Patulin accumulation in apples by Penicillium ... - Wiley Online Library

Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Patulin accumulation in apples by Penicillium expansum during postharvest stages H. Morales, S. Marıń, A. Rovira, A.J. Ramos and V. Sanchis Food Technology Department, University of Lleida, CeRTA-UTPV, Lleida, Spain

Keywords apple, patulin, Penicillium expansum, storage, temperature. Correspondence Vicente Sanchis, Food Technology Department, University of Lleida, CeRTAUTPV, Rovira Roure 191, 25198, Lleida, Spain. E-mail: [email protected]

2005/1277: received 26 October 2005, revised 23 June 2006 and accepted 3 August 2006 doi:10.1111/j.1472-765X.2006.02035.x

Abstract Aims: The aim of this study was to assess the opportunities of Penicillium expansum to develop and produce patulin in apples during cold storage and in the steps prior to processing of apple products. Methods and Results: Two lots of apples var. Golden with different ripeness degree were used. Half of each lot was fungicide treated. Apples were inoculated with P. expansum and stored at 1C for 6 weeks. The extent of lesions and patulin accumulation both at the end of cold storage and after 3 days at 20C were assessed. Short storage at 20C aimed to simulate the transport and storage steps at room temperature before processing. Lesion size significantly increased during the storage at 20C. An interaction between fungicide treatment and ripeness degree was found; efficiency of fungicide treatment was higher for ripe apples. Although lesions were evident after cold storage, no patulin was detected. Patulin was detected only when fruits were further stored at 20C. Neither ripeness degree nor fungicide treatment affected patulin accumulation. Conclusions: Cold storage periods of 6 weeks do not lead to patulin accumulation. Significance and Impact of the Study: Shortening preprocessing times at warm temperatures would result into a reduction in patulin content at initial steps of fruits entering the processing plants.

Introduction Patulin is a mycotoxin produced mainly by Penicillium expansum. Patulin levels in apple products are of great concern because of the severe acute and chronic effects caused by the toxin. Patulin’s acute toxic effects in human include nausea, vomiting, and other gastrointestinal trauma and accompanying kidney damage, and chronic patulin exposure has been shown to induce the formation of cancerous tumours and to cause genetic mutations and embryonic developmental defects (Dickens and Jones 1961; Mayer and Legaror 1969; Ciegler et al. 1976). The European Union (2001, Commission Regulation 1425/2003) has laid down maximum patulin levels of 30

50 lg kg)1 for apple juices, 25 lg kg)1 for solid apple products and 10 lg kg)1 for baby foods. Fungi are mainly responsible for decay in apples and pears kept in cold storage rooms. Among moulds isolated, P. expansum, causing blue mould, is one of the most common food-borne fungi on pome fruit and it causes between 70% and 80% of decay in stored fruit (Vinãs et al. 1993). In addition, it is regarded as the major producer of patulin. When apples invaded by P. expansum are used to elaborate apple products (e.g. fruit juices), these products will be likely contaminated with patulin. In this study, the capacity of P. expansum to develop and produce patulin during cold storage of apples was assessed. The potential influence of steps prior to

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derivative elaboration like transport or storage at room temperature was also taken into account. Materials and methods Isolates Three strains of P. expansum were obtained from apples stored at two different juice industries in Lleida (Spain) and had been proved to be patulin producers. The strains were stored in sloped potato dextrose agar (PDA) tubes at 5C. Apples Two different lots of sound apples var. Golden were kindly supplied by a packinghouse (Nufri, Mollerussa, Lleida, Spain). Two lots were chosen regarding ripeness (about 100 apples each lot). Determination of quality parameters such as colour, firmness, soluble solids and acidity as described later revealed that the lots differed mainly in hue angle and firmness. Luminosity, soluble solid concentration (SSC) and acidity were not significantly different for both lots. Average weight and caliber per apple were 226Æ175 g and 80Æ8 mm respectively. Half the apples of each lot were treated with a mixture of fungicides (in 1000 l water: 1 kg folpet 80% and 4 l of a formulation [14% (w/v) thiabendazole, 10% (w/v) imazalil]). In this way, the two lots of apples were divided into four different sub-lots: sub-lot 1 (ripe, treated apples), sub-lot 2 (ripe, untreated apples), sub-lot 3 (underripe, treated apples), and sub-lot 4 (underripe, untreated apples). Conidial suspensions and apple inoculation Petri dishes of PDA were point inoculated with the strains and incubated at 25C until sporulation. A conidial suspension was prepared in Tween-80 (0Æ005% (v/v) in sterile water and adjusted to 106 conidia ml)1. Apples were wounded 2 mm deep with a 2 mm diameter needle at the stem (top) and calyx (bottom) ends of the fruit. Both points were inoculated with 20 ll of the conidial suspensions. Each sub-lot was inoculated with the three different isolates of P. expansum plus a control (sterile water). Storage treatments Apples were stored for 6 weeks at 1C. After storage time, half the apples were kept at 20C during 3 days to simulate the interval of time that apples may be at room temperature before processing. The rest of the

Patulin in apples during postharvest

apples were directly analysed at the end of cold storage time. The whole experiment was designed as a full factorial one, with four factors to be tested on the spoilage caused by P. expansum: degree of ripeness, fungicide treatment, storage at room temperature and interspecific differences among P. expansum isolates. All treatments were repeated three times. Extent of lesions Both lesions at stem and calyx ends of each apple were measured. Two perpendicular diameters from each of the lesions were measured. After that the lesion at the top of the apple was removed and weighted to analyse patulin content. The remaining portion of the fruit was used to determine quality parameters. Quality parameters Quality parameters were determined for apples in each treatment at the end of the experiment in order to find out correlation among them and the extent of lesions and patulin accumulation. Colour was measured on two opposite sides on each fruit. Measurements were made with Macbeth color-Eye 3000 (Macbeth, New Windsor, NY, USA) colorimeter at the midpoint between the stem and the calyx end and the chromaticity values of the fruit in the L*a*b* space coordinates recorded (McGuire 1992). Hue angle [arctan (b/a)] was calculated. Firmness was measured on two opposite peeled sides using a penetrometer fitted with an 11 mm diameter probe. SSC was determined by measuring the refractive index of the juice resulting from the apples, and the data were expressed as percentage (g per 100 g fresh weight). Acidity was measured as follows: 10 ml of pulp juice was diluted with 10 ml H2O and titrated with a 0Æ1 mol l)1 NaOH solution. The acidity was expressed in grams of malic acid per litre of juice. Patulin analyses Distilled water was added to the decayed portion in 1 : 1 (w/w) relation (the same weight in water as portion weight). A ‘Polytron’ unit of Kinematica AG (Lucerne, Swizerland) was used to pulp it. Pectinase was added to puree and incubated during 1 h at 40C. In this way, emulsions were avoided during extraction. Following AOAC method 995.10 (Brause et al. 1996), patulin was extracted with ethyl acetate and then cleaned up by extraction with sodium carbonate solution. Extracted samples were dried with anhydrous sodium sulfate. After evaporation of ethyl


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acetate, patulin was resuspended in water pH 4 and determined by reversed-phase liquid chromatography (LC) with UV detection. Patulin is detected at UV at 276 nm wavelength. Patulin was expressed in nanograms of patulin in each assayed apple. The recovery rates obtained by spiking apple pieces with 10, 40 and 100 ng patulin g)1 in duplicate were 96Æ2%, 88Æ5% and 69Æ1%, respectively. Regarding repeatability the Sr of five determinations (25 ng g)1) carried out under repeatability conditions was 0Æ005. Finally the limit of detection of the analysis was 5 ng g)1. Statistical analysis of the results The assay was designed as a full factorial where factors analysed were ripeness, fungicide treatment, effect of incubation at 20C and importance of the strain used. Patulin concentrations and lesion diameters for each treatment were evaluated by analysis of variance using sas version 8Æ2 (SAS Institute Inc., Cary, NC, USA). Significant differences (P < 0Æ05) in parameters were tested by a Duncan’s Multiple Range Test. The significance of the correlation between apple quality parameters and both patulin content and lesion diameter and also between patulin accumulation and lesion size was assessed with the same program by using Pearson correlation coefficients at P < 0Æ05.

different isolates resulted in not significantly different lesion sizes (regardless of the remaining three factors; data not shown). Thus, the lesion diameter results presented in this section are independent of the strain considered. Significantly bigger sizes of lesions were reported when apples were kept at 20C than at the end of cold storage (Fig. 1). The observation of lesion diameters at the end of the cold storage period revealed that influence of fungicide treatment was highly significant: fungicide-treated apples had a mean lesion diameter smaller than untreated apples (3Æ31 mm vs 7Æ58 mm) (Table 1). The efficiency of fungicide treatment depended, however, on ripeness of apples, thus its effect could only be observed for ripe apples. For apples kept for 3 days at 20C after cold storage (Fig. 1), fungicide treatment was also significant but differences between lesion mean values were not as marked as in apples at the end of cold storage (26Æ15 mm for untreated apples vs 19Æ54 mm for treated apples). The efficiency of fungicide treatment also depended on ripeness of apples: for untreated apples, riper ones had significantly bigger lesions than underripe ones (28Æ58 mm vs 23Æ72 mm). On the other hand, for treated apples, the riper ones had smaller lesions (13Æ47 mm) than the underripe ones (25Æ61 mm). Patulin accumulation

Results Lesion diameters

B

(a)

(b)

30

30

25

25 Lesion diameter (mm)

Lesion diameter (mm)

No lesions were detected in control apples, neither after cold storage nor after 3 days at room temperature. The

Although lesions were evident, no patulin was detected in any of the apples at the end of cold storage, regardless of the remaining factors. For those apples that were kept for 3 days at 20C, no differences on patulin accumulation were found depending on any factor except for the differ-

20 C 15 10

A A

5

B

Untreated

AB

A

20 C 15 10 5

Untreated

0

0

Treated Underripe Ripe

Treated Underripe Ripe

Figure 1 Lesion diameters (mm, mean for three Penicillium expansum isolates) in apples. (a) At the end of cold storage time. (b) Apples kept for 3 days at 20C after cold storage.

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Table 1 Analysis of variance of lesion sizes (mm), both at the end of cold storage and after 3 days at 20C and significance of the assayed factors

End of cold storage

After 3 days at 20C MS

Source of variation

d.f.

MS

Ripeness (R) Strain (S) Fungicide treatment (F) S·R F·R S·F S·F·R

1 2 1 2 1 2 2

0Æ01 1Æ25 164Æ69 0Æ12 248Æ06 16Æ74 23Æ16

F 0Æ00 0Æ10 12Æ14** 0Æ01 18Æ29** 1Æ23 1Æ71

119Æ17 23Æ57 393Æ36 12Æ67 650Æ25 4Æ68 6Æ57

F 8Æ03** 1Æ59 26Æ51** 0Æ85 43Æ82** 0Æ73 0Æ64

Significance: **P < 0Æ01.

Table 2 Analysis of variance for total patulin (ng) as a dependent variable for apples stored for 3 days at 20C

Correlation between P. expansum spoilage and quality parameters

Source of variation

d.f.

Ripeness (R) Strain (S) Fungicide treatment (F) S·R F·R S·F S·F·R

1 2 1 2 1 2 2

Lesion diameter and patulin content were negatively correlated with firmness (Table 3). No significant correlation between patulin and lesion diameter after 3 days at 20C was found.

MS

F

947Æ99 1183Æ31 803Æ30 240Æ62 248Æ67 808Æ49 184Æ09

3Æ70 4Æ62** 3Æ14 0Æ94 0Æ97 3Æ16 0Æ72

ng patulin per apple

Significance: **P < 0Æ01.

60

S1

50 40 30

S2

20

S3

10

e rip

e ip nd

er

R

Untreated Treated

U

U Ri nd pe er rip e

U Ri nd pe er rip e

0

Figure 2 Patulin accumulation after 3 days at 20C for different ripeness grade, fungicide treatments and strain.

ent strains (Table 2), with mean levels of 30Æ602 ng of patulin per apple for strain 1, 19Æ175 ng of patulin per apple for strain 2 and 10Æ821 ng of patulin per apple for strain 3 (Fig. 2).

Table 3 Pearson correlation coefficients among lesion diameters, patulin accumulation and apple quality parameters

Lesion diameter Patulin content

Discussion Penicillium expansum is a psychrophilic fungus that grows well at 0C and even can grow at )2/)3C (Pitt and Hocking 1997). Thus, storage at low temperatures is not enough to avoid blue mould growth as shown in this study. Cold storage resulted, however, in the total prevention of patulin production. Although it has been reported that patulin can be produced in vitro by P. expansum at 0C (Pitt and Hocking 1997), in this assay no patulin was detected in fruits stored at 1C. Patulin production was evident only when apples were further kept at 20C. No differences were found among the strains tested in terms of apple lesions, thus the growth of P. expansum may be quite predictable. However, the ability to produce different amounts of patulin makes the problem more complex. Combinations of imazalil and folpet are commonly used in packinghouses to prevent blue mould and other pathologies in apples but their efficacy may depend on fruit conditions as shown in this study. Ripening of most climacteric fruits is characterized by softening of the flesh, enhanced colour development and increase in respiratory activity (Fallik et al. 2001). These processes make overripened fruits more sensitive to the attack of moulds in

Acidity

SSC

Luminosity

Hue angle

Firmness

Patulin

)0Æ23 )0Æ19

0Æ22 )0Æ12

)0Æ18 )0Æ19

0Æ08 0Æ16

)0Æ34* )0Æ43**

0Æ55** 1

Significance: *P < 0Æ05 and **P < 0Æ01. ª 2006 The Authors Journal compilation ª 2006 The Society for Applied Microbiology, Letters in Applied Microbiology 44 (2007) 30–35

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cold storage rooms and so, higher doses of fungicide may be required. In this study, we observed that in untreated fruits, lesions were more evident in those fruits classified as ripe both at the end of cold storage and further stored at 20C. However, when fungicide treated, ripe fruits had smaller lesions than underripe ones. Thus, the efficacy of fungicide in preventing the fruit from decaying was higher in ripe fruits. These results show the importance of adjusting the harvest date to enable apples to enter the packinghouses in optimal degree of ripeness. Few studies about the effects of folpet and imazalil combination on blue mould control were found. Current assays focus on new fungicides due to resistance that P. expansum develops to commonly used ones. It is a priority to discover the effects of new reduced risk chemicals. Fludioxinil was tested for its efficacy against blue mould not only as a preventive but also as a curative treatment when apples had been already infected (Errampali 2004; Errampalli et al. 2005). It was found that fludioxinil was very effective as co-treatment and postinoculation treatment. Although decay proceeds slowly at cold storage temperatures, rapid development occurs when the fruit is transferred to a warm environment (Fallik et al. 2001). Besides, as mentioned above, patulin accumulation occurs at these temperatures. The accumulation of patulin was independent of the pre-storage fungicide treatment. These results confirm that the lapse of time in which apples are taken out from storage room before being processed is critical in order to prevent patulin accumulation. Several studies have assayed the efficiency of some preprocessing methods to reduce patulin content in the fruit before processing. Removal of decayed tissue or washing before processing reduces levels of patulin in final products (Sydenham et al. 1995). However, some assays demonstrated that patulin is also found in sound tissues (Laidou et al. 2001), thus it is important to prevent patulin accumulation rather than trying to remove it from raw materials. Short preprocessing times at warm temperatures would result in higher quality of fruits entering the processing plants and therefore a reduction in patulin content at initial steps. Optimum patulin production by P. expansum has been reported in vitro at 25C (Pitt and Hocking 1997). The influence of temperature and time that apples are exposed to room temperature should be studied, as production of patulin may vary in a different way than growth of P. expansum. This study has focused on normal atmosphere cold storage conditions. However, storage at controlled atmosphere (CA) is becoming more and more common in packinghouses. Efficiency of fungicides and behaviour of P. expansum under CA conditions should be studied as well as influence of CA in metabolism of patulin and in 34

development of its accumulation at room temperatures after CA storage. Acknowledgements The authors are grateful to the Spanish Government (CICYT, Comisioń Interministerial de Ciencia y Tecnologıá, project AGL 2004-07549-05-01, and Ramon y Cajal program) and to the Catalonian Government (Projecte estrate`gic CeRTA 2005–2006, Seguretat bio`tica i abio`tica dels aliments) for their financial support. References Brause, A.R., Trucksess, M.W., Thomas, F.S. and Page, W.S. (1996) Determination of patulin in apple juice by liquid chromatography: collaborative study. J AOAC Int 79, 451–455. Ciegler, A., Beckwith, A.C. and Jackson, L.K. (1976) Teratogenicity of patulin adducts formed with cysteine. Appl Environ Microbiol 31, 664–667. Dickens, F. and Jones, H.C.H. (1961). Carcinogenic activity of a series of related lactones and related substances. Br J Cancer 15, 85–89. Errampali, D. (2004) Effect of fludioxinil on germination and growth of Penicillium expansum and decay in apple cvs. Empire and Gala. Crop Prot 23, 811–817. Errampalli, D., Northover, J., Skog, L., Brubacher, N.R. and Colluci, C.A. (2005) Control of blue mold (Penicillium expansum) by fludioxonil in apples (cv Empire) under controlled atmosphere and cold storage conditions. Pest Manage Sci 61, 591–596. European Union (2001) Commission Regulation 1425/2003 Amending Commission Regulation 466/2001 Setting Maximum Levels for Certain Contaminants in Foodstuffs. Brussels, Belgium, Official Journal of the European Communities, pp. L203/1–3. Fallik, E., Tuvia-Alaklai, S., Copel, A., Wiseblum, A. and Regev, R. (2001) A short hot water rinse and brushes: a technology to reduce postharvest losses – 4 years of research. In Proceedings of the 4th International Conference on Postharvest ed. Ben-Arie, R. and Philosoph-Hadas, S. Acta Horticult 553, 413–416. Laidou, I.A., Thanassoulpopoulos, C.C. and LiakopouloKyriakides, M. (2001) Diffusion of patulin in the flesh of pears inoculated with four post-harvest pathogens. J Phytopathol 149, 457–461. Mayer, V.W. and Legaror, M.S. (1969) Production of petite mutants of Saccharomyces cerevisiae by patulin. J Agric Food Chem 17, 454–456. McGuire, R. (1992) Reporting of objective color measurements. HortScience 27, 1254–1255. Pitt, J.I. and Hocking, A.D. (1997) Fungi and Food Spoilage. London, UK: Blackie Academic & Professional.


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Sydenham, E.W., Vismer, H.F., Marasas, W.F.O., Brown, N., Schlechter, M., Van der Westhuizen, L. and Rheeder, J.P. (1995) Reduction of patulin in apple juice samples – influence of initial processing. Food Control 6, 195–200.


Vinãs, I., Vela, E. and Sanchis, V. (1993) Capacidad productora de patulina de cepas Penicillium expansum procedentes de centrales hortofrutıćolas de Lleida. Rev Iberoam Micol 10, 30–32.


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