Diffusion of Mycotoxins in Fruits and Vegetables

Diffusion of Mycotoxins in Fruits and Vegetables

CHAPTER 5 Diffusion of Mycotoxins in Fruits and Vegetables Patrizia Restani Department of Pharmacological Sciences, University of Milan, Milano, Ita...

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CHAPTER

5

Diffusion of Mycotoxins in Fruits and Vegetables Patrizia Restani Department of Pharmacological Sciences, University of Milan, Milano, Italy

ABSTRACT This chapter reviews papers describing mycotoxin migration from rotten to unaffected parts of fruits and vegetables. It is known that mycotoxins can remain in fruits even when the fungal mycelium producing them has been eliminated and, depending on the characteristics of the fruit, mycotoxins can diffuse into sound tissues. Since second-quality fruits may be used to produce derivatives such as juices, jam, jelly, yoghurt, fruit salad, etc., a study of mycotoxin behavior in affected fruits is important to establish suitable means of protecting consumers from exposure to highly toxic substances such as ochratoxin A and patulin. Mycotoxin diffusion from moldy to unaffected parts of fruit and vegetables is reviewed in relation to: (a) the species of contaminating fungus; (b) the size of the rotten area; (c) the distance from the affected area and (d) the characteristic consistency and/or composition of fruit or vegetable pulp.

5.1. INTRODUCTION That mycotoxins can be present in fruits and vegetables is well known – see elsewhere in this book. In this chapter, factors affecting the diffusion of mycotoxins from the rotten to the unaffected area of fruits is discussed. This is particularly critical for the safety of consumers since parts of rotten fruits/ vegetables that are not rotten can be used in the preparation of fruit salad, juices, yoghurt, jam or jellies. It has been shown that contamination of fruit juices by mycotoxins results mainly from the use of poor-quality fruits. Of the large number of known mycotoxins, only a few are commonly found in fruits and vegetables: aflatoxins (produced by several Aspergillus species), ochratoxin A (by Penicillium and Aspergillus species), alternariol and derivatives (by Alternaria species), patulin (by Penicillium and Aspergillus species), citrinin (by Penicillium expansum) and trichothecin (by Trichothecium roseum) (Drusch and Ragab, 2003). 105

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Chapter 5 Diffusion of Mycotoxins in Fruits and Vegetables

Little has been published about the migration of mycotoxins in fruits and vegetables; and in those few publications this aspect was not always a primary goal of the research. This review will be organized along the following lines: – The role of fruit skin integrity in mycotoxin diffusion – Evidence for mycotoxin diffusion from rotten to unaffected areas – Factors affecting mycotoxin diffusion in fruits and vegetables.

5.2. ROLE OF FRUIT SKIN INTEGRITY IN MYCOTOXIN DIFFUSION A paper by Buchanan et al. (1975) concerning figs indicates that conidia of Aspergillus flavus may have penetrated fig skin even when the fruits do not present evident wounds. These authors showed that green fruits are resistant to attack by A. flavus but when they become ripe they lose this resistance because the skin is soft. Spores on the surface of ripe fruits were shown to infect and colonize the fruits, leading the authors to deduce that conidia can penetrate the skin even when it is intact. However, there might have been other causes for the penetration, the most probable being the presence of microscopic organisms such as insects or mites, which can produce minute skin wounds not visible to the eye, or the presence of juice exuded onto the fruit surface that allowed fungal colonization and mycotoxin penetration. Rapid fungal colonization and aflatoxin production continue until the growth of A. flavus is halted by adverse environmental conditions, such as the lack of moisture in dried fruit (Buchanan et al., 1975). In contrast to figs, oranges are contaminated by A. flavus only after the peel has been visibly attacked by other microorganisms, probably because citrus oil has antifungal properties (Varma and Verma, 1987).

5.3. EVIDENCE FOR MYCOTOXIN DIFFUSION FROM ROTTEN TO UNAFFECTED AREAS The diffusion of mycotoxins, mainly ochratoxin A and patulin, from a moldy area of a fruit to unaffected parts has been studied by various authors. The results (Table 5.1) indicate that damaged or moldy fruits can be contaminated with mycotoxins to some degree even after the rotten area has been removed. Engelhardt et al. (1999) analyzed different fruits after the removal of rotten tissue and found up to 2.71 mg/kg of ochratoxin A in cherries and up to 1.44 mg/kg in tomatoes and strawberries, while peaches and apples were contaminated to a lesser degree (0.59 and 0.41 mg/kg, respectively).

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Evidence for Mycotoxin Diffusion from Rotten to Unaffected Areas TABLE 5.1 Mycotoxin concentration in fruits after removal of rotten area. Maximum mycotoxin concentration detected (mg/kg)

Mycotoxin

Fruit considered

Ochratoxin A

Apple

0.41

Apricot

ND

Cherry

2.71

Nectarine

ND

Patulin

Peach

0.59

Strawberry

1.44

Tomato

1.44

Apple (with peel)

1166

Apple (without peel)

93

Pears

288 000a

Reference Engelhardt et al., 1999

Beretta et al., 2000

Laidou et al., 2001

ND = not detectable. a inoculated with Penicillium expansum.

Since patulin is one of the most frequently detected mycotoxin in fruits and their derivatives, most papers considered the migration of this mycotoxin in fruits and vegetables. Beretta et al. (2000) measured the migration of patulin in 26 apples with different-sized rotten areas containing active molds. To investigate whether the mycotoxin could migrate to areas not affected by rot, two identical portions of each naturally affected apple were analyzed after the removal of the rotten area, one with and one without the peel. Five out of 26 apples did not contain the mycotoxin, confirming that some apple molds are not capable of producing patulin, if the environmental conditions are not right. Figure 5.1 shows data from the remaining 21 moldy apples. The mycotoxin concentration in the rotten area was often very high: up to 113.3 mg/kg. Mycotoxins were detected in the unaffected portions of 17 out of 21 samples when the peel was not removed and 7 apples still presented detectable quantities of patulin after peeling. The content of patulin in unpeeled portions ranged from 0.05 to 1166 mg/kg, while that of the peeled part was between 0.44 and 93 mg/kg (Table 5.1 and Fig. 5.1). These data, and those concerning apple juices published by the same authors, show that if apple derivatives are prepared with low-quality fruits, the concentration of patulin could exceed the safe limits established by the international toxicological committees (50 mg/kg or mg/l). In fact, commercial apple juices containing pulp analyzed by these authors showed a patulin concentration of 0.68–1150 mg/kg (Beretta et al., 2000).

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Chapter 5 Diffusion of Mycotoxins in Fruits and Vegetables

Patulin concentration (µg/kg)

1000000 100000 10000 1000 100 10 1 1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21

Sample Rotten area

Unaffected area with peal

Unaffected area without peal

FIGURE 5.1 Patulin concentration (mg/kg) in the affected and unaffected areas of 21 rotten apples. Values lesser than were changed to 1 mg/kg in order to obtain a zero on the semilogarithmic scale. Data from Beretta et al., 2000.

5.4. FACTORS AFFECTING MYCOTOXIN DIFFUSION IN FRUITS AND VEGETABLES

5.4.1. PATULIN MIGRATION

AND

SIZE

OF THE

ROTTEN AREA

A common finding by different authors is that there is no direct correlation between the patulin concentration in apple/apple derivatives and the percentage of rottenness in the fruits (Beretta et al., 2000; Martins et al., 2002). In some cases, very high contents of patulin were detected in apples with a small affected area (Fig. 5.2) (Beretta et al., 2000). For instance, apple no. 5 showed 6.4 percent of rottenness but a very high content of patulin (93 mg/kg), also in the peeled unaffected area; by contrast, apple nos. 20 and 21, in which 50 percent of the apple was affected, contained only a few mg/kg of mycotoxin in the sound part of the fruit.

5.4.2. PATULIN MIGRATION CAN BE AFFECTED BY THE CONSISTENCY OF THE FRUITS Several authors have studied the diffusion of patulin from the rotten to unaffected areas. Some of them (Ostry et al., 2004; Rychlik and Schieberle, 2001; Taniwaki et al., 1992) found the diffusion of patulin in apples to be limited to a depth of 1–2 cm from the rotten area. Taniwaki et al. (1992) studied the migration of patulin in 22 apples, in an attempt to determine how much trimming of the rotten portion of fruit is needed to avoid the contamination of derivatives. The authors inoculated

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Factors Affecting Mycotoxin Diffusion in Fruits and Vegetables

% of rotten area

Patulin concentration (µg/kg)

100

10

1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Sample Rotten area (%)

Unaffected area without peal

FIGURE 5.2 Patulin concentration (mg/kg) in the unaffected area without peel versus size of rotten area (% of total fruit). Patulin concentrations <1 were changed to 1 mg/kg in order to obtain a zero on the semilogarithmic scale. Data from Beretta et al., 2000.

apples with 5-day-old mycelia of patulin-producing Penicillium expansum, inserting a 3 mm needle into each apple. The apples were then incubated at 25C until the diameter of the lesion reached 3.6–4.8 cm. After the development of lesions, the moldy area was removed with circular knives having a diameter depending on the extent of mold growth (3.6–4.8 cm in diameter and 10 cm in length). The cylinder obtained was then divided into 6 portions 1 cm thick, and their patulin content was measured (Fig. 5.3). The data from the same authors illustrated in Figs 5.4 and 5.5 show that: – the extent of the lesions (internal diffusion) in the 22 apples inoculated with P. expansum varies widely, even though the diameter of the superficial moldy areas was similar (3.6–4.8 cm); – the presence of lesions does not imply the presence of patulin; for example, at 3 cm from the inoculation point 41 percent of the apples showed a lesion, but only 2 out of 9 contained a detectable amount of patulin; – in portions with no lesion or even close to a lesion, patulin was undetectable or present at low concentration; – most of the patulin present in the apple tissue was found 1 cm from the patulin containing lesion. The authors concluded from these data that contamination of apple derivatives could be avoided by trimming and removing a 1 cm portion around and under the rotten area.

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Chapter 5 Diffusion of Mycotoxins in Fruits and Vegetables

Moldy area

Apple divided into 6 portions of 1 cm each FIGURE 5.3 Removal of apple portions to detect lesion extent and patulin migration (modified from Taniwaki et al., 1992).

100 90

Percentage of affected apples

80 70 60 50 40 30

ND 0.01–0.10 0.11–1.0 1.1–2.0 2.1–3.0 Average lesion size 3.1–4.0 (cm) 4.1–5.0

20 10 0 1

2

3

4

5

cm from point of inoculum FIGURE 5.4 Extent of lesions in six portions of apples inoculated with Penicillium expansum: percentage of affected apples versus distance from the inoculation point. (ND = undetectable) Data from Taniwaki et al., 1992.

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Factors Affecting Mycotoxin Diffusion in Fruits and Vegetables

100 90

Percentage of affected apples

80 70 60 50 40 30

< 6.5 6.5–100 101–1000 Patulin 1001–5000 concentration 5001–10000

20 10 0 1

2

> 10000

3

(µg/kg)

4

cm from the rotten area FIGURE 5.5 Migration of patulin in apples inoculated with Penicillium expansum and percentage of affected apples at different distances from the rotten area. Data from Taniwaki et al., 1992.

Figure 5.6 shows the results of Rychlik and Schieberle (2001), who examined patulin migration in different matrices, and revealed that the risk of mycotoxin diffusion is dependent on the consistency of the fruit or vegetable concerned. Migration was studied on apples, tomatoes, and bread experimentally infected by Penicillium expansum, and the extent of diffusion was examined after 7 days of incubation. Starting from the rotten area, which was

Patulin concentration (µg/kg)

100000 10000 1000 100

Apple Tomato Wheat bread

10 1

Apple Tomato Wheat bread

0 83700 52900 1600

1 1270 318

2 5 6500 5.4

3 1 2

4 1 450 1.4

cm from the rotten area

FIGURE 5.6 Patulin concentrations (mg/kg) at different distances from the rotten area of apple, tomato, and wheat bread crumbs infected by Penicillium expansum. Mycotoxin concentration in apples at 3 and 4 cm were <6  10–2 mg/kg; they were changed into 1 mg/kg in order to obtain a zero on the semilogarithmic scale. Data from Rychlik and Schieberle, 2001.

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Chapter 5 Diffusion of Mycotoxins in Fruits and Vegetables

scraped out, samples were cut in spherical segments 1 cm thick and patulin was quantified in each section. In apples with an initial concentration of 83.7 mg/kg in the affected tissue, the mycotoxin concentration rapidly dropped with distance, down to <0.0012 mg/kg at 3 cm from the rotten area. Unlike apples, tomatoes were penetrated easily by patulin: at 4 cm from the moldy tissue the concentration of patulin was still 0.450 mg/kg. After infection of wheat bread with Penicillium expansum and incubation for 7 days, the concentration of patulin in the layer containing fungal mycelium was 1.6 mg/kg; there was little diffusion and the contamination dropped sharply off with distance and was barely detectable 4 cm from the affected area. The authors concluded that, like ‘‘viscous’’ apple tissue, bread has also a consistency which reduces patulin diffusion. These results indicate that the consistency of the matrix is crucial in determining mycotoxin diffusion, which is facilitated by high water content and hindered by the presence of structure-forming polysaccharides. Thus, it has been shown that patulin penetrates more easily in water-rich fruits such as grapes, melons, tomatoes, and blueberries, while viscous matrices like those of apples and bread restrict patulin migration to a few centimeters from the rotten area.

5.4.3. PATULIN DIFFUSION IN FRUITS DEPENDS ON THE PARTICULAR PATHOGEN Laidou et al. (2001) considered the diffusion of patulin in the flesh of pears after infection with four different pathogens: Penicillium expansum, Aspergillus flavus, Stemphylium vesicarium, and Alternaria alternata. The inoculated fruits were incubated at room temperature (18–20C) for 2–22 days, depending on the strain inoculated. The infected fruits were then split longitudinally through the center of the lesion (5–20 mm in diameter) and were divided into four parts, from the tissue immediately below the lesions to the area around the carpel. These authors showed that the isolation frequency of all the fungi increased in all the sections as the lesion diameter increased. This was considered compatible with other data such as those by Thanassoulopoulos et al. (1990), who demonstrated a correlation between lesion diameter and the depth of penetration of Alternaria alternata in pears. Penicillium expansum and Aspergillus flavus showed higher frequencies of isolation at all depths than did Stemphylium vesicarium and Alternaria alternata. The two former fungi also showed more rapid penetration into the flesh; on the other hand, P. expansum and A. alternata penetrated the fruit flesh before there was any patulin diffusion, while the penetration of S. vesicarium lagged behind the diffusion of patulin. Patulin was detected in the flesh of pears in areas of 10–20 mm diameter from the rotten area when the fruit was inoculated with P. expansum, S. vesicarium or A. alternata, but not with A. flavus. The mycotoxin was detected even in apparently sound tissues (Fig. 5.7).

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Conclusions

Patulin concentration (ppm)

300 250 200 P. expansum S. vesicarium A. alternata

150 100 50 0

1

2

3

4

cm from the rotten area FIGURE 5.7 Patulin concentration (mg/kg) at different depths of pears inoculated with Penicillium expansum, Stemphylium vesicarium, and Aternaria alternata. Data from Laidou et al., 2001.

5.5. CONCLUSIONS The question raised by this short review is, what is the risk for consumers if a food manufacturer uses partially rotten fruits to derive a product, even if affected portions have previously been removed? The above data suggest several strategies to ensure safety in the use of fruits with limited moldy area(s): – the consistency of the fruit matrix should be considered: moldy fruits having a high water content and containing few structural polysaccharides must be avoided; – cutting off the rotten tissue up to a distance of 2 cm from the mold seems enough to remove most of the hazardous mycotoxin in most situations; – up to 99 percent of patulin can be eliminated by removing the moldy area and then suitably washing the fruit, e.g., by high-pressure water spraying (Acar et al., 1998; Lovett et al., 1974) – the initial patulin concentration in apples can be reduced 10-fold by combining washing and removal of the damaged area (Leggott et al., 2000); – patulin concentration can be significantly reduced by clarification (Go¨kmen et al., 2001) or ultrafiltration processes (Acar et al., 1998). Although these strategies can improve the quality of derivatives from secondquality fruits, the procedures are not easily achieved, and careful selection of fruit quality still remains the most appropriate procedure to protect consumers’ health. Moreover, elimination of the rotten area must include an unaffected area large enough to reduce the patulin level below 50 mg/kg, and this is not easily attained by automated processes. As shown by different authors, the use of second-quality fruit is acceptable only for the production of clear juices, in which the filtration process eliminates most of the mycotoxin with the fruit pulp.

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