthetic pathway in *Fusarium* species begins with the formation of a linear dimethylatedpolyketide and condensation of the polyketide with alanine, followed by a carbonyl reduction, oxygenations, and esterification with two propane-1,2,3-tricarboxylic acids (Desjardins & Proctor, 2007).

Fumonisin biosynthetic genes have been mapped to one locus in the *F. verticillioides* genome (Desjardins & Proctor, 2007).

Fig. 4. Chemical structure of fumonisin B1.

The basic chemical structure of fumonisins is a C-20 aliphatic chain with two ester linked hydrophilic side chains (Richard, 2007). The chemical structure of FB1 is 1,2,3- Propanetricarboxylic acid, 1,1N-[1- (12 amino-4,9,11-trihydroxy-2-methyltridecyl)- 2-(1 methylpentyl)-1,2-ethanediyl] Ester (Figure 4). FB2 is the C-10-deoxy analogue of FB1 and FB3 is the C-5-deoxy analogue of FB1(Yazar & Omurtag, 2008). The molecular mass of FB1 is 721 g/mol, while FB2 and FB3 have the same value of molecular mass (705g/mol). FB1 is soluble in water to at least to 20 mg/ml, and in methanol and acetonitrile-water. FB1 and

lower temperatures than the roasted products. Jackson et al. (1996) indicated that foods that reach greater temperatures than 150 ºC during processing may have lower fumonisin levels.

179

At a cellular level, the structural similarity between sphinganine and FB1 suggests that the action mechanism of this mycotoxin is mainly via the disruption of the sphingolipid metabolism. This mechanism is reflected in effects on cell growth and differentiation, in cell death (apoptosis) and carcinogenicity (Yazar & Omurtag, 2008). Fumonisins have often been found to be involved in liver and kidney toxicity; they have been shown to be hepatocarcinogenic in male rats and female mice and nephrocarcinogenic in male rats (Mazzoni et al., 2011). Purified FB1 has been shown to cause Equine Leukoencephalomalacia (ELEM) and Porcine Pulmunary Edema (PPE). In most animal species, the main target organs for FB1 are the liver and kidneys (Richard, 2007; Yazar & Omurtag, 2008; Wan Norashima et al., 2009). There is no carryover of fumonisins into milk in cattle and there appears to be little absorption of them in tissues (Richard et al., 2007). The high incidences of esophageal cancer in the Transkei region of South Africa, in northern Italy and in China have been linked to the ingestion of fumonisin contaminated maize; recent findings suggest that fumonisins might increase the risk of neural tube defects in populations that consume

large amounts of contaminated maize (Mazzoni et al., 2011; Yazar & Omurtag, 2008).

Patulin (PAT) was discovered in 1943 in relation to *P. griseofulvum* and *P. expansum*. The molecule was first studied as a potential antibiotic, but the subsequent research demonstrated its toxicological properties (Baert et al., 2007; Birkinshaw et al., 1943). PAT is produced by several species of *Aspergillus*, *Penicillium*, among these, *P. expansum* is the most relevant. In fact, almost all *P. expansum* isolates are PAT producers (Puel et al., 2010). This mycotoxins can be found in different food products and raw materials, but apples and apple by-products are of greatest concern regarding PAT accumulation: the frequency of contamination in other food resources and products is much lower than in apple processing

PAT has been reported to be mutagenic and to cause neurotoxic, immunotoxic, genotoxic and gastrointestinal effects in rodents; therefore, there is some concern that similar effects may occur in humans as a consequence of the long-term consumption of contaminated food

PAT, 4-Hydroxy-4H-furo[3,2-c]pyran-2(6H)-one, is a water-soluble unsaturated heterocyclic lactone (Figure 5). The biosynthesis involves a series of condensation and redox reactions. The pathway consists of approximately 10 steps, as suggested from several biochemical studies and from the identification of several mutants that are blocked at various steps in the PAT biosynthetic pathway. A cluster of 15 genes involved in PAT biosynthesis, containing characterized enzymes, a regulation factor and transporter genes, has recently been reported

PAT is a colourless and crystalline low-molecular weight compound, which is relatively heat resistant , with a melting point of 110 °C and a maximum UV absorption at 276 nm. It is soluble in water, ethanol, ethyl acetate, chloroform and acetone, while it is weakly soluble in ethyl ether and benzene and insoluble in petroleum ether, pentane and hexane (Pohland et al., 1982). PAT is unstable in a basic solution and stable in acidic media; in sulfurous

**5.3 Toxicity** 

**6. Patulin** 

(Moake et al., 2005).

(Puel et al., 2010).

or beverages (Hopkins, 1993).

FB2 are stable in methanol at −18 °C and degrade at 25 °C. However, in a mixture of acetonitrile/water (1:1) and at −25 °C, all fumonisins result to be stable (Wan Norashima et al., 2009).

### **5.1 Fungi**

Fumonisins are produced by a number of *Fusarium* species, notably *F. verticillioides* (formerly *Fusarium moniliforme=Gibberella fujikuroi), F. proliferatum, F. anthophilum, F. nygamai* as well as *Alternaria alternata* f. sp. *lycopersici* (Kumar et al., 2008; Sweeney & Dobson, 1998; Yazar & Omurtag, 2008). Recently, *Aspergillus niger* has also been found to produce fumonisins (i.e., fumonisins B2 and B4), and a new B-series fumonisin (FB6) has been identified from this fungus (Huffman et al., 2010).

Fumonisins found in food are produced mainly in the field; temperature and moisture conditions are important factors that affect *Fusarium* infection and toxin synthesis as is insect damage of corn ears and kernels (Richard, 2007; Yazar & Omurtag, 2008).

At a field level, two approaches are known to reduce infections and mycotoxin accumulation: pre-harvesting control strategies, which consist of crop practices designed to reduce the infection and the development of toxinogenic fungi (Nicholson et al., 2004; Wan Norashima et al., 2009; ) and the utilization of genetically resistant hybrids (Munkvold, 2003; Blandino & Rejneri, 2008). Direct fungal control with chemical or biological products (e.g. microbial antagonist or competitor) has only recently been considered (Mazzoni et al., 2011). Mycotoxin risk can be reduced by enhancing the resistence of insect attack, by inducing the process of detoxification pathway that inhibits the production of mycotoxins and increasing the resistance of the plant to infection by means of genetic engineering. A recent approach to the search for hybrids that are resistant to mycotoxin contamination consists in the obtaining of genetically modified hybrids which create the resistance action through transgenes (Blandino & Rejneri, 2008; Wan Norashima et al., 2009).

An increase in concentrations of fumonisins during storage does not appear to be a major problem. However, grains should be harvested without additional kernel damage, screened to remove broken kernels, dry stored and maintained at moisture concentrations < 14% (Richard, 2007).

#### **5.2 Food**

Fumonisins have been found to be a very common contaminant of corn-based food and feeds in Africa, China, France, Indonesia, Italy, the Philippines, South America, Thailand and the USA (Kumar et al., 2008).

In addition to corn or corn-based food and feeds, the occurrence of fumonisins has also been reported in other products, such as: rice and sorghum (CAST, 2003), wheat noodles, curry, beer and corn-based brewing adjuncts (Yazar & Omurtag, 2008).

Fumonisins B1 and B2 have been reported in "black oat" feeds from Brazil and forage grass in New Zealand. FB1 and FB2 have been found in rural areas of South Africa, in homegrown corn produced and consumed by the people living in those areas. Commercial corn based human foodstuff from retail outlets in several countries contains fumonisins (Wan Norashima et al., 2009).

Castelo et al. (1998) have reported that fumonisins found in artificially contaminated cornmeal samples are unstable under roasting conditions, but remain fairly stable during the canning and baking of corn-based foods because the canned and baked products reach

FB2 are stable in methanol at −18 °C and degrade at 25 °C. However, in a mixture of acetonitrile/water (1:1) and at −25 °C, all fumonisins result to be stable (Wan Norashima et

Fumonisins are produced by a number of *Fusarium* species, notably *F. verticillioides* (formerly *Fusarium moniliforme=Gibberella fujikuroi), F. proliferatum, F. anthophilum, F. nygamai* as well as *Alternaria alternata* f. sp. *lycopersici* (Kumar et al., 2008; Sweeney & Dobson, 1998; Yazar & Omurtag, 2008). Recently, *Aspergillus niger* has also been found to produce fumonisins (i.e., fumonisins B2 and B4), and a new B-series fumonisin (FB6) has been identified from this

Fumonisins found in food are produced mainly in the field; temperature and moisture conditions are important factors that affect *Fusarium* infection and toxin synthesis as is insect

At a field level, two approaches are known to reduce infections and mycotoxin accumulation: pre-harvesting control strategies, which consist of crop practices designed to reduce the infection and the development of toxinogenic fungi (Nicholson et al., 2004; Wan Norashima et al., 2009; ) and the utilization of genetically resistant hybrids (Munkvold, 2003; Blandino & Rejneri, 2008). Direct fungal control with chemical or biological products (e.g. microbial antagonist or competitor) has only recently been considered (Mazzoni et al., 2011). Mycotoxin risk can be reduced by enhancing the resistence of insect attack, by inducing the process of detoxification pathway that inhibits the production of mycotoxins and increasing the resistance of the plant to infection by means of genetic engineering. A recent approach to the search for hybrids that are resistant to mycotoxin contamination consists in the obtaining of genetically modified hybrids which create the resistance action through transgenes

An increase in concentrations of fumonisins during storage does not appear to be a major problem. However, grains should be harvested without additional kernel damage, screened to remove broken kernels, dry stored and maintained at moisture concentrations < 14%

Fumonisins have been found to be a very common contaminant of corn-based food and feeds in Africa, China, France, Indonesia, Italy, the Philippines, South America, Thailand

In addition to corn or corn-based food and feeds, the occurrence of fumonisins has also been reported in other products, such as: rice and sorghum (CAST, 2003), wheat noodles, curry,

Fumonisins B1 and B2 have been reported in "black oat" feeds from Brazil and forage grass in New Zealand. FB1 and FB2 have been found in rural areas of South Africa, in homegrown corn produced and consumed by the people living in those areas. Commercial corn based human foodstuff from retail outlets in several countries contains fumonisins (Wan

Castelo et al. (1998) have reported that fumonisins found in artificially contaminated cornmeal samples are unstable under roasting conditions, but remain fairly stable during the canning and baking of corn-based foods because the canned and baked products reach

damage of corn ears and kernels (Richard, 2007; Yazar & Omurtag, 2008).

(Blandino & Rejneri, 2008; Wan Norashima et al., 2009).

beer and corn-based brewing adjuncts (Yazar & Omurtag, 2008).

al., 2009).

**5.1 Fungi** 

(Richard, 2007).

and the USA (Kumar et al., 2008).

Norashima et al., 2009).

**5.2 Food** 

fungus (Huffman et al., 2010).

lower temperatures than the roasted products. Jackson et al. (1996) indicated that foods that reach greater temperatures than 150 ºC during processing may have lower fumonisin levels.

#### **5.3 Toxicity**

At a cellular level, the structural similarity between sphinganine and FB1 suggests that the action mechanism of this mycotoxin is mainly via the disruption of the sphingolipid metabolism. This mechanism is reflected in effects on cell growth and differentiation, in cell death (apoptosis) and carcinogenicity (Yazar & Omurtag, 2008). Fumonisins have often been found to be involved in liver and kidney toxicity; they have been shown to be hepatocarcinogenic in male rats and female mice and nephrocarcinogenic in male rats (Mazzoni et al., 2011). Purified FB1 has been shown to cause Equine Leukoencephalomalacia (ELEM) and Porcine Pulmunary Edema (PPE). In most animal species, the main target organs for FB1 are the liver and kidneys (Richard, 2007; Yazar & Omurtag, 2008; Wan Norashima et al., 2009). There is no carryover of fumonisins into milk in cattle and there appears to be little absorption of them in tissues (Richard et al., 2007). The high incidences of esophageal cancer in the Transkei region of South Africa, in northern Italy and in China have been linked to the ingestion of fumonisin contaminated maize; recent findings suggest that fumonisins might increase the risk of neural tube defects in populations that consume large amounts of contaminated maize (Mazzoni et al., 2011; Yazar & Omurtag, 2008).

### **6. Patulin**

Patulin (PAT) was discovered in 1943 in relation to *P. griseofulvum* and *P. expansum*. The molecule was first studied as a potential antibiotic, but the subsequent research demonstrated its toxicological properties (Baert et al., 2007; Birkinshaw et al., 1943). PAT is produced by several species of *Aspergillus*, *Penicillium*, among these, *P. expansum* is the most relevant. In fact, almost all *P. expansum* isolates are PAT producers (Puel et al., 2010). This mycotoxins can be found in different food products and raw materials, but apples and apple by-products are of greatest concern regarding PAT accumulation: the frequency of contamination in other food resources and products is much lower than in apple processing (Moake et al., 2005).

PAT has been reported to be mutagenic and to cause neurotoxic, immunotoxic, genotoxic and gastrointestinal effects in rodents; therefore, there is some concern that similar effects may occur in humans as a consequence of the long-term consumption of contaminated food or beverages (Hopkins, 1993).

PAT, 4-Hydroxy-4H-furo[3,2-c]pyran-2(6H)-one, is a water-soluble unsaturated heterocyclic lactone (Figure 5). The biosynthesis involves a series of condensation and redox reactions. The pathway consists of approximately 10 steps, as suggested from several biochemical studies and from the identification of several mutants that are blocked at various steps in the PAT biosynthetic pathway. A cluster of 15 genes involved in PAT biosynthesis, containing characterized enzymes, a regulation factor and transporter genes, has recently been reported (Puel et al., 2010).

PAT is a colourless and crystalline low-molecular weight compound, which is relatively heat resistant , with a melting point of 110 °C and a maximum UV absorption at 276 nm. It is soluble in water, ethanol, ethyl acetate, chloroform and acetone, while it is weakly soluble in ethyl ether and benzene and insoluble in petroleum ether, pentane and hexane (Pohland et al., 1982). PAT is unstable in a basic solution and stable in acidic media; in sulfurous

fruit in a stored batch, the greater the amount of PAT in the derived products. It has also been shown that the concentration of PAT in deck stored apples increases with storage time (FAO, 2003; Sydenham et al., 1997). In order to improve storage under refrigeration and in a controlled atmosphere against fungal growth and PAT production, additive treatments can be employed, including the use of sanitizers, natural or biological agents or a combination of the two (Chen et al., 2004). Another alternative is the use of polyethylene (PE) packages, with or without a controlled atmosphere, during storage and transport (Moodley et al., 2002). PAT can be reduced in stored apples through a washing stage with tap water, or tap water with active chlorine, or with highly pressurized water; the decrease percent depends on the initial amount of mycotoxin. The use of pressurized water makes it possible to remove the rotten parts of the fruit and also to reduce the fungal population, but it can also suspend and disperse PAT and spores in the washing water because it disturbs the rotten areas (Acar et al., 1998; Marin et al., 2006; Sydenham et al., 1997). Of all the apple products, apple juice is the most important source of PAT in the human diet throughout the world (World Health Organisation [WHO], 1995); the main steps of this production are

181

Fig. 6. Apple juice processing steps (modified from Sant'Ana et al., 2008)

PAT can be removed from juice by means of stirring or filtering through granulated activated carbon (Kadakal & Nas, 2002); the obtained percent of PAT reduction depends on the type of carbon, type of activation (physical or chemical), the solid content of the juice and the contact time (Leggott et al., 2001). As far as the heat treatments of juice, it is known that PAT is heat stable in acidic environments; nowadays various research and controversial results exist concerning the effect of the first pasteurization of the juice on the toxin (Kadakal & Nas, 2003). Experimental studies, on various combinations of temperature/time, generally demonstrate the heat stability of PAT to various time/temperature binomials (e.g. 80°C for 30 min, 100°C for 15 min at pH 2.0). Moreover, these studies show that if the contamination is high in the initial processing stages, it will be practically impossible to obtain significant reductions in the level of PAT. On the other hand, despite the studies showing no significant reduction in PAT in apple juice after pasteurization, the destruction of the spores of *P. expansum* reduces the risk of the subsequent production of this mycotoxin (FAO, 2003). Vacuum distillation is usually adopted for the concentration step of the juice and it can allow a reduction in the PAT level because of the time and temperature

summarized in Figure 6.

compound solutions, the instability is accompanied by the loss of biological activity (Harrison, 1989). It is stable at pH values ranging between 3,0 and 6,5: if the pH is higher, the lactone ring is opened and the toxic effect is lost (Janotovà et al., 2011).

Fig. 5. Chemical structure of patulin.

#### **6.1 Fungi**

PAT has been isolated from several species of *Penicillium*, *Aspergillus*, *Paecilomyces* and *Byssochlamys* (Puel et al., 2010). Recent studies based on HPLC-DAD (High Pressure Liquid Chromatography-Diode Array Detector) or LC-MS (Liquid Chromatography-Mass Spectometry) analysis of secondary metabolites, have established the reliable PAT producing species, which are listed in Table 5.


Table 5. Patulin producing fungi.

#### **6.2 Food**

Patulin-producing strains have been isolated from a variety of fruit and vegetables and both pastorized and unpastorized related products, but within the food industry, apples and apple products are of predominant concern as far as PAT contamination (Moake et al., 2005; Sant'Ana et al., 2008). PAT occurs mostly in apples evidently mould-damaged fruit, but sometimes fungal growth can occur internally, as a consequence of various kinds of damage, and can result in the occurrence of PAT in externally undamaged fruit. Therefore, apples must be handled adequately before and during processing to avoid all kinds of damage. It is also fundamental to reduce the possibility of contamination by eliminating mouldy fruit and taking particular care when cleaning containers (Codex, 2003b; Food and Agriculture Organisation of the United Nations [FAO], 2003). In terms of apple storage conditions, in general *P. expansum* shows psychrotrophic characteristics, in fact it is able to growth and produce PAT under refrigerated storage, but different strains show different capacities to produce PAT in different storage conditions (refrigeration and or controlled atmosphere) (Lovett et al., 1975; Paster et al., 1995; Taniwaki et al., 1989). The elimination of mouldy fruit is fundamental during storage because the greater the percentage of damaged

compound solutions, the instability is accompanied by the loss of biological activity (Harrison, 1989). It is stable at pH values ranging between 3,0 and 6,5: if the pH is higher,

O

O

PAT has been isolated from several species of *Penicillium*, *Aspergillus*, *Paecilomyces* and *Byssochlamys* (Puel et al., 2010). Recent studies based on HPLC-DAD (High Pressure Liquid Chromatography-Diode Array Detector) or LC-MS (Liquid Chromatography-Mass Spectometry) analysis of secondary metabolites, have established the reliable PAT

**Aspergillus, Clavati group (Varga et al., 2007)** *A. clavatus; A. giganteus; A. longivesica* **Penicillium** (Frisvad et al., 2004) *P. carneum; P. clavigerum; P. concentricum; P. coprobium; P. dipodomyicola; P. expansum; P. glandicola; P. gladioli; P. griseofulvum; P. marinum; P. paneum; P. sclerotigenum; P. vulpinum* **Paecylomyces** (Samson et al., 2009) *Paecylomyces saturatus* **Byssochlamys** (Samson et al., 2009) *B. nivea*

Patulin-producing strains have been isolated from a variety of fruit and vegetables and both pastorized and unpastorized related products, but within the food industry, apples and apple products are of predominant concern as far as PAT contamination (Moake et al., 2005; Sant'Ana et al., 2008). PAT occurs mostly in apples evidently mould-damaged fruit, but sometimes fungal growth can occur internally, as a consequence of various kinds of damage, and can result in the occurrence of PAT in externally undamaged fruit. Therefore, apples must be handled adequately before and during processing to avoid all kinds of damage. It is also fundamental to reduce the possibility of contamination by eliminating mouldy fruit and taking particular care when cleaning containers (Codex, 2003b; Food and Agriculture Organisation of the United Nations [FAO], 2003). In terms of apple storage conditions, in general *P. expansum* shows psychrotrophic characteristics, in fact it is able to growth and produce PAT under refrigerated storage, but different strains show different capacities to produce PAT in different storage conditions (refrigeration and or controlled atmosphere) (Lovett et al., 1975; Paster et al., 1995; Taniwaki et al., 1989). The elimination of mouldy fruit is fundamental during storage because the greater the percentage of damaged

the lactone ring is opened and the toxic effect is lost (Janotovà et al., 2011).

O

OH

Fig. 5. Chemical structure of patulin.

Table 5. Patulin producing fungi.

producing species, which are listed in Table 5.

**6.1 Fungi** 

**6.2 Food** 

fruit in a stored batch, the greater the amount of PAT in the derived products. It has also been shown that the concentration of PAT in deck stored apples increases with storage time (FAO, 2003; Sydenham et al., 1997). In order to improve storage under refrigeration and in a controlled atmosphere against fungal growth and PAT production, additive treatments can be employed, including the use of sanitizers, natural or biological agents or a combination of the two (Chen et al., 2004). Another alternative is the use of polyethylene (PE) packages, with or without a controlled atmosphere, during storage and transport (Moodley et al., 2002). PAT can be reduced in stored apples through a washing stage with tap water, or tap water with active chlorine, or with highly pressurized water; the decrease percent depends on the initial amount of mycotoxin. The use of pressurized water makes it possible to remove the rotten parts of the fruit and also to reduce the fungal population, but it can also suspend and disperse PAT and spores in the washing water because it disturbs the rotten areas (Acar et al., 1998; Marin et al., 2006; Sydenham et al., 1997). Of all the apple products, apple juice is the most important source of PAT in the human diet throughout the world (World Health Organisation [WHO], 1995); the main steps of this production are summarized in Figure 6.

Fig. 6. Apple juice processing steps (modified from Sant'Ana et al., 2008)

PAT can be removed from juice by means of stirring or filtering through granulated activated carbon (Kadakal & Nas, 2002); the obtained percent of PAT reduction depends on the type of carbon, type of activation (physical or chemical), the solid content of the juice and the contact time (Leggott et al., 2001). As far as the heat treatments of juice, it is known that PAT is heat stable in acidic environments; nowadays various research and controversial results exist concerning the effect of the first pasteurization of the juice on the toxin (Kadakal & Nas, 2003). Experimental studies, on various combinations of temperature/time, generally demonstrate the heat stability of PAT to various time/temperature binomials (e.g. 80°C for 30 min, 100°C for 15 min at pH 2.0). Moreover, these studies show that if the contamination is high in the initial processing stages, it will be practically impossible to obtain significant reductions in the level of PAT. On the other hand, despite the studies showing no significant reduction in PAT in apple juice after pasteurization, the destruction of the spores of *P. expansum* reduces the risk of the subsequent production of this mycotoxin (FAO, 2003). Vacuum distillation is usually adopted for the concentration step of the juice and it can allow a reduction in the PAT level because of the time and temperature

number count; it can also increase the frequency of defective embryos. Anomalies can include growth retardation, hypoplasia of the mesencephalon and telencephalon, and hyperplasia

183

At a cellular level, PAT is believed to cause cell damage by forming adducts with thiolcontaining cellular components (Barhoumi & Burghardt, 1996); in fact, many enzymes with a sulfhydryl group in their active site are sensitive to PAT. PAT has also been shown to induce intra- and intermolecular protein cross-links (Fliege & Metzler, 1999). Finally, PAT can interact directly with DNA and RNA inhibiting transcription and translation (Lee &

Abramson, D.; House, J.D. & Nyachoti, C.M. (2005). Reduction of deoxynivalenol in barley

Abrunhosa, L.; Robert R.; Paterson, M. & Venâncio, A. (2010). Biodegradation of Ochratoxin

Acar, J.; Gökmen, V. & Taydas, E. E. (1998). The effects of processing technology on the

Barhoumi, R. & Burghardt, R.C. (1996). Kinetic Analysis of the Chronology of Patulin- and

Bakirci, I. (2001). A study on the occurrence of aflatoxin M1 in milk and milk products

Bankole, S.A.; Adenusi, A.A.; Lawal, O.S. & Adesanya, O.O. (2010). Occurrence of aflatoxin

Barbiroli, A.; Bonomi, F.; Benedetti, S.; Mannino, S.; Monti, L.; Cattaneo, T. & Iametti, S.

Battacone, G.; Nudda, A. & Pulina, G. (2010). Effects of Ochratoxin A on Livestock

Baxter, E.D.; Slaiding, I.R. & Kelly, B. (2001). Behavior of ochratoxin A in brewing*. Journal of* 

Bennett, J.W. & Klich, M. (2003). Mycotoxins. *Clinical Microbiology Review*, Vol. 16, pp. 497–516. Bezuidenhout, S.C.; Gelderblom, W.C.A.; Gorst-Allman, C.P.; Horak, R.M.; Marasas, W.F.O.;

A for food and feed decontamination. *Toxins*, Vol. 2, pp. 1078-1099.

*International Journal of Food Microbiology,* Vol. 119, pp. 170–181.

produced in Van province in Turkey. *Food Control*, Vol. 12, 47-51.

Nigeria. *Food control*, Vol. 21, pp. 974-976.

Production. *Toxins*,Vol. 2, pp. 1796-1824.

*Communication,* Vol. 11, pp. 743-745.

Milk. *Journal of Dairy Science*, Vol. 90, pp. 532-540.

*the American Society of Brewing Chemists*, Vol. 59, pp. 98–100.

by treatment with aqueous sodium carbonate and heat. *Mycopathologia*, Vol. 160,

patulin content of juice during commercial apple juice concentrate production. *Zeitschrift fur Lebensmittel-Untersuchung und -Forschung A*, Vol. 207, pp. 328–331. Baert, K.; Devlieghere, F.; Flyps, H.; Oosterlinck, M.; Ahmed, M.M.; Rajković, A.; Verlinden,

B.; Nicolaï, B.; Debevere, J. & De Meulenaer, B. (2007). Influence of storage conditions of apples on growth and patulin production by *Penicillium expansum*.

Gossypol-lnduced Cytotoxicity in Vitro. *Fundamental and Applied Toxicology*, Vol. 30,

B1 in food products derivable from 'egusi' melon seeds consumed in southwestern

(2007). Binding of Aflatoxin M1 to Different Protein Fractions in Ovine and Caprine

Spiteller, G. & Vleggaar, R. (1988). Structure elucidation of the fumonisins, mycotoxins from *Fusarium moniliforme*. *Journal of the Chemical Society, Chemical* 

and/or blisters of the mandibular process (Smith et al., 1993).

Roschenthaler, 1987).

pp. 297–301.

pp. 290-297.

**7. References** 

exposition. Possibly PAT transformation occurs, while PAT removal to volatile phase is unprobable. As regard that, the results obtained in various studies are controversial, and in some cases show a certain reduction while in other no changes are observed (Kadakal & Nas, 2003; Leggott et al., 2000). The PAT levels in formulated juices may be affected by adding ingredients such as ascorbic acid, thiamine hydrochloride, pyridoxine hydrochloride and calcium pantothenate (Yazici & Velioglu, 2002). Nevertheless, the use of these additives has some limitations; as regard ascorbic acid, its use is influenced by the storage conditions and if it is oxidized, no further degradation of PAT is observable (Drusch et al., 2007). Other possible additives are sulphur dioxide, sodium benzoate and potassium sorbate (Lennox & McElroy, 1984; Roland et al., 1984), but the current demand for healthy food, free of additives, could result in an impediment to the use of such techniques. Thus, it is preferable to use treatments that guarantee the elimination/inactivation of the ascospores of the heat resistant fungi (such as filtration with diatomaceous earth) than to apply these additives.

It can be said that, although the juice manufacturing process stages are capable of reducing the amount of PAT in the final products to a certain extent, the incidence of this mycotoxin throughout the World confirms its stability to some degree; when faced with the techniques currently in use, only the adoption of adequate controls to reduce the incidence of fruit damage and rot, during pre-harvest, harvest and post-harvest, can lead to an important reduction in the final product, whether it is fruit for direct consumption or one of the various fruit products (Sant'Ana et al., 2008).

#### **6.3 Toxicity**

The health risks of PAT for humans include acute and chronic symptom and effects at a cellular level.

Some of the acute toxic signs that have consistently been reported are agitation, convulsions, dyspnea, pulmonary congestion, edema, and ulceration, hyperemia and distension of the gastro intestinal tract (WHO, 1995). Sub-acute toxicity has also been indicated: PAT is recognized to mainly induce gastrointestinal disorders; it has mainly been studied in rats, where it has been shown to induce weight loss, gastric and intestinal changes and alterations in the renal function (Puel et al., 2010).

PAT is genotoxic; most assays carried out with mammalian cells have been positive while those with bacteria have mainly been negative. Some studies have indicated that PAT impairs DNA synthesis. These effects might be related to the ability PAT to react with sulphydryl groups and to induce oxidative damage (Liu et al., 2007). The IARC has placed PAT in group 3, as "not classifiable as to its carcinogenicity to humans" (IARC, 1986). PAT can also alter the immune response of the host (Oswald & Comera, 1998). Several in vitro studies have demonstrated that PAT inhibits various macrophage functions. PAT has also been found to reduce the cytokine secretion of IFN-γ and IL-4 by human macrophages and of IL-4, IL-13, IFN-γ, and IL-10 by human peripheral blood mononuclear cells and human T cells (Luft et al., 2008; Wichmann et al., 2002). In vivo studies using mice have indicated variable effects of PAT on the immune system, such as an increased number of splenic T lymphocytes and depressed serum immunoglobulin concentrations (Escoula et al., 1988; Paucod et al., 1990). As regard humans, exposure to PAT, at levels that are consistent with potential human exposure in food, would not be likely to alter immune responses (Llewellyn et al., 1998). When injected into the air cell of chick eggs, PAT is found embryotoxic, depending on the age of the embryo, and teratogenic (Ciegler et al., 1976). PAT can induce a reduction in the protein and DNA content, in the yolk sac diameter, crown rump length, and somite

exposition. Possibly PAT transformation occurs, while PAT removal to volatile phase is unprobable. As regard that, the results obtained in various studies are controversial, and in some cases show a certain reduction while in other no changes are observed (Kadakal & Nas, 2003; Leggott et al., 2000). The PAT levels in formulated juices may be affected by adding ingredients such as ascorbic acid, thiamine hydrochloride, pyridoxine hydrochloride and calcium pantothenate (Yazici & Velioglu, 2002). Nevertheless, the use of these additives has some limitations; as regard ascorbic acid, its use is influenced by the storage conditions and if it is oxidized, no further degradation of PAT is observable (Drusch et al., 2007). Other possible additives are sulphur dioxide, sodium benzoate and potassium sorbate (Lennox & McElroy, 1984; Roland et al., 1984), but the current demand for healthy food, free of additives, could result in an impediment to the use of such techniques. Thus, it is preferable to use treatments that guarantee the elimination/inactivation of the ascospores of the heat resistant fungi (such as filtration with diatomaceous earth) than to apply these additives. It can be said that, although the juice manufacturing process stages are capable of reducing the amount of PAT in the final products to a certain extent, the incidence of this mycotoxin throughout the World confirms its stability to some degree; when faced with the techniques currently in use, only the adoption of adequate controls to reduce the incidence of fruit damage and rot, during pre-harvest, harvest and post-harvest, can lead to an important reduction in the final product, whether it is fruit for direct consumption or one of the

The health risks of PAT for humans include acute and chronic symptom and effects at a

Some of the acute toxic signs that have consistently been reported are agitation, convulsions, dyspnea, pulmonary congestion, edema, and ulceration, hyperemia and distension of the gastro intestinal tract (WHO, 1995). Sub-acute toxicity has also been indicated: PAT is recognized to mainly induce gastrointestinal disorders; it has mainly been studied in rats, where it has been shown to induce weight loss, gastric and intestinal changes and

PAT is genotoxic; most assays carried out with mammalian cells have been positive while those with bacteria have mainly been negative. Some studies have indicated that PAT impairs DNA synthesis. These effects might be related to the ability PAT to react with sulphydryl groups and to induce oxidative damage (Liu et al., 2007). The IARC has placed PAT in group 3, as "not classifiable as to its carcinogenicity to humans" (IARC, 1986). PAT can also alter the immune response of the host (Oswald & Comera, 1998). Several in vitro studies have demonstrated that PAT inhibits various macrophage functions. PAT has also been found to reduce the cytokine secretion of IFN-γ and IL-4 by human macrophages and of IL-4, IL-13, IFN-γ, and IL-10 by human peripheral blood mononuclear cells and human T cells (Luft et al., 2008; Wichmann et al., 2002). In vivo studies using mice have indicated variable effects of PAT on the immune system, such as an increased number of splenic T lymphocytes and depressed serum immunoglobulin concentrations (Escoula et al., 1988; Paucod et al., 1990). As regard humans, exposure to PAT, at levels that are consistent with potential human exposure in food, would not be likely to alter immune responses (Llewellyn et al., 1998). When injected into the air cell of chick eggs, PAT is found embryotoxic, depending on the age of the embryo, and teratogenic (Ciegler et al., 1976). PAT can induce a reduction in the protein and DNA content, in the yolk sac diameter, crown rump length, and somite

various fruit products (Sant'Ana et al., 2008).

alterations in the renal function (Puel et al., 2010).

**6.3 Toxicity** 

cellular level.

number count; it can also increase the frequency of defective embryos. Anomalies can include growth retardation, hypoplasia of the mesencephalon and telencephalon, and hyperplasia and/or blisters of the mandibular process (Smith et al., 1993).

At a cellular level, PAT is believed to cause cell damage by forming adducts with thiolcontaining cellular components (Barhoumi & Burghardt, 1996); in fact, many enzymes with a sulfhydryl group in their active site are sensitive to PAT. PAT has also been shown to induce intra- and intermolecular protein cross-links (Fliege & Metzler, 1999). Finally, PAT can interact directly with DNA and RNA inhibiting transcription and translation (Lee & Roschenthaler, 1987).

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**11** 

195

*Spain* 

**Microbial Pectic Enzymes** 

Abigaíl F. da Silva1 and Tomás G. Villa2

 **in the Food and Wine Industry** 

Carmen Sieiro1, Belén García-Fraga1, Jacobo López-Seijas1,

*1Department of Functional Biology and Health Sciences, University of Vigo* 

*2Department of Microbiology and Parasitology, University of Santiago de Compostela* 

Pectins are polysaccharides ubiquitous in the plant kingdom and constitute the major component of plant cell walls. The pectinases are a group of related enzymes capable of degrading pectin. Therefore, this group of enzymes have been used for decades in the food and winemaking industry for the processing of fruit juices (Mohnen, 2008; Prade et al., 1999;

The pectinases are synthesized by plants and microorganisms, the latter being used for industrial production. Microorganisms are used to produce many enzymes of industrial interest in processes relatively inexpensive and environmentally friendly. Moreover, enzymatic catalysis is preferred over other chemical methods since it is more specific, less aggressive and generates less toxicity (Hoondal et al., 2002; Lara-Márquez et al., 2011). Advances in biotechnology, especially in the fields of molecular biology and microbial genetics, have led to major advances in enzyme technology and have allowed, in many cases, the development of new producing strains and microbial enzymes. The production of

This article reviews the characteristics of pectic substances, the types and mode of action of enzymes which degrade them and the main applications of commercial preparations of microbial pectinases in the food and winemaking industry, followed by a review of new microorganisms and pectolytic enzymes, evaluating new approaches to their production,

Pectic substances are polysaccharides of high molecular weight, with a negative charge, appearing mostly in the middle lamella and the primary cell wall of higher plants, found in the form of calcium pectate and magnesium pectate. They are formed by a central chain containing a variable amount although in high proportion of galacturonic acid residues linked through α-(1-4) glycosidic bonds partially esterified with methyl groups (Fig. 1). This molecule is known as pectin, while the demethylated molecule is known as polygalacturonic acid or pectic acid. Several L-rhamnopyranosyl residues may be attached to the main chain through its C-1 and C-2 atoms. In addition, galacturonate residue may be

**1. Introduction** 

Ribeiro et al., 2010).

marketing and use.

**2. Pectic substances** 

pectinases may also benefit from these technologies.

