biology 116 (2007) 260ndash265wwwelseviercomlocateijfoodmicro

International Journal of Food Micro

Cold and ambient deck storage prior to processing as acritical control point for patulin accumulation

Heacutector Morales Sonia Mariacuten Xavier Centelles Antonio J Ramos Vicente Sanchis

Food Technology Department University of Lleida CeRTA-UTPV Rovira Roure 191 25198 Lleida Spain

Received 3 August 2006 received in revised form 12 December 2006 accepted 16 January 2007

Abstract

Patulin a mycotoxin produced primarily by Penicillium expansum is currently of great concern because of its undesirable effects in humanhealth It has been proven that patulin can damage organs and tissues in animals and some studies revealed carcinogenic and teratogenic effectsPatulin is found mainly in low quality apples diverted to production of apple by-products Apples from cold storage or recently harvested (usuallyground harvested or low quality apples) are stored under ambient conditions (deck storage) until they are processed The present assay studies theconsequences of this type of storage in development of lesions and patulin accumulation

The assayed factors were the size of lesions when apples were taken out from cold storage time the apples stayed at room temperature aftercold storage (as a simulation of deck storage) and intraspecific differences between 2 isolates of P expansum A sublot of P expansum inoculatedapples was cold stored until lesions achieved concrete sizes Then apples were either transferred to a 20 degC storage room for 0 to 5 days or wereanalysed for patulin immediately The rest of the apples were directly stored at 20 degC Each treatment had three replicates

Increase of lesion size with time at 20 degC depended on initial lesion size after cold storage Bigger lesions were always achieved in apples withbigger initial lesion size Initial lesion size and time at 20 degC significantly influenced patulin accumulation No significant amounts of patulin werefound in apples with lesions up to 2 cm after cold storage Patulin amounts significantly increased on the 2nd day at 20 degC day and remainedconstant until the 5th day Patulin accumulation tended to be higher when initial lesions were bigger

The assay showed the influence of apple quality (measured as overall lesion size) after cold storage on patulin accumulation during deckstorage as well as the importance of duration of deck storage Quality of apples entering the processing plant should be assessed in order toprogram deck storage and minimise patulin accumulation Predictive models of patulin accumulation as a function of time at room temperatureand apple quality should be a useful tool to elaboratorscopy 2007 Elsevier BV All rights reserved

Keywords Penicillium expansum Patulin Cold storage Deck storage

1 Introduction

Patulin (4-hydroxy-4H-furo(32c)pyran-2(6H)-one) a my-cotoxin produced primarily by Penicillium expansum is afrequent contaminant of apples and apple products Animalorgans affected by patulin administration included kidneyand intestine (Gopalakrishnan and Sakthisekaran 1991Speijers et al 1988) In addition it is considered to be acarcinogen and teratogen in certain animal models (Osswaldet al 1978 Ciegler et al 1976) Current studies point topatulin as potentially genotoxic with the ability to induce

Corresponding author Tel +34 973 702535E-mail address vsanchistecaludles (V Sanchis)

0168-1605$ - see front matter copy 2007 Elsevier BV All rights reserveddoi101016jijfoodmicro200701004

oxidative DNA damage in human cells which is consideredto play a role in mutagenesis and cancer initiation (Liu et al2003)

Within the food industry apples and their respective by-products are of the greatest concern for patulin accumulationWhile a variety of other food sources and products havedemonstrated patulin andor contamination with patulin-pro-ducing fungi the frequency of these events is much less thanthat of the apple industry (Moake et al 2005) Patulincontamination within apple products poses a serious healthrisk to consumers particularly children whom a USDA surveyhas shown to consume increased levels of apple products duringthe first year of life placing them at increased risk for patulintoxicity (Plunkett et al 1992)

261H Morales et al International Journal of Food Microbiology 116 (2007) 260ndash265

In general the fungal growth and the production of patulinare associated with damaged fruit (Sydenham et al 1995)Although patulin can be present in apparently sound applesintended for direct consumption it is typically found at higherlevels in lower quality fruit destined for processing into juicecider sauces purees and jellies (Doores 1983) Duringprocessing some steps can reduce patulin content The removalof decayed or damaged fruit or the trimming of mouldy portionsof apples prior to processing can reduce patulin levels Washtreatments alone cannot ensure that patulin is completelyremoved as some patulin may diffuse to the sound tissue (Mariacutenet al 2006) Postpressing treatments such as the addition ofascorbic acid to cider (Brackett and Marth 1979) or thefiltration of cider through bed of charcoal and polymers(Sydenham et al 1995 Acar et al 1998 Huebner et al2000) resulted in small to modest (10 to 60) reductions inpatulin levels Sydenham et al (1997) showed that freshlypressed juice prepared from apples subjected to deck storage formore than 7 days might be expected to contain in excess of50 ng patulin gminus1

Thus although patulin can be reduced in some processingsteps it is important to avoid accumulation of patulin in theapples before processing It has been reported that no patulin isproduced during a short cold storage (6 weeks) in decayedapples with lesions smaller than 35 cm (Morales et al in press-a) and that patulin is primarily produced when apples are furtherstored at room temperature

In South Africa limited cold storage and processingcapacities may result in apples being stored in the open forconsiderable periods of time Given the climate of the regionthe exposure of apples to the prevailing conditions increasesthe possibility of patulin contamination (Sydenham et al1995) During production of apple products patulin isaccumulated before processing begins So it may be morepractical to identify these points in order to reduce patulincontamination rather than trying to destroy patulin during juiceproduction Usually process-grade apples are ground har-vested or apples that after cold storage are not suitable forfresh market because of their low quality (there is evidentspoilage) Due to seasonal nature of apples and capacity ofprocessing plants these low quality apples are often deckstored FAO (2001) has identified open storing of apples as aCCP in terms of patulin control as during this period woundedor damaged fruit may be infected by P expansum and developblue mould or existing P expansum spoilage will increaseconsiderably

This study reports on apple quality after cold storage(measured as lesion diameter) and the time apples remain atroom temperature in deck storage (up to 5 days) on loss ofquality and patulin accumulation

2 Materials and methods

21 Isolates

Two isolates of P expansum (UdlTA 372 and UdLTA 378)isolated from cold stored apples in Lleida (Spain) were used

They had been proven to be patulin producers The isolates werestored on plugs of potato dextrose agar (PDA) submerged indistilled water at 4 degC in the Fungal Collection of the FoodTechnology Department of Lleida University

22 Apples

In total 144 apples var Golden from the 2005 season wereused Average weight and diameter were 19570 g and 781 mmrespectively They were stored at 1 degC until used

23 Conidial suspensions and apple inoculation

Petri dishes of potato dextrose agar (PDA) were pointinoculated with the isolates of P expansum and incubated at25 degC until sporulation occurred A conidial suspension wasprepared in Tween 80 in sterile water (0005) and adjusted to106 conidia mlminus1

All 144 apples were surface disinfected in a 2 solution ofsodium hypochlorite for 1 min and rinsed with distilled waterThen they were divided into 2 lots of 72 units and each applewas weighted and coded Then they were wounded to 2 mmdepth with a 2 mm diameter needle on the side of the apple halfway between stem and calyx Each lot was inoculated with 20 μlof the conidial suspensions of either isolate 372 or 378

24 Apple cold storage and incubation at 20 degC

Thirty six apples (18 inoculated with isolate UdLTA 372and 18 inoculated with isolate UdLTA 378) were not coldstored but kept at 20 degC for 1 2 3 4 or 5 days or wereanalysed for patulin immediately Three replicates for eachcondition were done The rest of the apples (108) were storedin a storage room at 1 degC and 85ndash90 humidity until fungalgrowth was observed From this moment lesion size wasmonitored daily in order to remove individual apples fromcold storage when the size of the lesion achieved 05 cm2 cm or 4 cm Eighteen apples for each lesion size and isolatewere obtained in this way Apples were transferred to 20 degCstorage room as the lesion reached the desired diameter Theywere stored at this temperature for 1 2 3 4 or 5 days ordirectly kept apart for patulin analysis without storing at20 degC Three replicates for each lesion size and time at 20 degCwere done In this way three replicates per isolate and time at20 degC were obtained Apple lesions were measured whenremoved from 20 degC storage

25 Sample acquisition

In order to analyse patulin accumulation necrosed tissuewas removed from the apple For that purpose a cylindricalcork borer was used with a diameter of at least 1 cm biggerthan the lesion diameter The cylinder of apple flesh thusobtained was cut 1 cm below the necrosed tissue allowing forpatulin that may have diffused to the sound tissue Thecylinder thus acquired was weighed and frozen at minus25 degC untilpatulin extraction

Fig 1 Lesion diameter growth at 20 degC after cold storage X Apples with noapparent initial lesion diams Apples with 05 cm initial lesion Apples with 2 cminitial lesion Apples with 4 cm initial lesion

Fig 2 Average patulin accumulation for all initial lesion sizes after cold storageas a function of time of storage at 20 degC Different letters mean significantdifferences

262 H Morales et al International Journal of Food Microbiology 116 (2007) 260ndash265

In summary the assay consisted in 3 apples per storage timeat 20 degC (0 1 2 3 4 or 5) times 4 different lesion sizes (0 05 2 and4 cm) times 2 isolates (372 and 378) = 144 apples

26 Patulin extraction and analyses

Distilled water was added to each sample in 11 ww ratioand the sample homogenised using a ldquoPolytronrdquo unit ofKinematica AG (Switzerland) The pureacutee obtained wasenzymatically treated (at 40 degC for 1 h) with pectinase for theremoval of pectin and centrifuged at 6000 rpm for 5 minSupernatant (5 ml) was taken for patulin analyses FollowingAOAC method 99510 (Brause et al 1996) patulin wasextracted with ethyl acetate and then cleaned up by extractionwith sodium carbonate solution Extracts were dried withanhydrous sodium sulfate After evaporation of ethyl acetatepatulin was resuspended in water adjusted to pH 4 with glacialacetic acid and determined by reversed-phase LC with UVdetection A solution of water and tetrahydrofuran (100+08)was used as mobile phase Patulin was detected at UVat 276 nmwavelength Patulin was expressed in ng gminus1 The recoveryrates obtained by spiking apple pieces with 10 40 and 100 ngpatulin gminus 1 in duplicate were 962 885 and 691respectively Regarding repeatability the Sr of five determina-tions (25 ng gminus1) carried out under repeatability conditions was0005 Finally the limit of detection of the analysis was 5 nggminus1

27 Statistical analysis of the results

Non-linear regression was used to adjust growth of fungi at20 degC to a sigmoidal Gompertz model For that purposeStatgraphics Plus 51 (StatPaint Inc Herndon V USA) wasused

The whole assay was designed as a full factorial one Factorstested on the spoilage caused by P expansum were size oflesions when apples entered the incubation room at 20 degC timeapples remained at 20 degC and interspecific differences betweenboth P expansum isolates All treatments were repeated threetimes Variability of patulin concentrations and lesion diameterswas evaluated by analysis of variance using the SAS version 82

(SAS Institute Inc Cary NC USA) Significant differences(Pb005) among levels were tested by Duncans MultipleRange Test

3 Results

31 Effect of storage at 20 degC after cold storage on lesion size

Average time taken to achieve different lesion sizes at coldstorage was 22 days for 05 cm lesion 35 days for 2 cm lesionsand 60 days for 4 cm lesions

A significant increase in apple lesion size when stored at20 degC was observed (Pb00001) Differences between bothisolates in growth at 20 degC were small (P=00419) andresponses to the other factors assayed did not depend on theisolates assayed Growth curves for each initial lesion size wereobtained with the Gompertz model with an average growth ofboth isolates (Fig 1)

The ANOVA test showed that the increase in lesion size withtime depended on initial lesion size and interacted with days at20 degC (Pb00001 for both) Fungal growth during the first daysat 20 degC apparently had the same behaviour for sound applesand for apples with initial lesion size of 05 cm A significantincrease was not found till the 2nd day at 20 degC Howeverlesion size achieved at the 5th day was significantly bigger inapples with initial lesion size of 05 cm than in apples with noapparent fungal growth (35 cm vs 19 cm) When initial lesionsize was 2 cm rot size increased significantly every day till 5 cmat the 5th day When initial lesion was 4 cm rot sizesignificantly increased every day except from the 3rd to the4th day and lesion size at the end of room storage was thebiggest (66 cm) For all 5 days the larger the initial lesion thegreater the increase of rot during the 5 days at 20 degC This ismore evident when the influence of the initial lesion size wasstudied for each day at 20 degC Duncan test showed that for eachday lesion size was significantly different depending on initiallesion size so that bigger lesions were always achieved inapples with bigger initial lesion size Lag phase was estimatedthrough the Gompertz model A lag phase of 18 days wasobserved in apples with no apparent initial lesion whereasapples with blue mould when stored at 20 degC started exponentialgrowth after 076 days regardless of lesion size and isolate

Fig 3 Average patulin accumulation for apples stored for 0 1 2 3 4 5 days at20 degC as a function of initial lesion size after cold storage a) Isolate 372 and b)isolate 378 Different letters mean significant differences

263H Morales et al International Journal of Food Microbiology 116 (2007) 260ndash265

32 Patulin accumulation

ANOVA test showed that factors affecting patulin accumula-tion were days at 20 degC (P=00028) and lesion size when appleswere taken out of the cold storage room (Pb00001) with nosignificant interaction between them in this case There was nosignificant difference between isolates (P=0561) but there was aslight interaction between isolate and initial lesion size(P=00494)

Average production of patulin in all the apples stored at20 degC (including all the initial lesion sizes) significantlyincreased on the 2nd day From this moment patulin remainedstatistically constant (Fig 2)

Fig 4 Patulin accumulation for different lesion sizes after cold storage as afunction of time of storage at 20 degC diams Apples with no apparent initial lesionApples with 05 cm initial lesion size Apples with 2 cm initial lesion size bullApples with 4 cm initial lesion size Different letters in the same day meansignificant differences Rings cluster observations with the same letter

An LS means test was carried out to study the interactionbetween initial lesion size and isolate Results showed thatpatulin accumulation was not influenced by the isolate exceptfor apples with initial lesion of 4 cm and stored for 1 day at20 degC Nevertheless behaviour of both isolates was the same(Fig 3a and b) so this slight interaction is not to be taken intoaccount for further results Apples with no evident fungalgrowth and with a 05 cm initial lesion contained the leastamount of patulin (107 ng patulin gminus1) compared with appleswith initial lesion of 2 cm and 4 cm whose average content ofpatulin was 502 ng patulin gminus1

Although the interaction between initial lesion size and timeat 20 degC was not significant some differences among days at20 degC were observed When apples were taken out from thestorage room at 1 degC (0 days at 20 degC) patulin accumulationwas significantly different depending on the lesion size Nopatulin was found either in apples without evident fungalgrowth or in apples with a 05 cm lesion diameter Apples with2 cm lesion had no significant patulin accumulation whereasapples with 4 cm lesion accumulated 253 ng patulin gminus1 After1 day at 20 degC however patulin was detected in all samplesregardless of initial lesion size Those apples with initial lesionsize of 4 cm accumulated significantly greater amounts ofpatulin than the rest (368 ng patulin gminus1 vs 648 ng patulin gminus1)After 48 h at 20 degC no factors significantly affected patulinaccumulation which averaged 356 ng patulin gminus1 From thistime significant differences in patulin accumulation tended tobe between apples with no initial lesion and the rest Thus onthe 3rd and 4th days apples with no initial lesion hadsignificantly smaller amounts of patulin than the rest (9 ngpatulin gminus1 vs 649 ng patulin gminus1 at the 3rd day and 100 ngpatulin gminus1 vs 446 ng patulin gminus1 at the 4th day) Initial lesion

Fig 5 Plot of expected patulin as a function of initial lesion size and time at20 degC

264 H Morales et al International Journal of Food Microbiology 116 (2007) 260ndash265

size did not significantly affect patulin accumulation on the 5thday by which time average accumulation was 412 ng patulingminus1

The tendency of patulin was to increase with time andexcept for those apples without initial lesion it seemed to reacha peak and decrease afterwards The bigger the initial lesion theearlier the peak of patulin seemed to appear (Fig 4)

Patulin accumulation as a function of initial lesion size andtime at 20 degC has been plotted in Fig 5 which shows theamount of patulin that may be expected in such stored applesFor example apples with initial lesion of 05 cm stored till 1 dayat 20 degC are not expected to contain patulin but if storage isextended up to 200 ng patulin gminus1 may accumulate If storage isfurther prolonged to approx 28 days patulin may accumulateup to 400 ng patulin gminus1 In this same time if initial lesion was4 cm more than 800 ng patulin gminus1 may be accumulated Thusif apples must be deck stored for more than 2 days it isnecessary that the initial lesion is smaller than 025 cm to assureno patulin accumulation

4 Discussion

When apples are infected with P expansum spores it is quitedifficult to avoid fruit spoilage even if fruits are stored at lowtemperatures or controlled atmosphere conditions (Jacksonet al 2003 Morales et al in press-ab) In this assay all fruitsinoculated with P expansum developed blue mould at 1 degC innormal atmosphere before 20 days The above mentionedauthors also showed that growth of P expansum during coldstorage is not prevented although it is dramatically reduced Inour assay apples inoculated and stored directly at 20 degCdeveloped lesions of almost 2 cm in 5 days whereas the time toachieve this same lesion size at 1 degC was 35 days

Jackson et al (2003) reported that patulin accumulationoccurs during cold storage In this study patulin was detected inapples with 4 cm lesion after cold storage On the other handand in agreement with Morales et al (in press-ab) no patulinwas detected in apples with lesion size up to 2 cm at the end ofcold storage Apple quality state is reported to play an importantrole in fruit spoilage and patulin accumulation (Sydenham et al1995 1997 Jackson et al 2003 Moake et al 2005) The mainfinding of this study is in accordance with these assays

Storage at room temperature after cold storage leads to arapid development of decayed tissue and patulin accumulationFallik et al (2001) and Morales et al (in press-ab) reportedsimilar conclusions The bigger lesions were observed in appleswith bigger initial lesion through a 5-day period at 20 degC So inthis period of time (longer than desirable) a deceleration ofgrowth was not appreciable Thus the bigger the lesions arewhen apples are kept out from cold storage the shorter the timethey should remain in stand by at room temperature Factorssuch as temperature and humidity influence the initial stages ofinfection (Doores 1983) Different conditions than thoseassayed that may occur during deck storage may result intomore differences between apparently sound apples (in which alag phase is observed) and those with initial blue moulddevelopment

Sometimes apples used for juice elaboration are not onlythose rejected from packinghouses after cold storage Groundharvested apples or apples with evident lesion when harvestedare directly diverted to apple processing plants Usually theseapples are not cold stored but directly deck stored Accordingto our results the non-desirable effect of deck storage maybecome more noticeable as fungal growth may not bedecelerated by previous cold storage Besides during appleharvest season it is expected that apples will stay in stand by atroom temperature for longer due to limited capacity ofprocessing plants

Noncold stored apples with no apparent spoilage had smallerlesion till the 4th day at 20 degC However long term storage at20 degC tended to equal patulin accumulation regardless of initialapple quality state Jackson et al (2003) reported that ciders madeby nonstored tree-picked apples did not contain patulin in anycase On the other hand patulin was detected in some batches oftree-picked apples air stored for 4 to 6weeks at approximately 0 to2 degC

Unfortunately in most cases quality of apples supplied toapple juice elaborators is difficult to assess as they are quiteheterogeneous and different lesions sizes can be found in thesame lot As seen in this study even if the lot is apparentlysound patulin contamination can appear in short periods of timeif they are slightly wounded and stored at room temperature Inour results regardless of the initial lesion sizes patulinaccumulation significantly increased after 48 h at 20 degC Thisis in agreement with results obtained by FAO (2001) in whichbulk storage is qualified as a CCP (Critical Control Point) and itrecommends not to extend this kind of storage for more than48 h However this lapse of time should change depending onthe quality state of the apples and other factors such astemperature or relative humidity

As a conclusion reducing to minimum deck storage oravoiding it should improve apple quality and patulin accumu-lation before processing begins As it is quite impossible tosucceed in processing in a continuous way from harvest or coldstorage to plant elaboration industries should assess quality ofapples entering the processing plant and adjust stand by timeaccording to it in order to minimise patulin accumulationPredictive modelling of patulin accumulation as a function ofapple quality (measured as overall lesion size) and time at roomtemperature would be an efficient tool for apple productindustries to program deck storage and elaboration

Patulin accumulation seemed to achieve a maximum after atime at 20 degC depending on initial lesion size Possibledegradation of patulin or dilution when necrosed tissueincreases should be studied

Acknowledgements

This work was supported by the Spanish Government(CICYT Comisioacuten Interministerial de Ciencia y Tecnologiacuteaprojects AGL 2004-07549-C05-01ALI and AGL 2004-06413ALI and Ramon y Cajal program) and the CatalonianGovernment (Projecte estrategravegic CeRTA 2005-2006 Seguretatbiogravetica y abiogravetica dels aliments)

264 H Morales et al International Journal of Food Microbiology 116 (2007) 260ndash265

size did not significantly affect patulin accumulation on the 5thday by which time average accumulation was 412 ng patulingminus1

The tendency of patulin was to increase with time andexcept for those apples without initial lesion it seemed to reacha peak and decrease afterwards The bigger the initial lesion theearlier the peak of patulin seemed to appear (Fig 4)

Patulin accumulation as a function of initial lesion size andtime at 20 degC has been plotted in Fig 5 which shows theamount of patulin that may be expected in such stored applesFor example apples with initial lesion of 05 cm stored till 1 dayat 20 degC are not expected to contain patulin but if storage isextended up to 200 ng patulin gminus1 may accumulate If storage isfurther prolonged to approx 28 days patulin may accumulateup to 400 ng patulin gminus1 In this same time if initial lesion was4 cm more than 800 ng patulin gminus1 may be accumulated Thusif apples must be deck stored for more than 2 days it isnecessary that the initial lesion is smaller than 025 cm to assureno patulin accumulation

4 Discussion

When apples are infected with P expansum spores it is quitedifficult to avoid fruit spoilage even if fruits are stored at lowtemperatures or controlled atmosphere conditions (Jacksonet al 2003 Morales et al in press-ab) In this assay all fruitsinoculated with P expansum developed blue mould at 1 degC innormal atmosphere before 20 days The above mentionedauthors also showed that growth of P expansum during coldstorage is not prevented although it is dramatically reduced Inour assay apples inoculated and stored directly at 20 degCdeveloped lesions of almost 2 cm in 5 days whereas the time toachieve this same lesion size at 1 degC was 35 days

Jackson et al (2003) reported that patulin accumulationoccurs during cold storage In this study patulin was detected inapples with 4 cm lesion after cold storage On the other handand in agreement with Morales et al (in press-ab) no patulinwas detected in apples with lesion size up to 2 cm at the end ofcold storage Apple quality state is reported to play an importantrole in fruit spoilage and patulin accumulation (Sydenham et al1995 1997 Jackson et al 2003 Moake et al 2005) The mainfinding of this study is in accordance with these assays

Storage at room temperature after cold storage leads to arapid development of decayed tissue and patulin accumulationFallik et al (2001) and Morales et al (in press-ab) reportedsimilar conclusions The bigger lesions were observed in appleswith bigger initial lesion through a 5-day period at 20 degC So inthis period of time (longer than desirable) a deceleration ofgrowth was not appreciable Thus the bigger the lesions arewhen apples are kept out from cold storage the shorter the timethey should remain in stand by at room temperature Factorssuch as temperature and humidity influence the initial stages ofinfection (Doores 1983) Different conditions than thoseassayed that may occur during deck storage may result intomore differences between apparently sound apples (in which alag phase is observed) and those with initial blue moulddevelopment

Sometimes apples used for juice elaboration are not onlythose rejected from packinghouses after cold storage Groundharvested apples or apples with evident lesion when harvestedare directly diverted to apple processing plants Usually theseapples are not cold stored but directly deck stored Accordingto our results the non-desirable effect of deck storage maybecome more noticeable as fungal growth may not bedecelerated by previous cold storage Besides during appleharvest season it is expected that apples will stay in stand by atroom temperature for longer due to limited capacity ofprocessing plants

Noncold stored apples with no apparent spoilage had smallerlesion till the 4th day at 20 degC However long term storage at20 degC tended to equal patulin accumulation regardless of initialapple quality state Jackson et al (2003) reported that ciders madeby nonstored tree-picked apples did not contain patulin in anycase On the other hand patulin was detected in some batches oftree-picked apples air stored for 4 to 6weeks at approximately 0 to2 degC

Unfortunately in most cases quality of apples supplied toapple juice elaborators is difficult to assess as they are quiteheterogeneous and different lesions sizes can be found in thesame lot As seen in this study even if the lot is apparentlysound patulin contamination can appear in short periods of timeif they are slightly wounded and stored at room temperature Inour results regardless of the initial lesion sizes patulinaccumulation significantly increased after 48 h at 20 degC Thisis in agreement with results obtained by FAO (2001) in whichbulk storage is qualified as a CCP (Critical Control Point) and itrecommends not to extend this kind of storage for more than48 h However this lapse of time should change depending onthe quality state of the apples and other factors such astemperature or relative humidity

As a conclusion reducing to minimum deck storage oravoiding it should improve apple quality and patulin accumu-lation before processing begins As it is quite impossible tosucceed in processing in a continuous way from harvest or coldstorage to plant elaboration industries should assess quality ofapples entering the processing plant and adjust stand by timeaccording to it in order to minimise patulin accumulationPredictive modelling of patulin accumulation as a function ofapple quality (measured as overall lesion size) and time at roomtemperature would be an efficient tool for apple productindustries to program deck storage and elaboration

Patulin accumulation seemed to achieve a maximum after atime at 20 degC depending on initial lesion size Possibledegradation of patulin or dilution when necrosed tissueincreases should be studied

Acknowledgements

This work was supported by the Spanish Government(CICYT Comisioacuten Interministerial de Ciencia y Tecnologiacuteaprojects AGL 2004-07549-C05-01ALI and AGL 2004-06413ALI and Ramon y Cajal program) and the CatalonianGovernment (Projecte estrategravegic CeRTA 2005-2006 Seguretatbiogravetica y abiogravetica dels aliments)

265H Morales et al International Journal of Food Microbiology 116 (2007) 260ndash265

References

Acar JV Gokmen V Taydas EE 1998 The effects of processingtechnology on the patulin content of juice during commercial apple juiceconcentrate production Zeitschrift fuer Lebensmittel-Untersuchung undForschung A European Food Research and Technology 2007 328ndash331

Brackett RE Marth EH 1979 Ascorbic acid and ascorbate causedisappearance of patulin from buffer solutions and apple juice Journal ofFood Protection 42 864ndash866

Brause AR Trucksess MW Thomas FS Page W 1996 Determination ofpatulin in apple juice by liquid chromatography collaborative study Journalof AOAC International 79 451ndash455

Ciegler A Beckwith AC Jackson LK 1976 Teratogenicity of patulin andpatulin adducts formed with cysteine Applied and EnvironmentalMicrobiology 3 664ndash667

Doores S 1983 The microbiology of apples and apple products CRC CriticalReviews in Food Science and Nutrition 19 983ndash989

Fallik E Tuvia-Alkalai S Copel AWiseblurm A 2001 A short hot water rinseand brushes a technology to reduce post-harvest lossesmdash 4 years of researchIn Ben Arie R Philosoph-Hadas S (Eds) Proceeding of the 4th InternationalConference on Postharvest Acta Horticulturae vol 553 pp 413ndash416

FAO Food and Agriculture Organisation of the United Nations 2001 Manualon the application of the HACCP system in mycotoxin prevention andcontrol FAO Food and Nutrition Paper vol 73

Gopalakrishnan VK Sakthisekaran D 1991 Effect of patulin on albuminfraction on plasma proteins studied in rats Biochemistry International 25461ndash475

Huebner HJ Mayura K Pallaroni CL Ake CL Lemke SL Herrera PPhillips TD 2000 Development and characterization of a carbon-basedcomposite material for reducing patulin levels in apple juice Journal of FoodProtection 63 101ndash110

Jackson LS Beacham-Bowden T Keller SE Adhikai C Taylor KTChirtel SJ MErker RI 2003 Apple quality storage and washingtreatments affect patulin levels in apple cider Journal of Food Protection 66618ndash624

Liu B Yu F Wu T Li S Su M Wang M Shih S 2003 Evaluation ofgenotoxic risk and oxidative DNA damage in mammalian cells exposed tomycotoxins patulin and citrinin Toxicology and Applied Pharmacology191 255ndash263

Mariacuten S Morales H Hasan HAH Ramos AJ Sanchis V 2006 Patulindistribution in the tissue of Penicillium expansum-contaminated Fuji andgolden apples Food Additives and Contaminants 23 1316ndash1322

Moake MM Padilla-Zakour OI Worobo RW 2005 Comprehensivereview of patulin control methods in foods Comprehensive Reviews inFood Science and Food Safety 1 8ndash21

Morales H Mariacuten S Rovira A Ramos AJ Sanchis V in press-a Patulinaccumulation by Penicillium expansum during postharvest stages Letters inApplied Microbiology doi101111j1472-765X200602035x

Morales H Sanchis V Rovira A Ramos AJ Mariacuten S in press-b Patulinaccumulation in apples during postharvest effect of controlled atmospherestorage and fungicide treatments Food Control doi101016jfoodcont200610008

Osswald H Frank HK Komitowski D Winter H 1978 Long-term testingof patulin administered orally to SpraguendashDawley rats and Swiss mice Foodand Cosmetics Toxicology 16 243ndash247

Plunkett LM Turnbull D Rudricks JV 1992 Differences between adultsand children affecting exposure assessment In Guelian PS Henry CJOlis SS (Eds) Similarities and Differences Between Children and AdultsILSI Press Washington pp 79ndash94

Speijers GJ Franken MA Van Leeuwen FX 1988 Subacute toxicity studyof patulin in the rat effects on the kidney and the gastro-intestinal tract Foodand Chemical Toxicology 26 23ndash30

Sydenham EW Vismer HF Marasas WFO Brown NL Schlechter MVan Der Westhuizen L Rheeder JP 1995 Reduction of patulin in applejuice samples mdash influence of initial processing Food Control 6 195ndash200

Sydenham EW Vismer HF Marasas WFO Brown NL Schlechter MRheeder JP 1997 Influence of deck storage and initial processing onpatulin levels in apple juice Food Additives and Contaminants 14429ndash434